Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Adebowale, Adeyemi Philips
Doctor of Philosophy - PhD
University of British Columbia
Breadfruit (Artocarpus altilis Parkinson (Fosberg)) and its hybrids (Artocarpus altilis Ã Artocarpus mariannensis) are underutilized despite their significant potential. One barrier to broader acceptance may be the intense flavour present in some varieties, which has been described as âgreen tea-likeâ and is considered unpalatable by some individuals. A previous study identified varieties with more intense flavour profiles. The objective was to identify the chemical fingerprint responsible for the intense flavour in breadfruit varieties, examine commercial breadfruit flour from different geographical locations to determine whether they belong to mild or intense flavour varieties, and make recommendations to remediate the flavour intensity during processing to improve the utilization of breadfruit flour. I compared the physicochemical properties and phytochemical compositions of three breadfruit varieties with intense flavour profiles and three with mild flavour profiles. Total flavonoids using rutin equivalent (RE) were significantly higher in the mild varieties (0.48 ± 0.11 mg/RE 100g) compared to the intense varieties (0.17 ± 0.08 mg/RE 100g). Similarly, total protein content was higher in the mild varieties (32.66 ± 2.73 mg/100g) compared to the intense varieties (21.12 ± 2.42 mg/100g). Untargeted chemical analyses using high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) identified about 200 putative phytochemicals unique to the intense varieties. Data suggest that phenylpropanoids, vitamin C, L-amino acids, and flavonoids interact with taste receptors responsible for astringency, bitterness, sourness, and umami, contributing to the flavour intensity. Commercial breadfruit flour (acronyms as ADS and SIHCL) from similar geographical locations, exhibited comparable metabolite profiles. Based on the findings, I recommend several strategic approaches to mitigate the flavour intensity, including the use of sodium bisulfite, encapsulation of target compounds through cyclodextrins, and the use of spices to modulate the compounds responsible for the intense flavour. The adoption of these techniques in processing breadfruit will likely improve its acceptance across diverse cultures.
Flavour Chemistry in Breadfruit (Artocarpus altilis Parkinson (Fosberg)) and Its Hybrids (Artocarpus altilis Ã Artocarpus mariannensis) by Adeyemi Philips Adebowale M.Sc., University of Ibadan, Ibadan, Nigeria, 2019 B.Tech., Federal University of Technology, Akure, Nigeria, 2014 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) May 2025 © Adeyemi Philips Adebowale, 2025 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a dissertation entitled: Flavour Chemistry in Breadfruit (Artocarpus Altilis Parkinson (Fosberg)) and Its Hybrids (Artocarpus altilis Ã Artocarpus mariannensis) submitted by Adeyemi Philips Adebowale in partial fulfillment of the requirements of the degree of Doctor of Philosophy in Biochemistry and Molecular Biology Dr. Susan J. Murch, Chemistry, Irving K. Barber Faculty of Science Supervisor Dr. Wesley F. Zandberg, Chemistry, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Micheal K. Deyholos, Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Lauren A.E. Erland, Agriculture, University of the Fraser Valley, Abbotsford Supervisory Committee Member Dr. Kevin Hanna, Centre for Environmental Assessment Research University Examiner Dr. James J. Harynuk, Chemistry, University of Alberta, Edmonton, Alberta External Examiner iii Abstract Breadfruit (Artocarpus altilis Parkinson (Fosberg)) and its hybrids (Artocarpus altilis Ã Artocarpus mariannensis) are underutilized despite their significant potential. One barrier to broader acceptance may be the intense flavour present in some varieties, which has been described as âgreen tea-likeâ and is considered unpalatable by some individuals. A previous study identified varieties with more intense flavour profiles. The objective was to identify the chemical fingerprint responsible for the intense flavour in breadfruit varieties, examine commercial breadfruit flour from different geographical locations to determine whether they belong to mild or intense flavour varieties, and make recommendations to remediate the flavour intensity during processing to improve the utilization of breadfruit flour. I compared the physicochemical properties and phytochemical compositions of three breadfruit varieties with intense flavour profiles and three with mild flavour profiles. Total flavonoids using rutin equivalent (RE) were significantly higher in the mild varieties (0.48 ± 0.11 mg/RE 100g) compared to the intense varieties (0.17 ± 0.08 mg/RE 100g). Similarly, total protein content was higher in the mild varieties (32.66 ± 2.73 mg/100g) compared to the intense varieties (21.12 ± 2.42 mg/100g). Untargeted chemical analyses using high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) identified about 200 putative phytochemicals unique to the intense varieties. Data suggest that phenylpropanoids, vitamin C, L-amino acids, and flavonoids interact with taste receptors responsible for astringency, bitterness, sourness, and umami, contributing to the flavour intensity. Commercial breadfruit flour (acronyms as ADS and SIHCL) from similar geographical locations, exhibited comparable metabolite profiles. Based on the findings, I recommend several strategic approaches to mitigate the flavour intensity, including the use of sodium bisulfite, encapsulation of target compounds through cyclodextrins, and the use of spices to modulate the compounds responsible for the intense flavour. The adoption of these techniques in processing breadfruit will likely improve its acceptance across diverse cultures. iv Lay Summary Breadfruit (Artocarpus altilis Parkinson (Fosberg)) is a food crop with rich nutritional compositions and industrial purposes. Despite its potential, these benefits have not been utilized at optimal levels. However, variety exists in breadfruit with an array of different traits, especially the flavour intensity. Therefore, I hypothesized that there are chemicals in some breadfruit varieties that are not present in others. The goal of this work is to determine the chemical influence on the intense flavour properties in breadfruit using validated and standardized analytical methods for the analysis. Additionally, I analyzed breadfruit flour samples from different geographical locations to explore regional variations. The results revealed that several compounds such as flavonoids,, vitamin C, tannins, proteins, and their interaction can cause the relative difference in the flavour intensity of breadfruit. Finally, this thesis proposes methods for food producers to remediate flavour intensity, which could be applied to improve the flavour profile of breadfruit for future consumption and industrial use. v Preface Chapter 1 is where I critically reviewed the literature, summarized, and graphed all of the data that explains the diversity and variability of breadfruit and its peculiarities. Also, I explored for studies that explained mass spectrometry methods and their applications. This was supervised by Dr. Susan J. Murch and Dr. Wesley F. Zandberg. Some parts of this chapter were presented as a guest lecture to third year analytical chemistry class (CHEM 311) in UBCO: Adebowale, A. P. Zandberg, W. F. & Murch, S. J. (2024). Mass Spectrometry Methods and Applications. Guest Lecture. CHEM 311, Irving K. Barber Faculty of Science, University of British Columbia, Kelowna, BC, 04 March 2024. Chapter 2 is a work I conducted in Dr. Susan J. Murchâs Lab at the University of British Columbia, Okanagan on breadfruit flour and tissue identified as Mild and Intense Flavour Varieties. The samples used was organized and collected by Dr. Maxwell Jones and Dr. Susan J. Murch. Furthermore, past students from the Dr. Susan J. Murch Lab: Dr. Maxwell Jones, Dr. Ying Liu, and Abisola Z. Kehinde provided data that formed the background for the determination of assays to study the phytochemical and physicochemical properties of the Mild and Intense Flavour Varieties. I conceived and designed the study. I performed all the experimental work in Dr. Susan J. Murchâs Lab and the UBCO Chemistry Teaching Labs. Part of the results of this output was presented at NHPRS conference in Vancouver, British Columbia and being prepared for publication: Adebowale, A. P. Zandberg, W. F. & Murch, S. J. (2024). Discovery of Flavour Chemistry in Breadfruit. NHPRS/CHFA conference. 03 to 05 April 2024. Chapters 3 and 4 are based on an untargeted metabolomics approach through standardized protocol at Dr. Susan J. Murch Labâs in conjunction with Supra Research & Development using ultra-high pressure liquid chromatography (UHPLC) and gas chromatography (GC) tandem mass spectrometry (MS/MS). I designed the studies alongside Dr. Susan J. Murch. Dr. Lauren A.E.s Erland assisted with the sample preparation for UHPLC-MS/MS, and R.T. Giebelhaus supported sample preparation, method validation, analysis, and interpretation of GC-MS/MS data. In addition, Sarah Marie Lyons, Alisha Greene, Seamus Riordan-Short and Ryan Hayward at Supra Research & Development provided technical assistance for the instruments and data collection. vi Part of the results of this was presented at NHPRS/CHFA conference in Vancouver, British Columbia. The findings of the chapters are being prepared for publication: Adebowale, A. P. Erland, A. E. Sarah, L. M., Ragone, R., & Murch, S. J. (2025). Metabolomics-Guided Discovery of Flavour Chemistry in Breadfruit (Artocarpus altilis Parkinson (Fos)) and Its Hybrids (Artocarpus altilis Ã Artocarpus mariannensis). Chapter 5 is a work on ten commercial breadfruit flour. It was designed alongside Dr Susan J. Murch. The commercial breadfruit samples were shipped from countries in different continents where breadfruit is commercialized such as Africa, Asia, the Caribbean, the Pacific Island, and North America. All experimental work, data analysis and interpretation were performed by me in Dr Susan J. Murchâs Lab and the UBCO Chemistry Teaching Labs. A version of Chapter 5 will be prepared for publication. Chapter 6 focused on the techniques that would remediate the flavour intensity in breadfruit. I put together the methods. The methods focused on the metabolites that signal flavour intensity in breadfruit. A version of Chapter 6 will be prepared for publication: Adebowale, A. P. & Murch, S. J. (2025). Flavour Intensity in Commercial Breadfruit Flour from Different Locations and Its remediation (Manuscript in process). Presentations: Data in this thesis was presented at the Three Minutes Thesis 2024 Competition: Adeyemi P. Adebowale and Susan J. Murch (2024). False Signal. 3MT. Irving K. Barber Faculty of Science, University of British Columbia, Kelowna, February 26, 2024. Data in this thesis was also presented at the 8th Chemistry and Biology Graduate Symposium: Adebowale, A. P. & Murch, S. J. (2024). Flavour Intensity in Commercial Breadfruit Flours from Different Geographical Locations and Its Remediation. Annual Chemistry and Biology Graduate Symposium. Irving K. Barber Faculty of Science, University of British Columbia, Kelowna, BC, November 22, 2024. Generative Artificial Intelligence (GAI) was used for checking grammatical errors. In addition, it assisted in univariate, multivariate and functional analyses on Metaboanalyst ver 6. s vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................ xi List of Figures .............................................................................................................................. xii List of Symbols and Abbreviations .......................................................................................... xvi Acknowledgements ......................................................................................................................xx Dedication .................................................................................................................................. xxii Chapter 1: Introduction and Literature Review.........................................................................1 1.1. Taxonomy and Germplasm Collections........................................................................... 2 1.2. Origin of Breadfruit, Inspiration for Transfer and, Its Propagation ................................. 4 1.3. Diversity and Nomenclature of Breadfruit ...................................................................... 8 1.4. Breadfruit Metabolites Composition and Antioxidative Potential................................. 12 1.5. Economic Importance of Breadfruit and Environmental Effects .................................. 14 1.6. Breadfruit for Food Security .......................................................................................... 17 1.6.1. Handling and Processing of Breadfruit ................................................................... 17 1.7. Challenges Affecting the Widespread Use of Breadfruit .............................................. 20 1.8. Flavour and Its Formation in Food Crops ...................................................................... 21 1.8.1. Sensorial Properties of Breadfruit Products and Acceptability ........................ 25 1.9. Volatile Compounds Discovered in Breadfruit.............................................................. 28 1.10. Chromatographic Separation and Techniques ............................................................. 30 1.11. Mass Spectrometry....................................................................................................... 32 1.11.1. Mass Spectrometry Methods................................................................................. 34 1.11.2. The Application of Metabolomics ....................................................................... 39 1.12. Mass Spectrometry Data Processing through Software ............................................... 42 1.13. Statistical Modeling for Metabolomics Data .............................................................. 43 1.14. Study Rationale ............................................................................................................ 45 1.14.1. Thesis Objectives .................................................................................................. 46 viii Chapter 2: Understanding the Flavour Chemistry of Breadfruit Varieties through Phytochemical Assays ............................................................................................................. 48 2.1. Synopsis of Chapter ....................................................................................................... 48 2.1.1. Objectives ............................................................................................................... 48 2.2. Materials and Methods ................................................................................................... 49 2.2.1. Breadfruit sample collection and preparation ......................................................... 49 2.2.2. Reagents .................................................................................................................. 51 2.3. Chemical Analyses......................................................................................................... 51 2.3.1. Acidity Determination ............................................................................................ 51 2.3.2. Brix and D-Glucose analysis ................................................................................... 52 2.3.3. Reducing Sugars Determination ............................................................................. 52 2.3.4. Total Phenol and Flavonoid Content ...................................................................... 53 2.3.5. 1,1-diphenyl-2-picrylhydrazyl (DPPH) Determination for Antioxidant Potential . 54 2.3.6. Vitamin C Determination ........................................................................................ 54 2.3.7. Total Protein Analysis............................................................................................. 55 2.3.9. L-amino acids Determination .................................................................................. 56 2.3.10. Correlation-Based Network .................................................................................. 57 2.4. Results ........................................................................................................................ 57 2.4.1. Preliminary Data Analysis of Breadfruit Cultivars ................................................. 57 2.4.2. Breadfruit Morphological Attributes ...................................................................... 58 2.4.3. Physicochemical Properties .................................................................................... 61 2.5. Discussion ...................................................................................................................... 68 2.6. Conclusion and Further Analysis ................................................................................... 72 Chapter 3: Metabolomics-guided Discovery of Flavour Chemistry in Breadfruit Varieties using Ultra-High Pressure Liquid Chromatography â Mass Spectrometry ........................73 3.1. Synopsis ......................................................................................................................... 73 3.2. Objectives ...................................................................................................................... 73 3.3. Materials and Methods .................................................................................................. 73 3.3.1. Breadfruit Sample Collection and Preparation ....................................................... 73 3.3.2. Reagents .................................................................................................................. 74 3.3.3. Liquid Chromatography â High Resolution Mass Spectrometry ........................... 74 ix 3.3.4. Data Processing and Exporting ............................................................................... 75 3.3.5. Detection of Significant Features and Functional Analysis .................................... 75 3.3.6. Synthetic Biotransformation .................................................................................. 76 3.4. Results ............................................................................................................................ 76 3.5. Discussion ...................................................................................................................... 95 3.6. Conclusion and Further Analysis ................................................................................... 98 Chapter 4: Metabolomics-guided Discovery of Flavour Chemistry in Breadfruit Varieties using Gas Chromatography Tandem Mass Spectrometry ...................................................100 4.1. Synopsis of Chapter ..................................................................................................... 100 4.2. Materials and Methods ................................................................................................. 100 4.3. Results .......................................................................................................................... 103 4.4. Discussion .................................................................................................................... 106 4.5. Conclusion and Further Analysis ................................................................................. 108 Chapter 5: Understanding the Flavour Chemistry of Commercial Breadfruit Flour from Different Geographical Locations ........................................................................................109 5.1. Synopsis ....................................................................................................................... 109 5.2. Materials and Methods ................................................................................................. 110 5.3. Results ......................................................................................................................... 113 5.4. Discussions .................................................................................................................. 120 5.4.1. Geographical Profiling of Commercial Breadfruit Flour...................................... 120 5.5. Conclusion and Further Analysis ................................................................................. 123 Chapter 6: Strategic Techniques in Mediating Flavour Intensity in Breadfruit .............125 6.1. Synopsis of Chapter ..................................................................................................... 125 6.2. Methods ...................................................................................................................... 125 6.2.1. Flavour Techniques for Improved Acceptability in Breadfruit ............................ 125 6.2.1.1. Modification of Polyphenols.......................................................................... 126 6.2.1.2. Encapsulation through Cyclodextrins ........................................................... 128 6.2.1.3. Incorporation of Receptor Blockers and Modifiers ...................................... 129 6.2.1.4. Enzymatic Modifications and Fermentation .................................................. 130 6.2.1.5. Size Reduction ............................................................................................... 131 6.2.1.6. Use of Spices.................................................................................................. 131 x 6.2.1.7. Adsorbent Resins ........................................................................................... 131 6.2.1.8. Extraction of Sub-Critical Water ................................................................... 132 6.3. Industrial Application Technique for Intense Breadfruit .......................................... 132 Chapter 7: Summary .................................................................................................................134 Bibliography ...............................................................................................................................138 Appendix .....................................................................................................................................173 xi List of Tables Table 1-1. The Classification of Breadfruit Systematics with their Scientific and Common Nameâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦....â¦â¦â¦2 Table 1-2. Breadfruit Vernacular Namesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦8 Table 1-3.Summary of Breadfruit Bligh Named Cultivars Linked to Some Geographical Region.11 Table 1-4. Summary of Literature Search about Processing of Breadfruit in Different Geographical Regionsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..18 Table 1-5. Summary of Literature Search about Breadfruit, Flavour and Sensory Using Web of ScienceTM and Google ScholarTMâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦â¦â¦â¦â¦â¦26 Table 1-6. Volatile Compounds Discovered in Breadfruitâ¦â¦â¦â¦â¦.â¦â¦â¦...â¦â¦â¦â¦â¦â¦.28 Table 1-7. Strengths and Weaknesses of Orbitrap and Triple Quad Mass Spectrometric Methodsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..39 Table 1-8. Strengths and Weaknesses of the Statistical Conceptsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦44 Table 2-1. Breadfruit (Artocarpus altilis and A. altilis Ã A. mariannesis) selected for flavour intensityâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦.50 Table 2-2. HPLC gradient elution for the breadfruit amino acid studyâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..56 Table 2-3. Breadfruit morphological quality propertiesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.59 Table 3-1. Quality control for the features in the Mild and Intense varietiesâ¦â¦â¦â¦â¦â¦â¦â¦...77 Table 3-2. Features Identified as significant by the Partial Least Squares-Discriminant Analysis (PLS-DA) for intenseâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.80 Table 3-3. Bio transformed Metabolites in the Mild and Intense from The Flavonoidsâ¦â¦â¦â¦.94 Table 4-1. Features of Total Ion Chromatogram (TIC) for the Reference Standard Mix â¦â¦â¦103 Table 5-1. List of Commercial Breadfruit Flour and their Originsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦110 Table 5-2. Summary of the Flavour Intensity of Breadfruit Commercial Flour with different Geographical Locationsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...124 Table 6-1. Application of Cyclodextrins for Flavour and Taste Improvementsâ¦â¦â¦â¦â¦â¦â¦128 xii List of Figures Figure 1-1. Artocarpus altilis (Parkinson) Fosberg (breadfruit) collection. This contains 326 accessioned trees from over 120 cultivars in NTBG Breadfruit Institute Germplasm Repository, Hana, Maui, Hawaii. Photo Credit: Susan J. Murchâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦4 Figure 1-2. Summary for Publication and Citations for Breadfruit. Times breadfruit cited and published over time from 1922 - 2024 (Source: web of science database â 606 publications and 6,564 citations). ............................................................................................................................... 7 Figure 1-3. Breadfruit Domestication and Development Across the Pacific Islands. The yellow and grey colour show the change in the breadfruit as human migrate from Melanesia to Polynesia. The change in the seed show its disappearance towards East Polynesia. (Figure created on Biorender)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..11 Figure 1-4. Breadfruit Vodka with the Gold Award Word Spirits Competitionâ¦â¦â¦â¦â¦â¦â¦15 Figure 1-5. Processing of breadfruit and Breadfruit flour packaged in an exhibition in Samoa...20 Figure 1-6. Breadfruit in Canada â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦.21 Figure 1-7. The Summary of the Breadfruit Taste Compounds, and the Flavour Intensityâ¦â¦â¦.22 Figure 1-8. Protein Interaction with Flavour Compounds in Food Matrix (Modified from Reineccius, 2006; Wang and Arntfield, 2017). Figure created on Biorender â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.24 Figure 1-9. Electrospray Ionization in Positive Mode. Figure created on Biorenderâ¦â¦â¦â¦â¦34 Figure 1-10. Quadrupole Mass spectrometer (Figure created on Biorender)â¦â¦â¦â¦â¦â¦â¦â¦35 Figure 1-11. Schematic for Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometerâ¦â¦â¦38 Figure 2-1. The workflow for the mild and intense flavour varieties of breadfruit (Picture created on Biorender)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.50 Figure 2-2. Nutritional Composition of Breadfruit (A. altilis, A. altilis x A. mariannensis). (A) minerals, (B) vitamins, (C) Total Dietary Fiber, (D) Carbohydrates. Blue bars represent the means for the mild cultivars and green bars represent means for Intense cultivars. Error bars represent the standard error of the means. Significance difference (p-0.05) is indicated using t-test with between the meansâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦58 xiii Figure 2-3. Growth of Different Varieties of Breadfruit (Artocarpus Altilis) and Its Hybrids (Artocarpus altilis Ã Artocarpus mariannensis) determined by geographical origin and season (Jones et al., 2010). Season, climate and origin did not differ for the mild and intense flavour varieties chosen for this study. (a) Male inflorescence of selected mild and Intense breadfruit varieties, (b) Climatic conditions of the genetic origins of selected breadfruit varieties with different in flavour intensities. Bar represents the standard error of 6 replicates within each treatmentâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦60 Figure 2-4. Physicochemical Analyses of Mild and Intense Flavour Varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The spectrophotometric analyses were performed on Microplate Reader. The flavonoids, proteins, soluble solids, and DPPH exhibit significant differences between the groups. (a) pH, (b) Titrable Acidity (potentiometric method, %malic acid), (c) D-glucose (Megazyme assay), (d) Brix - soluble solids, (e) Total Phenols (gallic acid equivalent), (f) Total Flavonoids (rutin equivalent), (g) Total Protein content (Bicinchoninic acid assay), (h) Vitamin C - Iodine Titration, (i) DPPH (2,2-diphenyl-1-picrylhydrazyl), (j) Reducing sugars. Bars represents Mean±Standard error calculated from the nine replicates in each variety. Yellow denotes increased in mild and blue denotes increased in intenseâ¦â¦â¦â¦â¦â¦â¦â¦â¦.63 Figure 2-5. L-amino acids Determination in the Mild and Intense Breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) through high performance liquid chromatography (HPLC)methodâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...65 Figure 2-6. (a) Total Tannins in Mild and Intense Flavour Varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). (b) Sodium Bisulfite Treatment (0.03 %) to the Mild and Intense Breadfruit (c) Sodium Bisulfite Treatment (0.3 %) to the Mild and Intense Breadfruitâ¦66 Figure 2-7. Possible Proteins and Phenols Structural Changesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..72 Figure 3-1. Significant features for the Mild and Intense flavour varieties through partial least squares-discriminant analysis (PLS-DA) (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The PLS-DA identified features Variable Importance in Projection (VIP) scores. This analysis was performed on MetaboAnalyst Ver. 6.0. (a) scores plot for the mild and intense, (b) Features identified between the groups, (c) accuracy of the componentsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦78 Figure 3-2. Detecting the significant features for the polar metabolites in Mild and Intense Flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) through chemometrics analyses on metabonalystver6.0. Filtering, normalization, transformation, and scaling of data normalization were performed prior to the analyses. The significant features correlate across the analyses (a) Volcano plot with the Fold change between the groups showed 159 significant features upregulated and 57 significant features downregulated. (b) Heatmap cluster analysis (c) Pattern hunter analysis, Pearson r as a choice of distance measureâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦81 xiv Figure 3-3. Principal Component Analysis for the significant in the PLS-DA, Volcano plot and Pattern Hunter between the Mild and Intense flavour for the polar metabolitesâ¦â¦â¦â¦â¦â¦â¦83 Figure 3-4. Summary of Significance Analysis of Microarray (SAM) between the Mild and Intense flavour for the polar metabolitesâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.84 Figure 3-5. Untargeted metabolomics profiling for the polar metabolites through LC-MS/MS in the Mild and Intense Flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensisâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..86 Figure 3-6. Untargeted metabolomics analysis for unique pathways for the intense flavour varieties (Meinpadahk, Porohiti and Meiarephe)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦88 Figure 3-7. Untargeted Metabolomics Analysis for Unique Pathways for The Intense Breadfruit varieties (Meinpadahk, Porohiti and Meiarephe)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦90 Figure 3-8. Functional analysis for the mild and intense breadfruit varieties through orthogonal partial least squares-discriminant analysis (OPLS-DA) for the Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis)â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦â¦.92 Figure 3-9. Untargeted metabolomics profiling for the polar metabolites through LC-MS/MS in the Mild and Intense flavour breadfruit varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦...93 Figure 3-10. Relative Intensity of Metabolites through the Synthetic Biotransformation in the Mild and Intense flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.95 Figure 4-1. Method of preparation of breadfruit (Artocarpus altilis) and its hybrids (Artocarpus altilis Ã Artocarpus mariannensis) samples for the non-polar metabolites untargeted analysis by Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS)â¦â¦â¦â¦â¦â¦â¦.â¦â¦â¦â¦101 Figure 4-2. Determining the significant non-polar metabolites between the Mild and Intense Flavour Varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) (Mild<Intense)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦.105 Figure 4-3. Clustering analysis for the significant metabolites between the Mild and Intense breadfruit varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis)â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦106 Figure 4-4. Formation of Off-Flavour in the Mild and Intense Breadfruit Varietiesâ¦â¦â¦â¦â¦108 xv Figure 5-1. The Workflow for Tannins Determination in Breadfruit Using Spectrophotometric Approach on a Microplate Readerâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦111 Figure 5-2. Principal Components Analysis of Intense Breadfruit Varieties with Ten Commercial Breadfruit Flour. (a) PC1 vs PC2 scores plot (b) PC1 vs PC2 loading plot (c) PC1 vs PC3 scores plot (d) PC1 vs PC3 loading plotâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..114 Figure 5-3. Acidity, Sugars, and Total Soluble Solids in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locationsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..117 Figure 5-4. Total Protein in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locationsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..118 Figure 5-5. Scavenging Activity in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locationsâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...â¦119 Figure 6-1. Enzymatic Conversion of Phenols to O-quinone (bitter compound) through Polyphenol oxidase. Figure created on Biorenderâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦...127 Figure 6-2. The encapsulation and stability of targeted compound in the taste bud release in the Intestines. Guest â the targeted compound. Host â cyclodextrins. Figure created on Biorenderâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..129 Figure 6-3. Proposed Schematic Workflow For Reducing The Intense Flavour In Breadfruit. SBS â sodium bisulfite, SCW- subcritical waterâ¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦â¦..â¦â¦133 xvi List of Symbols and Abbreviations The following is a list of abbreviations and symbols employed in this thesis: º Degrees °C Degree Celsius % Percent > Greater than â Approximately equal λ Lambda MΩ Megaohms µg Microgram, 10â6 g µL Microlitre, 10â6 L µm Micrometre, 10â6 m µM Micromolar, 10â6 M Aa Artocarpus altilis ACN Acetonitrile ADAP Automated Data Analysis Pipeline AGC Automatic Gain Control Ala Alanine Am A. mariannensis ANOVA Analysis of variation AOAC Association of Official Analytical Chemistsâ¢ AQC 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate Arg Arginine Asn Asparagine Asp Aspartic acid Aux Auxiliary °B Degrees brix BCA Bicinchoninic acid BF Breadfruit BSA Bovine serum albumin C Carbon C18 Octadecyl-functionalized silica Ca Calcium CaCO3 Calcium carbonate CO2 Carbon dioxide C-trap Curved linear trap Cys Cysteine Da Dalton DDA Data Dependent Acquisition xvii DPPH 2,2-Diphenyl-1-picrylhydrazyl ESI Electrospray ionization EI Electron Impact EtOH Ethanol FAO Food Agriculture Organization FDA U.S. Food and Drug Administration FSM Federation States of Micronesia g Gram GA Gibberellic acid GAE gallic acid equivalent GC-MS Gas chromatography-mass spectrometry GC-MS/MS Gas chromatography-tandem mass spectrometry GIV 2,2,3-trimethylcyclopentyl Glu Glutamic acid Gly Glycine GOPOD Glucose oxidase/peroxidase GUI Graphical user interface GSEA Gene Set Enrichment Analysis HCD High energy collisional induced cell HCl Hydrochloric acid HEPES 4-(2-Hydroxyethyl)-piperazine ethane sulfonic acid HESI Heated electrospray ionization His Histidine HPLC High-performance liquid chromatography HRMS High resolution mass spectrometry Int Intense Flavour Varieties IAA Indole acetic acid IUPAC International Union of Pure and Applied Chemistry kg Kilogram, 103 g kV Kilovolts KEGG Kyoto Encyclopedia Biomolecules of Genes and Genomes KI Potassium iodide KIO3 Potassium iodate Ile Isoleucine L Litre LAB Lactic Acid Bacteria Leu Leucine LC-MS Liquid chromatography mass spectrometry LC-MS/MS Liquid chromatography-tandem mass spectrometry Lys Lysine xviii m Metre M Molar, moles/litre m/z mass-to-charge MALDI Matrix-assisted laser desorption MCT Micro-centrifuge MeOH Methanol Met Methionine min Minutes Mild Mild Flavour Varieties mL Millilitre, 10â3 L mm millimetre mM Millimolar, 10â3 M MM Millimetre Ã millimetre MRM Multiple reaction monitoring MS Mass spectrometer MS/MS Tandem mass spectrometry MSI Metabolomics Standards Initiative N Normality N North Na Sodium NaCl Sodium chloride NaâSâOâ Sodium thiosulfate NIST National Institute of Standards and Technology. NTBG National Tropical Botanical Garden O2 Oxygen OPLS-DA Orthogonal partial least squares discriminant analysis PCA Principal component analysis Phe Phenylalanine PLS-DA Partial least squares discriminant analysis ppm Parts per million PPO Polyphenol oxidase Pro Proline PVDF Polyvinylidene difluoride pVACs pro-vitamin A carotenoids QqQ Triple Quadrupole r Pearsonâs correlation coefficient RE Rutin equivalent RF Radio frequency ROS Reactive oxygen species rpm Revolutions per minute xix rt Retention time RSD Relative standard deviation SAM Significance analysis of microarray Ser Serine SDG Sustainable development goal St.Dev Standard deviation SWE Sub-critical water extraction SVG St. Vincent and the Grenadines TA Titrable Acidity TE Trolox equivalent Thr Threonine ToF Time of Flight TRIS Tris(hydroxymethyl)-aminomethane Trp Tryptophan Trolox (Â±)-6-Hydroxy-2,5,7,8- tetramethylchromane-2-carboxylic acid TTFF Trees That Feed Foundation TSS Total soluble solids Tyr Tyrosine UHPLC Ultra high-performance liquid chromatography UPLC Ultra performance liquid chromatography V Volume Val Valine Ver Version VIP Variable Importance in Projection VOCs Volatile organic compounds W West WHO World Health Organization WoSâ¢ Web of Science databaseâ¢ xx Acknowledgements I would like to appreciate the collective efforts of the following people who have assisted directly or indirectly during my PhD program, and respectfully acknowledge the traditional, and unceded territory of the Syilx Okanagan people where my program was conducted: First and foremost, I would like to express my deepest gratitude to my supervisor Dr. Susan J. Murch for the rare support and mentoring in the last three and half years. She really stood by me and made sure that things worked whenever there was turbulence. Indeed, she was magical with her methods, and brilliance to manage circumstances and scenarios. Thank you for believing in me. It was such an amazing and fulfilling experience, I will surely live to remember her for the rest of my life, and even thereafter. Also, I would like to thank my co-supervisor, Dr. Wesley F. Zandberg, for his kind support, and the confidence he has in me throughout the journey. I am also grateful to the rest of my supervisory committee members: Dr Michael K. Deyholos, and Dr Lauren AE. Erland for their continued support, and advice throughout the graduate program. Likewise, I express my sincere thank you to Dr. Emmanuel T. Osei, and Dr. Kirsten Wolthers for graciously accepting to be the external examiner and neutral chair during my comprehensive exam. I would like to extend my gratitude to the past and present members of the Plant Secondary Metabolite Analytical Research Team (PlantSMART) Lab who have immensely assisted me during this research or whose works in the lab provided a platform for me to succeed: Dr. Paul Shipley, Dr. Dianne Ragone, Dr. Maxwell Jones, Dr. Ying Liu, Dr. Stephanie Bishop, Abisola Kehinde, Ryland Giebelhaus, Julia Solonenka, Tom Head, Simona Mastroianni, Pawanjit Sandhu, Daniel Gaudet, Hall Barbara, Ieva Zigg, Alisha Greene, Karine Jarzecki, Trevor Fox, Georgia Chalmers, and Olivia Carlson. I acknowledge the support of the staff of Supra Research and Development: Dr. Rob OâBrien, Sarah Mary Jones, Seamus Riordan-Short, and Ryan Hayward. Additionally, the staff in the University of British Columbia, Okanagan (UBCO) Teaching Chemistry Lab always allowed me to use the instruments and wares, in particular Michele Cannon, Emma Mitchell, and Eric Dennis. I thank the Department of Chemistry, the College of Graduate Studies (CoGS), UBCO for the rare opportunity to have worked as sessional instructor/lecturer, and financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC), and through the awards xxi of scholarships that have supported my studies and research in the last three years and half. In addition, I am grateful to lecturers of the department and the colloquium series with interesting and informative lectures which helped me in shaping various parts of my thesis. I would like to extend my appreciation to my colleagues in other labs on campus for their assistance with chemicals and reagents we do not have in our lab, or through their encouragement. Additionally, I recognize the uplifting words, and continued encouragement from my past students in CHM 1X3 and CHM 211âJared Brown, Courtney Fornasero, Fern Deyholos, Jack Verdurmen, Mathew Bourget, Sophie Vanslyke, Elizabeth Mckenzie, Alexander Niazi, Tory Taylor, Suroor Ajani, Kevin Loro, and Samuel Akinlusi. Every time we cross paths on campus; their words serve as motivation and lift my spirit. Also, I would like to acknowledge Bob Murch, and Dr. Spencer Murch for their love and kindness they extended to me. Sincerely, they always make me feel at home anytime I am with them. I appreciate my former supervisors in Nigeria for their guidance, invaluable advice, and showing me the way to food and nutrition security as an area of research to pursueâProfessor G.O. Adegoke, Professor A. A. Badejo, and Dr. F. O. Adeboyejo. In addition, I thank Nigerian Breweries plc, Lagos, and Ibadan Brewery, and West African Seasoning Company Limited, Lagos Plant, Nigeria (Ajinomoto Nigeria) for granting me the rare opportunity to work prior to my PhD; this experience provided a flavour and taste background knowledge to this thesis. Finally, this would not have been possible without the huge moral support I enjoyed from my parents: Mr. and Mrs. Margaret, Ebenezer Adebowale, and my elder brothers: Tunji, Banjo Adebowale. Also, I will not forget my amiable friends and colleagues, Dr. Jude Okolie, Dr. Peter Ohemeng, Oladimeji Adewusi, and Ikenna Okehie; they really supported me with everything humanly possible within their reach for me to succeed. It is difficult to list all but none is forgotten. Thank you...Thank youâ¦Thank youâ¦ xxii Dedication I dedicate this thesis to Almighty God, the giver of life and wisdom. To Him alone be the glory. Great is thy faithfulness! 1 Chapter 1: Introduction and Literature Review The adoption of underutilized crops, which are crops not fully used to their potential or capacity, is critical to addressing global food and nutrition security challenges. One of such crops with significant potential to contribute to this goal is breadfruit (Turi et al., 2015a). Breadfruit (Artocarpus spp., Moraceae) is a traditional staple in the Pacific Islands, and is increasingly consumed in various countries worldwide due to its nutritional value, and sustainability as a local food source. It is important to recognize that flavour plays a crucial role in the acceptability of food, regardless of its nutritional composition, and other desirable qualities (Liem et al., 2019). Specifically, the variations in the sensory attributes of breadfruit have not been adequately explored, and the chemistry of the flavour compounds in breadfruit has received limited attention. To the best of my knowledge, there is a lack of detailed and explicit information on the metabolites responsible for the flavour intensity in breadfruit. Therefore, this thesis aims to investigate the flavour chemistry of breadfruit through physicochemical and phytochemical assays, as well as metabolomics profiling of both volatile and non-volatile compounds in breadfruit, and commercial breadfruit flour. The findings from this research will provide insights into the metabolites, and their interactions that contribute to flavour formation in breadfruit. Furthermore, technological approaches will be recommended to producers for mitigating the flavour intensity, thereby encouraging the use of breadfruit flour in food applications. Conversely, in order to build a comprehensive understanding of this study, and its connection to the differences in flavour chemistry, it is essential to explore underlying information related to breadfruit, including its taxonomy, origin, morphology, variability, nomenclature, domestication, mode of propagation, nutritional composition, and the flavour characteristics of its commercialized products. To provide a broader and more objective perspective, it is equally important to extend this understanding to the general field of flavour chemistry in foods. Additionally, it is necessary to clarify the principles, capabilities, and reasons for the use of tools, and standardized protocols, such as mass spectrometry, along with their methods and applications. This chapter aims to address these foundational aspects before delving into the specific objectives of the thesis. 2 1.1. Taxonomy and Germplasm Collections The Greek words âartosâ (bread) and âkarposâ (fruit) are put together to name the fruit, breadfruit (Jones et al., 2011a). Artocarpus altilis, formerly referred to as Artocarpus incisa and Artocarpus communis, belongs to the Moraceae (mulberry/fig) family (Fosberg, 1960; Jarrett, 1959). The Pacific Islanders have been cultivating breadfruit (Artocarpus altilis (Parkinson) Forsberg) for at least 3000 years ago (Zerega et al., 2004). For centuries, the Indigenous people have selected over 300 different cultivars. A. camansi Blanco (resulting in Artocarpus. altilis), Artocarpus. mariannensis Trecul or Artocarpus altilis Ã A. mariannensis (hybridization) make up the cultivars on these Islands (Jones et al., 2011a; Zerega et al., 2004). Furthermore, the variety of breadfruit are seeded and seedless, the seeded are commonly found in and around New Papua Guinea, and throughout Melanesia, whereas seedless cultivars are mostly found in the Polynesia Islands. The taxonomic classification of Artocarpus altilis is described in Table 1-1, and the draft genomes for twoâArtocarpus sp; breadfruit (Artocarpus. altilis) and jackfruit (Artocarpus. heterogenous)âhave also been released (Ragone, 2001; Murch et al., 2007; Sahu et al., 2019; Williams et al., 2017). Table 1-1. The Classification of Breadfruit Systematics with their Scientific and Common Name (Modified from Deewa et al. 2023) Rank: Scientific Name and Common Name Domain: Eukaryota Kingdom: Plantae Subkingdom: Viridaeplantae Infrakingdom: Streptophyta Division: Magnoliophyta Infradivision: Angiospermae Class: Dicotyledonae Super order: Rosanae Order: Urticales Family: Moraceae - Mulberries Genus: Artocarpus Species: Artocarpus altilis The Moraceae â often called the mulberry family or fig family â are a family of flowering plants comprising about 38 genera and >1100 species. The family includes well-known plants such as fig, banyan, breadfruit, jackfruit, mulberry, and Osage orange. The family is characterized as 3 having milky or sometimes watery latex. Leaves are mostly alternate, simple, entire, and pinnately veined. The genus Artocarpus comprises tropical plants primarily cultivated in Asia, particularly in South and Southeast Asia (Boonyaketgoson et al., 2020). This genus is a rich source of (PF) and more than 300 PFs have been isolated (Ye et al., 2019). Hakim et al. (2006) reported that flavonoids, stilbenoids, and arylbenzofurons are common phenolic compounds found in Artocarpus species, along with their chemical compositions. In line with these findings, Chaurasia and Pandey, (2022) discovered that the main classes of phytochemicals which were detected in abundance among the five Artocarpus species were flavonoids, glycosides, tannins, saponin, alkaloids, steroids, and triterpenoids. But these are just the metabolite classes, with sub-classes exist (Harborne, 1998). The texture is always smooth and skin colour is light green or yellow-green where A. mariannensis is heavily seeded, having dark skin when ripe and pulp is yellow more than A. altilis or A. camansi. (Jones et al., 2011a). For over hundreds or thousand years ago, two groups of interspecific hybrids of A. altilis and A. mariannensis with proximity have been bred (Fosberg, 1960; Ragone, 2007). The earlier hybrid is like the parent A. mariannensis, while the other hybrid is like A. altilis and A. camansi (Jones et al., 2011a). The ex situ germplasm has been established in order to preserve the genetic diversity of breadfruit (Ragone, 2007). Based on morphological attributes, the cultivars have been named and identified by leaf shape, or skin texture (Ragone, 1991; Ragone, 2004). However, they lack proper cultivar identifications (Ragone, 2007). As of today, the Breadfruit Institute (BFI) of the National Tropical Botanical Garden (NTBG) boosts of the largest active germplasm collection in the world in Kahanu Garden, Hana, Hawaiâi with ~330 accessions of breadfruit (A. altilis and A. altilis and A. mariannensis), breadnut (A. camansi Blanco), and dugdug (A. mariannensis TrÄcul) gathered throughout the world (Figure 1-1) (Ragone, 1997; Murch et al., 2008; Jones et al., 2011a). All the trees represent a cultivar in the germplasm, and they are conserved to avoid extinction. Based on the presence of seeds, size, skin, flesh texture, leaf, and fruiting season, due to these differences, the people in Pacific Island have cultivated hundreds of cultivars (Zerega et al., 2006). The genus Artocarpus has over 50 species and belongs to the Moraceae Family. There are thousands of cultivated varieties of A. altilis, A. camansi, A. mariannensis, and hybrids of these species. In previous ethnobotanical research, it was revealed that 132 cultivars in Vanuatu, 130 cultivars in Pohnpei, 70 cultivars from Fiji, over 40 and 30 cultivars in Samoa, and Tahiti, respectively (Fownes and Raynor, 1993; 4 Ragone et al., 2004). However, many of the cultivars have never been scientifically described. In particular, the distribution of breadfruit outside of the Pacific stemmed from about 10 cultivars of Polynesia origin, through Captain Bligh, and the French contemporaries in the later part of 18th century, and moved to their territory in Mauritius, and the Caribbean (Leakey and Roberts-Nkrumah, 2015; Powell, 1977; Ragone, 1997). Over the millennia, hundreds of traditional breadfruit cultivars have been named by the Pacific Islanders, with 539 distinct names from Vanuatu alone (Labouisse, 2016). In places like Samoa, cultivars are named with a generic name âuluâ together with a description of the fruit (Ragone et al. 2004), while Maâafala which is the most widely cultivated could referred to as âulu maâfalaâ (Ragone et al., 2004) similarly to the naming in Pohnpei; Mein-padahk or Mei-uhpw (Ragone and Raynor, 2009). Several ex situ germplasms have been established to preserve breadfruit genetic diversity (Ragone, 2007). Murch et al. (2008) and Shi et al. (2007) noted that natural disasters, disease, and other deleterious circumstances could fall on the ex situ germplasm collections. In addition, about 60 % of the plants do not survive cross-border propagation (Murch et al., 2008). According to their findings, it was demonstrated that the in vitro collection could assist in adding a measure of security to the NTBG collection. Figure 1-1. Artocarpus altilis (Parkinson) Fosberg (breadfruit) Collection. This contains 326 accessioned trees from over 120 cultivars in NTBG Breadfruit Institute Germplasm Repository, Hana, Maui, Hawaii. Each tree represent each cultivar of breadfruit. (Photo Credit: Susan Murch) 1.2. Origin of Breadfruit, Inspiration for Transfer and, Its Propagation In Westerâs article "The Breadfruit: A Plea for the Preservation of Varieties" published in 1922, Wester reported that Europeans first encountered breadfruit in the Marquesas in 1595 and 5 later found it across the Pacific archipelagoes. It was noted that many of the varieties recorded in earlier times are no longer known today. Wester also recounts a historical interpretation from Tahiti during the period 1788â1791, where inhabitants of the city were said to have eaten Araea, a type of red earth, during times of hardship. According to the mythical and fantastic story the writer cites, a man, moved by compassion for his sick son who could not eat the red earth, prayed to his god to take his life so that he might be transformed into food for his son. The prayer was answered: after his death, the man was buried, and from his body sprouted the "staff of life", that produced the seedless breadfruit that enabled his son to survive. Breadfruit, A. altilis (Parkinson) Fosberg was introduced to the Philippines during the 17th century by the Spanish before it was known to be out of Oceania in early 1700s (Ragone, 1997, Smith et al. 1992), and this has been adopted as a staple food by the Pacific Islanders majorly in Oceania (Melanesia, Micronesia and Polynesia), Jamaica and the Caribbean Islands for over 2000 - 3000 years still being cultivated (Zerega et al., 2004). It was described by colonial plantation owners as bland, reliable, abundant, and nearly labour-free source of energy for enslaved. Several reports were pitched to Sir Joseph Banks. Drawing on D. Mackayâs interpretation in his essay "Banks, Bligh and Breadfruit" (1974), it is noted that in July 1784, Hinton East stated that breadfruit, in contrast to the plantain, which was then among the most important provisions offered to the enslaved, "would be raised with infinitely less labour" (Mackay, 1974). Mackay also explained that Valentine Morris was claimed to have been the first to directly recommend breadfruit to Joseph Banks, a point that is supported by earlier accounts recorded by (Hawkesworth, 1773). Earlier before Banks, the French philosopher, Voltaire took the next tentative step by suggesting that breadfruit was 'a real treasure' which, if transplanted successfully into our climates, would soon surpass the 'conventional riches, even to the ends of the earth (Voltaire, 1769). Morrissey (2017) clarified that, in the Caribbean not all the breadfruit came from Tahiti, the Island of Timor now part of Indonesia contributed to the wide variety of plants delivered by Captain Bligh, when they sailed to the Caribbean with other fruit trees such as: mango, guava, rose apple, and Jackfruit from the gardens of St. Vincent and Bath, Jamaica. The West Indian beneficiaries shared equally the surviving trees and other from Timor that contains 300 breadfruit trees. Therefore, the Caribbean are now grateful to Tahiti and the Island of Timor through which 6 they are enjoying the fruits they currently have. The Jamaicans took the breadfruit suckers with them to Africa, following the failure of missionaries to make an impact in Africa in the early 1900. The Jamaicans missionaries were seen as a bridge connecting with their ancestral, and cultural roots while sharing the gospel. In 1843, the Moravian mission, the Baptists and Presbyterians were tasked to evangelize in the Gold Coast, Cameroun, and Nigeria. Before their departure, amidst tears and hugs they stated the myth: âWhen we go to Africa, we are not in a foreign country, Africa is our country and our home. Our grandfathers and great-grandfathers were taken from there and brought here. We go there to witness the grace of God not only to the European, but also to the African and our prayer is that the eyes of Africans whom we regard as our brothers may be opened to see Jesus Christ as Saviour of the World.â The mission sailed from Kingston Port, on board were donkeys, horses, mules and agricultural seeds and cuttings. These included breadfruit trees, mango, cocoa, coffee, guava, yam, cassava, plantains, and cocoyam. Through this way, the breadfruit suckers from Tahitian made their way to West Africa 50 years later. In addition, Fielding (2024) explained the transfer from the Pacific to the Caribbean. In Tahiti during the first voyage of the Endeavour by Joseph Banks, he hypothesized that the breadfruitâs effortlessness of cultivation might have divine implication, demonstrating the exemption of the Tahitian people from the Holy curse that destined humans to âearn their bread with the sweat of their brow. The appeal for breadfruit to be brought to the Caribbean Islands had nothing to do with the distinctiveness of its taste, but rather with its effortless and abundant productivity. The planters' interest in breadfruit was primarily economic, rather than gastronomic, and was driven by their reading of 'glowing reports from Pacific voyagers,' which emphasized breadfruit's productivity rather than its flavour (DeLoughrey, 2007). Due to the food insecurity ravaging the Island, the planters needed to increase their food imports. The British Caribbean imported grains in such abundance that the US came to be collectively known as the âbread coloniesâ (Schlebecker, 1987). In response to the emergence of an independence movement in thirteen of Englandâs American colonies, the British government imposed restrictive trade controls, severely limiting commerce between the North American mainland and its Caribbean Island holdings and requiring alternate, local food sources to feed. 7 As at the last search (16 May 2024 in the web of science; search item: breadfruit) a total of 606 papers have been published in one century. In Figure 1-2, from 1922 to 2006 there were 177 papers published in 84 years whereas >358 papers, which is ~67 percent of the current total papers were published in only 15 years. The sharp increase happened between 2006 â 2007, during this period was when Murch et al. (2007) made a significant advancement in tissue culturing the cells of this plant to ease the planting of breadfruit, and propagation to mitigate the reduction in the number breadfruit trees damages as a result of the reported several deaths, and other deleterious events that the plant is exposed to during its propagation, and ultimately, to avoid the extinctions that may occur if care is not taken. To that effect, >250, 000 trees have been planted, and distributed in >50 countries to date (TTFF, 2022). Since then, numerous works have discussed the history, propagation, and utilization of breadfruit products in ensuring food security. From the literature published in this period (1922 - 2022), the history and propagation of breadfruit have been extensively discussed by these authors, where some variation may exist in the way the stories were narrated, but key important details acknowledged by these authors has been extracted and summarized as a background for the purpose of this study. The many varieties of breadfruit available exhibit diverse metabolic profiles, making their identification a potential challenge. Figure 1-2. Summary for Publication and Citations for Breadfruit. Times breadfruit cited and published over time from 1922 - 2024 (Source: web of science database â 606 publications and 6,564 citations). 8 1.3. Diversity and Nomenclature of Breadfruit Bligh collected the names of breadfruit during an ill-fated expedition in 1789, namely: Apeere, Awanna, Eroroo, Mire, Ore, Patteah, Powerro, and Roweah (Morrissey, 2017; Leakey and Roberts-Nkrumah, 2015). It is believed that the sets in 1793 were among the same eight cultivars in the first voyage, HMS Bounty by Captain Bligh. In the St. Vincent Botanical Garden where some of the plants introduced by Bligh were planted, there have been differences in the number of cultivars. Roberts-Nkrumah, (1997) reported 25 cultivars, while earlier Leakey et al. (1977) reported five seedless varieties of breadfruit found. Seven breadfruit types was said to have been found (Andrew and Mason, 1991). As the utilization of foods increases, the knowledge may disappear due to its primarily oral means of transmission. Interestingly, by understanding traditional knowledge, the Republic of Marshall were able to discover two neglected local cultivars which were threatened with extinction due to its small-size, and low production as compared to other cultivars (Englberger et al. (2012). In addition, Navarro et al. (2007) reported that owing to this means there was a loss in genetic diversity in relation to the loss in the traditional knowledge. The following are the names of breadfruit in other languages (Table 1-2). Table 1-2. Breadfruit Vernacular Names (Modified from Daley et al. (2022); Akouêhou et al. (2006)). Country/Island Vernacular Names of Breadfruit Spain Ãrbol de pan, fruta de pan, pana, pana de pepita, panapén Brazil Ãrvore-do-pão; fruta-pão-de-caroço; pão-de-massa; pão-de-pobre France Arbre Ã pain, arbre veritable, châtaignier â grain. Cook Islands Kuru Cambodia khanaôr sâmloosakéé Cuba castaño del malabar; mapén; palo de pan Dominica Yam paine blanc, yam paine common, yam paine juane Portugal Fruta-pão India Bakri chajhar, eema panasa, nirphanas, irppla, kada chakka, gujjekai Ghana Bofrot, afen, efe Jamaica Banbran, banjam, macca, man bread, prickly walled, St. Kitts, yellow heart Kiribati te mai Grenada Green skin, pika Haiti Châtaignier des Antilles; jaquier; meat fruit; pti-lâarbre Ã pain; rimier Hawaii Ulu Fiji Kulu, uto, uto mbutho, uto ndina 9 Country/Island Vernacular Names of Breadfruit Guatemala arbol de mazapán Indonesia Sukun (seedless), timbul (seeded), kelur (seeded), Malaysia Kelor (seeded), kelur, sukun (seedless), timbul Micronesia mei Nicaragua Fruta de pan Philippines Kamansi/camansi (seeded), rimas (seedless) Papa New Guinea Kapiak Puerto Rico Lavapé; nmapén; pana forastera Pohnpei Mei, mein Nigeria Gbere, jakole, barafuta, ukwa, ofon, ediang Republic of Benin Berefutu, tevitin, belefututin, akutapanWun Sri Lanka Erapillakai; rata del St. Vincent Black breadfruit, butter breadfruit, butterheart, cocobread, captain bligh, common, creole, england, dessert, floater, hogpen hope marble, kashee bread, koshi, lawyer caine, lulu, massa, old wind, ready roast, Samoa Ulu Solomon Islands Bia; nimbalu Tonga Mai; mei Thailand Sa-ke, khanun-sampalor Vanuatu Beta Venezuela Frutepan; pan del año Vietnam Sake Henceforth, the documentation of traditional knowledge of breadfruit is imperative to improve its utilization, production, and biodiversity. In the study carried out by Daley et al., it was found that the respondents who were knowledgeable used different approaches to identify, name, and classify breadfruit varieties using an array of descriptors such as pulp color, skin texture, fruit size, and leaf characteristics, due to their unique features (Daley et al., 2020). Furthermore, many of the vernacular names are reflective mainly of their morphology, cooking, and eating quality. Cultivars or types with excellent eating quality usually have a yellow, soft, and smooth texture when cooked, and are often described as having a great mouthfeel. Cultivars with good-eating quality generally have cream to light yellow pulp and are described as having good mouthfeel and flavour. Poor eating quality among breadfruit cultivars is generally associated with dry pulp and poor flavour. These cultivars usually have white to cream-colored pulp. Words such as âstringy,â âbarky,â and âstranyâ were often used to describe the mouthfeel of those cultivars considered to 10 have poor eating quality. Some cultivars were found to be easy to roast or boil in St. Vincent and Jamaica. Consequently, since naming varies from country to country, it is possible that cultivars with the same name may be morphologically different, or vice versa. Therefore, detailed information in screening these cultivars is required to accommodate the variability that might come from the naming system (Daley et al., 2022). To assemble a database for breadfruit, it is imperative to have detailed information on the species' diversity, their geographical distribution, and the methods used to identify the uniqueness of each accession (Jones et al., 2011a). There has been a gap in the use of standardized methods to investigate these characteristics. Ragone (1995) recommended the use of standardized measurements in describing plant species due to the high variability in breadfruits, and Ragone and Wiseman (2007) later developed standardized morphological characteristics that would accommodate the variability. Jones et al. (2011a) investigated the 221 breadfruit trees domiciled in the NTBG in Hawaii across 31 Pacific Islands providing fingerprint information that could be used to identify cultivars and clarify local nomenclatures. One of the well-known changes in breadfruit morphology is the reduction that occurred during the migration to the east Oceania (Ragone, 1997), and the most pronounced were exclusively found in East Polynesia, indicating domestication pressure on starchy preference in the region (Ragone 2001; Zerega et al., 2004, 2006). Additionally, Sweeney et al. (2007) also noted the changes in the coloration of the pulp from yellow to white in Polynesian cultivars (Figure 1-3). Jones et al. (2011a) compiled findings using Discriminant models in analysing cultivar from Melanesia, Western Polynesia, Eastern Polynesia, Micronesia, which are significantly distinct. Moreover, Jones et al. demonstrated the use of multi-access software in identifying cultivars amidst the vernacular names that may cause ambiguity and allowed the creation of gene banks to determine cultivars with superior nutritional and sensory qualities for propagation and use in various food applications. Consequently, with detailed information about its morphology, the uniqueness can be leveraged to solve food and nutrition insecurity depending on the available cultivar. 11 Figure 1-3. Breadfruit Domestication and Development Across the Pacific Islands. The yellow and grey colors illustrate changes in breadfruit as humans migrated from Melanesia to Polynesia. The central plant represents breadfruit. The gradual disappearance of seeds toward East Polynesia is shown through seed variation. (Figure created using BioRender). The Caribbean remains the second home to the growing of breadfruit outside Oceania. It is difficult to differentiate between these cultivars due to their single introduction from East Polynesia (Tahiti) where the seedless triploid dominates. In January 1793, the seedless cultivars landed in the Caribbean from the St. Vincent and the Grenadines (SVG). However, the record of the cultivars was not kept. Curiously, Audi et al. (2023) studied how many lineage groups are in the Caribbean with a focus on SVG, and investigated which cultivars were originally introduced by Bligh, how they relate to other regions (Table 1-3). It was found that most of the Caribbean breadfruit are similar to Micronesian (similar to the West Polynesia), and a subset of triploid seedless from East Polynesia (Labouisse, 2016; Navarro et al., 2007; Zerega et al., 2015). Table 1-3. Summary of Breadfruit Bligh Named Cultivars Linked to Some Geographical Regions (Audi et al., 2022). Region Cultivar Source Description Asia and Caribbean Sucoom Beejee Timor, Sunda Islands or Melanesia small, and very inferior with distinctive pebbly skin that resembles Annona muricata. it is uncommon, does not have a particularly desirable taste and is not actively cultivated East Polynesia Maire SVG Leaves of this cultivar resemble the fern 12 Region Cultivar Source Description Jamaica Macca SVG Leaves with deep sinuses, thin, non-overlapping leaf lobes, and small, round fruit. Caribbean Cocobread SVG Desirable for cooking and the cultivar with the largest fruits West Polynesia Patteah SVG Large yellowish, pale, green ellipsoid fruit with raised points on the skin and a large oblong core with aborted seeds. The availability of breadfruit depends on the cultivar but is generally associated with the humid months, July, and August in Pohnpei. The breadfruit season for consumption varies from year to year, from one part of the island to another. The fruiting season and trees productivity is not influenced by humans; therefore, breadfruit was considered âthe fruit of the hands of spiritsâ in Pohnpei (Lawrence, 1964; Fownes and Raynor, 1991). In order to understand the factors that contribute to the phenology of the species, Jones et al. (2010) investigated the variation in the seasonality of genetically-diverse groups of breadfruit trees growing in a single location and demystified how seasonality may be affected by location. This study provided a predictive tool in which cultivars like Maâafala with an earlier seasonality, if planted in Trinidad where the fruiting season is from June to September can extend the season to March to July (Roberts-Nkrumah, 2007). In addition, this approach can be applicable to other cultivars with complementary season profiles. This strategy would ensure that fruiting of breadfruit in all-year round. Planting a combination of breadfruit cultivars with complementary seasons can provide a sustainable and continued food resource for many parts of the world (Jones et al., 2010). 1.4. Breadfruit Metabolites Composition and Antioxidative Potential The protein content in the âMaâafalaâ cultivar of breadfruit is around 7.6 %, which is more than what is present in many tropical staplesâ food crops, for example, rice which has 7.4 % and others including corn, wheat, soybean, potato, and pea. Maâafala has a complete set of essential L-amino acids (568 mg/g protein) required for human health, having a particularly high concentration of phenylalanine, leucine, isoleucine, and valine. The essential L-amino acids present happen to be more than many other staple food crops, among which are soybean (443 mg/100g protein), corn (413 mg/g protein), rice (374 mg/100g protein), wheat (336 mg/100g protein), yellow pea (366 13 mg/100g protein), and potato (326 mg/100g protein) (Liu et al., 2015). Depending on the cultivar, breadfruit has about 84 % carbohydrates, containing about 60 % starch, giving it potential to meet the ever-increasing market for industrial starch (Oladunjoye et al., 2010), and having a greater diversity of starch genes than what is obtainable in other plant species (Sahu et al., 2020). The carbohydrates content of cooked breadfruit is around 18.1 â 37 g of the total carbohydrates, which is comparable to other cooked starchy food crops, for example taro (34.60 g/100g), plantain (31.15 g/100g), white rice (28.59 g/100g), potato (21.55 g/100g), and sweet potato (20.71 g/100g) (Turi et al., 2015b). The fruit crop is a good source of vitamins, including pro-vitamin A, vitamin C, riboflavin, niacin and thiamine, and some minerals such as calcium (283 â 1491 μg/g), iron (6.2 â 21.2 μg/g), cobalt (3.6-31 ng/g), potassium (7.5 - 16.2 mg/g), and magnesium (630 â 2281 μg/g). These vitamins and minerals concentration have been found to be greater than those in corn, wheat, and rice making it to have a safe and high-quality alternative to these staples (Jones et al., 2013; Jones et al., 2011a; Ragone 2006; Turi et al., 2015a). In addition, breadfruit contains high levels of resistant starch, dietary fiber of low glycemic index, that could play a significant role in diabetes control (Baba et al., 2016; Kehinde et al., 2022; Turi et al., 2015a). It has been reported that the number of undernourished people has reached ~1.02 billion (FAO, 2009a). Breadfruit is rich in nutritional composition, having more macro and micronutrients than many staples food crops (Kehinde et al., 2022; Liu et al., 2015, 2020). Breadfruit is a sustainable food with wide diverse crops, poised to meet the diverse demands in the food industry. With its rich pro-vitamin A carotenoids (pVACs), gluten-free nature, and low glycemic index, it stands out as a versatile food suitable for individuals with various health conditions such as anaemia, blindness, cancer, celiac, obesity, and diabetes. For example, anaemia; a health condition caused by iron deficiency is reported to affect one-quarter of the world's population. Also, an estimated 250 000 â 500 000 children who are vitamin A-deficient become blind every year, and half of them die within 12 months of losing their sight. Flour produced from the cultivar âMeionâ, contains an iron amount equivalent to whole wheat flour and approaches that are found in fortified wheat flour. Moreover, it is predicted that by 2045, approximately 1 in 8 adults worldwide will be grappling with one or more cases of obesity, cancer, and diabetes, paving the way for a multibillion-dollar market for breadfruit. Nonetheless, the high amylose content starch, high levels of pVACs, crude fiber, vitamin C, and minerals in breadfruit would regulate blood sugar levels or delay onset of diabetes in adults. With cultivar ââSamoan 1â containing significantly more pVACs 14 than other cultivars, therefore, this would provide 62 â 84 % of the vitamin A requirement of an adult male or 43 â 48 % of the requirement of a pregnant woman (Jones et al., 2013). Many fruits around the world are known for their antioxidant properties, and one of these is breadfruit. Specifically, breadfruit has been found to contain higher levels of total polyphenols, total anthocyanins, and total antioxidant capacity compared to some commonly consumed fruits and vegetables in Fiji Island (Lako et al., 2007). Moreover, Englberger et al. (2007) reported the quantified content of phytochemicals, such as polyphenols and carotenoids, in several breadfruit cultivars. In addition, Handa et al. (2008) posited that several compounds have been identified from breadfruit, with over 70 compounds derived from the phenylpropanoid pathway, which have demonstrated notable biological activities, such as anti-tumor effects, and the inhibition of leukemia cells. Furthermore, breadfruit flour has been discovered to have low levels of antinutrients, including saponin, trypsin inhibitors, and phytic acid (Ijarotimi and Aroge, 2005). As a potential food source to mitigate diabetes, this have been examined by the glycemic index (GI), breadfruit flour and cooked have GI 64 ± 7 and 60 ± 9 lower than wheat flour and white bread (Ramdath et al., 2004; Widanagamage et al., 2009). In addition, a comparison to 100 % glucose standard, breadfruit demonstrated a close value range, GI 47 ± 5 and 72 ± 8 which further showed to be a low to moderate GI for diabetic patients. Turi et al. (2015) stated that 100 g of breadfruit contain 7.37 g of fiber, higher than the fiber content of rice and potatoes. Diets with substantial amounts of fiber regulate insulin sensitivity. The slow digestion of breadfruit due to the high amount of amylose content of breadfruit starch regulate blood level sugar (Akanbi et al. 2011; Nwokocha & Williams, 2010). Martini et al. (2010) noted that delay onset of diabetes in adults can happen by consuming food richer in components such as pVACs. 1.5. Economic Importance of Breadfruit and Environmental Effects Sydney Parkinson was among the first Europeans to describe the uses of the breadfruit tree (Parkinson, 1773), encountered in Tahiti where it is called âThe Staff of Life.â. In his work, Parkinson noted that the capacity of breadfruit as an essential food source for the Tahitians. Drawing from this and other recommendations, the utilization of locally grown food crops has the potential to strengthen their local economy; interestingly, breadfruit happens to be cultivated in the most economically-challenged countries in the world. The development of the global market for the breadfruit by making its high-quality food products likewise tertiary products such as latex, 15 ethanol, and carbon would assist the less developed countries income generation (Jones, et al., 2011b; Lucas & Ragone, 2012; Lincoln et al., 2019). A recent report shows that in 2021/2022, about 787.4 million metric tons of wheat were consumed globally (Statista, 2022). The protein content in the âMaâafalaâ cultivar of breadfruit offers a relatively more digestible form than that of wheat flour, which could be transformed as a healthier protein isolate and provide sought after functional properties. Alternative proteins, which include plant-based protein, would add $1.1 trillion to the global economy by 2050. Furthermore, depending on the cultivar, breadfruit starch has shown superior functional properties than traditional starch sources like broken rice and cassava. According to Maximize Market research, the global starch market is expected to grow from USD 61 billion in 2024 to USD 95 billion in 2032. Breadfruit starch presents an enticing alternative, potentially disrupting the market landscape. The wide acceptance of the double-gold medal Mutiny Island Breadfruit Vodka underscores breadfruitâs potential in the beverage sector, which has aligned with the flourishing craft vodka market, expected to rise by $760 million from 2022 to 2027 (Figure 1-4). The climate models tested with breadfruit predict a modest ~4.5 % decrease, validating its resilience to future climate change as opposed to other industrial-based raw material crops (Maximize Market Research, 2024). Based on this unique diversity that exists in its nutritional profile, tastes, and flavours; deliberate actions by the growers for additional improved varieties, and rightful selection of cultivar by the processor in the industry would enable it to be a global superfood, now and even better in the future. Figure 1-4. Breadfruit Vodka with the Gold Award Word Spirits Competition in San Francisco (Photo Credit: Todd Manley). 16 Consequently, as breadfruit could serve as suitable replacement for wheat or commonly consumed grains in making food products, the economics of breadfruit could also reach billions of dollars. Most of the regions where breadfruit is grown are among the struggling nations in the world such as Nigeria etc. continue the importation of grains from developed nations like US, Canada, UK etc. Further exacerbating poverty in these regions (Jones et al., 2011b). Nigeria is among the economically struggling nations, with over 80 million people living in extreme poverty. In 2021 alone, the country spent â¦1.29 trillion on durum wheat importationâan amount that could have covered the annual minimum wage for approximately 1.5 million people (NBS, 2021). Similarly, most of the agricultural-related greenhouse gas (GHG) come from rice, cows, soils etc. (US EPA, 2020). The country is now 6th largest producer of rice after China, India, US, Indonesia, and Pakistan (WPR, 2022), and among the largest producer of GHG emissions in Africa (Statista, 2022). Hence, the need to adopt the cultivation of breadfruit in this region contributes a lesser GHG to the environment. The burning of rice stalks release GHG, and land used for the cultivation of rice can harbor microbes that GHG (Devkota et al., 2022). On the other hand, breadfruit reduces the CO2, one of the major GHGs in the environment, through photosynthesis for their own food or store it as biomass (Jones et al., 2011a). In addition, breadfruit trees can live up to 100 years, this will reduce the release of CO2 that come frequently from annual crops during harvesting or uprooting (Abbas et al., 2017). With improved cultural practices, Nigeria can produce >100 million metric tons of breadfruit yearly (Amusa et al., 2002). Numerous industrial potentials of breadfruit starch have been identified in previous studies as drug tablet formulation (Adebayo et al., 2008), bioethanol production (Betiku and Taiwo, 2015), coatings for post-harvest storage (Bezerra et al., 2019), stabilizer in low-fat products for the oil-in-water emulsion (Anwar et al., 2020), and nanoparticles of good consumer value (Andrade et al., 2019). The senescence leaves have been used in numerous ways to treat diseases. In Taiwan, fevers and liver disease are cured with the leaves, whereas in the Caribbean, it is used to control diabetes by reducing the high-blood pressure (Whistler, 1988). Erland et al. (2022) determined the impact of environmental conditions on fruit and nutritional characteristics on Maâafala cultivar in four Hawaiian Islands discovered that climate was found to have a significant relationship with proximate qualities, particularly fat, fiber, starch, and protein 17 content of fruit. Starch enzymes have been found to be sensitive to abiotic factors which may be a mechanism for these differences in starch physiochemical characteristics 1.6. Breadfruit for Food Security Food prices have been one of the challenges with the increase in hunger and number of undernourished people in the world with the record of over 1.02 billion people (FAO, 2008; 2009a). Food security has been defined as the availability of sufficient, safe nutritious food to maintain a healthy and active lifestyle (World Bank, 2007). Breadfruit is one of the 35 priority crops in the International Treaty on Plant Genetic Resources for Food and Agriculture for the actualization of food independence and food security (FAO, 2009b). The probable yield of the breadfruit has been reported to surpass some of the most consumed food crops in the world, for example rice, wheat, corn, and wheat (FAO, 2009c; Calpe, 2007; Sauerborn, 2002). Due to its perishable nature, beginning to lose its quality after 5 daysâ post-harvest, breadfruit is often fresh (Worrell et al., 2002). An ideal approach to mitigate this loss is to extend the shelf-life stability and make it suitable in making food products is to process the fresh breadfruit into flour (Mayaki et al., 2003). Importantly, turning the fruit into value-added products would be helpful in the better utilization of the fruit to avoid its natural perishability (Ragone, 2002, 2007). Furthermore, the food crop has been projected to be on the global market once it has been properly processed through regulatory and meet up with world market forms. In 2016, the Food Drug and Administration (FDA), has granted âGenerally Recognized as Safeâ status to breadfruit in the United States (FDA, 2016). 1.6.1. Handling and Processing of Breadfruit Breadfruit is a versatile food with edible leaves, seeds, flowers, and pulp. However, breadfruit has a short shelf life and can easily deteriorate due to its high respiration rate, thereby predisposing it to biotic factors such as flies and mealybugs, requiring that the breadfruit is processed to conserve its quality. Molimau-Samasoni et al. (2019) explained the use of heat and air has been a promising way to prevent pest actions. The storage of harvested breadfruit 13 ± 1 ºC with a relative humidity of 85 â 95 % and minimal exposure to ethylene prolongs the shelf-life of breadfruit. There have been some other suggestions in the post-handling of the breadfruit; it was discovered that the use of semi-permeable coating reduced, off-odors and greater discoloration. 18 Furthermore, controlling the atmosphere with 5 % CO2 and 5 % O2 at 16 ºC prolongs the quality attributes for more than three weeks than the untreated with one week, and maintain the skin coloration during this period (Kader, 2001; Worrel et al., 2002; Maharaj and Sankat, 2004). The processing of breadfruit is different across the geographical locations (Table 1-4). In Pohnpei, the type of fruit determines the method of preparation. Different cultivars give different types of results (Ragone, 2002). The traditional knowledge of preparing breadfruit is dying with the farmers. It has been recommended that the common practices in the Islands of Pohnpei and Federation of Micronesia should be documented before it is too late (MacMillan, 1946; Fosberg, 1946). The commonly used methods are frying, boiling, pounding, and mashing. Interestingly, this method was introduced regarding the improvement in sensory properties, and reduction in loss of nutrients (Olaoye et al., 2007). In the Pacific, traditional breadfruit can be dried under the sun for about a week, diced, and cooked into paste to make snacks and curries. This treatment will halt any enzymatic changes, fermentation that may occur and growth of bacteria and fungi (Famurewa et al., 2016; Ragone, 2011). Another method of preservation is the use of semi-anerobic fermentation where peeled breadfruit are put in pit between 5 to 7 days. Starch will be degraded into carbon dioxide and lactic acid, afterwards, the lactic further undergoes another fermentation to form butyric and acetic acids, therefore, the end-product with sourdough-like taste could last year (Ragone, 2002). Presently, breadfruit pulp is commonly used as flour or in isolated starch variant. These forms are stable and found applications in formulated food products (Figure 1-5ab). Mechanical drying at 40 ºC to 60 ºC has improved the drying process from week to few hours. The process mildly affects the nutritional, antioxidant, and functionality as compared to the sun drying approach (Famurewa et al., 2016). Sari and Lestari (2017) discovered that the optimum breadfruit flour production in terms of appearance was achieved with a temperature of 60 ºC and a drying time of 100 min. Notably, temperature above this can lead to low gelatinization or starch retrogradation of breadfruit flours (Huang et al., 2019). Table 1-4. Summary of Literature Search about Processing of Breadfruit in Different Geographical Regions Region Common Breadfruit Dishes/Products Reference Barbados Breadfruit bowl and meat. Picked with pudding and sauce. Roasted or boiled with butter. Yelluh Meat (2024). 19 Region Common Breadfruit Dishes/Products Reference Hawaii Baking in earth ovens (imu) or broiling over hot coals and prepare as poi âulu. Roasted as kükini. transformed into piele âulu (pudding of coconut milk and breadfruit cooked in earth ovens). Malo et al. (1951); Buck (1959) Jamaica Roasted in an open flame until black all rounds. Ackee in puddings and stuffed in breadfruit. Slices canned in brine Thompson et al. (1974) Pohnpei Mahr produced in large pits Ragone and Raynor (2009) Trinidad and Tobago Boiled and steamed â âoil downâ Roberts-Nkrumah, L.B. and Badrie, N. (2005) Polynesia Ferment breadfruit in local oven Balick, (2009) Philippines Cooked as a vegetable and eaten by dipping into sugar, vinegar; or cooked into coconut milk. Coronel, (1983) Republic of Benin Baked, fried, or with banana plantain for fufu Assogba et al. (2008) Samoa Breadfruit is cooked with coconut milk and chopped onions. Baked and served with coconut milk as a dipping sauce Kiribati Biscuits- soak in seawater and turn to the dough and dry Tusialofa, 1992 Micronesia Fermented breadfruit Balick, (2009) Melanesia Fermented breadfruit Balick, (2009) New Papua Guinea Nigeria Boiled cookies, fried Omobuwajo, 2003 Ghana Snack Appiah et al. 2011 Guam Boiled, baked, fried or dried Elevitich et al. 2014; Ragone, 2006 20 Figure 1-5. Processing of Breadfruit Its Flour Packaged in an Exhibition in Samoa (Photo Credit: Susan Murch). 1.7. Challenges Affecting the Widespread Use of Breadfruit There are several factors mitigating breadfruitâs acceptance, both locally and internationally. Propagating breadfruit remains a challenge. Although some cultivars produce seeds, they do not survive desiccation (Zerega et al., 2004). Root suckers, root cuttings, and air layering have traditionally been used for clonal propagation (Ragone, 1997, 2006). In 1789, 678 trees survived out of 2,000 during the infamous voyage by Captain Bligh from Tahiti to the Caribbean (Powell, 1977). Transporting breadfruit through shipments could spread disease, and approval is needed before it can be allowed into countries (Murch et al., 2008), which slows down progress in meeting global demands but remains suitable for small-scale propagation (Moustache and Moustache, 2007; Roberts-Nkrumah, 2007). However, a solution has been provided by Murch et al. (2007; 2008); yet, it is possible that some people in other parts of the world are still not abreast of the principles behind tissue culturing, or do not have access to the plants distributed globally through this earlier initiative. There are now breadfruits in places like Canada, where they are not cultivated or common (Figure 1-6ab). As humans migrated across the Pacific Islands, several cultivars became native to specific islands, giving rise to many cultivars with different phenotypes (Zerega et al., 2004). Furthermore, some of these islands are in remote places, for 21 example, the nearest landmass to Hawaiʻi is 2,000 miles away, making the expansion of certain breadfruit cultivars challenging. Therefore, there is a need for a detailed description of the cultivars to be planted, as these cultivars may vary in their phenotypes and chemical compositions (Ragone, 2007). Additionally, one of the attributes of breadfruit limiting its acceptance by consumers is the taste of the food crop, and ultimately, the flavour that they perceive from these cultivars. Figure 1-6. Breadfruit in Canada. (a) breadfruit plants at the UBCO Plant Growth Facility. (b) breadfruit in a commercial shop in Kelowna shipped from Colombia. 1.8. Flavour and Its Formation in Food Crops Flavour is one of the senses that involves the perception stimulated when a substance in the mouth reacts chemically with taste receptor cells located on taste buds in the oral cavity, mostly on the tongue. Volatile compounds are responsible for aroma, whereas non-volatiles compounds determine taste. Flavour results from the interaction of specific chemicals with specific receptors on the tongue, giving the experience of sweet, sour, salty, bitter, and umami (Figure 1-7) (Czerny et al., 2011; Gravina et al., 2013). According to the Society of Sensory Professionals, responses to chemical components are integrated together and processed into an overall package that the brain can recognize as flavour. Flavour formation depends on developmental, physiological, and environmental cues (Kader, 2008). Fruits have many chemical compositions, with compounds 22 such as esters, alcohols, aldehydes etc., with significant taste and flavour perception. These are biosynthesized through a restricted number of major biochemical pathways, for example, sugars, L-amino acids, lipids, carotenoids, and terpenoids. (Goff & Klee, 2006; Sanz et al., 1997). Figure 1-7. The Summary of the Breadfruit Taste Compounds, and the Flavour Intensity. F-Fructose; G-Glucose; Na-Sodium. The shaded portion indicate each perception area on the taste bud. Primarily, esters, aldehydes, saturated and unsaturated short-chain fatty acids are majorly derived via the lipoxygenase pathway. It is believed that volatile formation from L-amino acids and fatty acids is mainly due to the decarboxylases activity and lipoxygenase pathways, while alcohol dehydrogenase and acyltransferases are selectively involved in the interconversion of aldehydes, resulting in the synthesis of a wide range of esters, and corresponding alcohols to the aldehydes in many fruits. Other key volatile compounds that could influence flavour are ketones from carotenoids, monoterpenes, and sesquiterpenes from either carotenoids or glycosylated compounds (Souleyre et al., 2014; Yauk et al., 2017; Defilippi et al., 2005; Gonda et al., 2010). Peleg et al. (1999) discovered that the bitterness and astringency from tannins and catechins could influence off-flavours. Irrespective of the saponin skeleton, whether it would be sweet, or bitter depends on some other constituents attached (Heng, 2005). Hufnagel and Hofmann (2008) noted the impact of some phenolic acids such as caffeic, vanillin, p-coumaric and ferulic acids together with their ethyl esters in the formation of bitterness and astringency. Crucially, the phytochemistry of these stated compounds, such as aldehydes, alcohols, ketones, monoterpenes, sesquiterpenes, esters, tannins, saponins, and phenolic acids in relation to flavour role is still not well understood. 23 The Society of Sensory Professionals had suggested that to study these complex traits, it is advisable to partition the details to what they can be evaluated, understood, and acted upon. Thus, âflavour intensityâ is one of the notable attributes that can be used in this regard. One of the many fruits reported that have divergent and unclear views in the formation of its flavour is breadfruit. There are several compounds that have an affinity for volatile compounds, such as protein. The affinity can be considered as multi-factorial, related to the protein amino acid profile, protein overall conformation, and the stereochemistry of the flavour compound. In other words, different aroma compounds, based on their chemical structure and functional group, could possess different binding affinities to proteins due to the nature, and type of interactions involved (Wang and Arntfield, 2017). As a flavour compound could possess multiple functional groups which react differently with proteins, this could make the nature of interaction more complicated (Figure 1-8). Most flavour chemicals bind reversibly with protein via hydrophobic interactions with its non-polar interior region due to the presence of a non-polar compound. Compared with physicochemical interactions via non-covalent forces, chemical bonding via covalent linkages is more specific and associated with specific groups on proteins (e.g., -NH2, -SS-, and -SH) (van Ruth and Rozzen, 2002; Reineccius, 2006). One of the most famous examples is Schiff-base formation between aldehyde and ε-amino group of lysine residue (lys-NH2 + R-CHO â lys-N=CH-R + H2O) (Preininger, 2006). As a consequence, intensity (potency) and character of the desired aroma profile can be altered. A higher number of sulfhydryl groups from cysteine and disulfide groups from cystine in proteins could also promote changes which result from sulfhydryl disulfide interchange reaction. Mechanisms of binding, the interactions between protein and flavours are affected by the intrinsic properties of proteins and flavours and how these parameters react to environmental factors (extrinsic factors) such as temperature, pH, nature, and concentration of salts as well as protein modification. 24 Figure 1-8. Proteins with Flavour Compound Interaction (Modified from Reineccius, 2006; Wang and Arntfield, 2017). Figure created on BioRender. Usually, off-flavours are linked very closely to lipids compared to proteins and carbohydrates. Lipid oxidation not only lessens the nutritional benefits of products due to the changes in essential fatty acids and vitamins but also negatively affects the sensory qualities such as flavour, texture, and color, which impacts on the overall consumer acceptance (Shahidi and Abad, 2019; Hossain et al., 2020). Lipid oxidation is a very complex phenomenon that undergoes a variety of reactions and produces a wide range of compounds. Fatty acids, mainly polyunsaturated fatty acids (PUFAs), react with molecular oxygen and produce primary oxidation products (hydroperoxides) via free radical mechanisms. These compounds are highly unstable due to a weak oxygenâoxygen bond and are believed to make no contribution to odor and aroma development, but they break down rapidly and produce a wide variety of secondary constituents, which are responsible for the formation of off-flavours (Shahidi and Abad, 2019). Among these compounds, aldehydes seem to be the major compounds contributing to flavour development due to their low odor threshold and higher concentration. The most common aldehydes generated from lipid oxidation are n-alkanals, 4-hydroxy-2-alkenals, 2-alkenals, and malondialdehyde (MDA). Lipid oxidation can occur in various pathways including autoxidation, photooxidation, thermal 25 oxidation, and enzyme-catalyzed oxidation (Domínguez et al., 2019). Lipids oxidation products (e.g., aldehydes) can interact with Maillard interaction intermediates and contributes to flavour formation. The Maillard reaction develops a wide range of chemical constituents belonging to oxazole, thiophene, furan, thiazole, pyrazine, and pyridine, among others. Compounds such as peptides, L-amino acids, thiamine, carbohydrates, nucleotides, and lipids in food promote the development of flavour. Hence, the generation of aroma volatiles is basically related to the breakdown and oxidation of lipids, the Maillard reaction, and the degradation of vitamins, mainly thiamine. The formation of Strecker aldehydes through the degradation of L-amino acids and lipid oxidation is another example of developing aroma compounds (Kerler et al., 2010). These compounds react with Maillard reaction-derived carbonyls and form intermediates, which further break down into flavour compounds. Apart from L-amino acids, phenolic compounds may promote the generation of Strecker aldehydes. For example, L-amino acids and quinones originating from phenolics could produce flavour precursor volatile aldehydes through Strecker degradation (Rizzi, 2006). The same study also reported that L-amino acids (e.g., phenylalanine and methionine) were capable of interacting with phenolic compounds (e.g., chlorogenic acid, caffeic acid, epicatechin, and catechin), and developing strecker aldehydes (e.g., phenylacetaldehyde and methional) in a ferricyanide-based model system. 1.8.1. Sensorial Properties of Breadfruit Products and Acceptability There are several cultivars of breadfruit, and each cultivar tastes different and differs in flavour (Table 1-5). Wester, (1922) described breadfruit is edible when boiled, roasted, and baked; retains its uncooked palatable flavour, which could serve as alternative to sweet potato. The fragrance of one breadfruit can occupy a room. Breadfruit from composite wheat flour and 10 % breadfruit is acceptable but 20 % breadfruit flour will need the supplementation of an additive (Olatunji and Akinrele, 1978). Furthermore, it was also demonstrated that 10 % breadfruit flour and 5 % whey for bread, and 10 % breadfruit and 5 % soy protein were rated most for flavour, colour, and texture. Nochera and Caldwell (1992) studied the extent by which breadfruit can be used to reduce the dependence on wheat flour in making food products that would be acceptable, and recommended substituting with up to 10 %. Due to the inclusion as composite flour, Iwaoka et al. (1994) examined the volatile and non-volatile compounds in either the fresh or cooked breadfruit. It was revealed that volatile constituents in the raw breadfruit are cis-3-hexenol, 3-26 hydroxy-2-butanone, 1,2-cyclohexanediol, and 2-pentanone while cooked breadfruit contains mostly ethyl acetate and ethanol (Table 1-5). Experimental composite flour of wheat and breadfruit flour was more acceptable than the commercial prawn. A decline in the overall acceptability was observed when there was an increase in breadfruit flour added to the wheat flour during the evaluation of biscuits produced from this composite flour (Omobuwajo, 2003). In a study conducted in Trinidad on the sensorial qualities of some breadfruit cultivars, involving more than 200 breadfruit buyersâ vendors and food-service operators, it was observed that the taste is the paramount quality in its acceptability (Roberts-Nkrumah and Badrie, 2005). Weaning foods from soy-breadfruit flour was significantly different to the control food samples (Ijarotimi and Aroge, 2005). The University of Hawaii and NTBG conducted a sensory evaluation of 20 different cultivars which were carefully selected as they were known to be popular in the Pacific Islands. The evaluation revealed that the cultivars differ in their flavour intensity (Ragone and Cavaletto, 2006). In another study, significant differences were observed in both taste and texture of doughnuts made from wheat and breadfruit flour, with the acceptability of the doughnut decreases with the addition of breadfruit flour (Oke et al., 2017). Table 1-5. Summary of Literature Search about Breadfruit, Flavour and Sensory Using Web of ScienceTM and Google ScholarTM Source Product Findings Abrajano et al. 2021 Polvorón (Bread) Breadfruit Polvoron was highly acceptable having the desired appearance aroma, color, taste, texture, and general acceptability. Adebowale et al. 2008 Pounded yam The sensory panelist ratings showed that 60:40 % instant yam - breadfruit flour paste possess sensory. qualities very close to the traditional pounded yam. Adegunwa et al. 2024 Cookies 75:20:5 plantain-breadfruit-cinnamon flour had the highest overall acceptability along with improved nutritional properties. Ajifolokun, and Adeniran, 2015 20 % of breadfruit co-fermented with cassava yields a novel food product that compares favorably with 100 % cassava gari in terms of microbial and sensory characteristics Arinola et al. 2024 Biscuits Biscuits produced from breadfruit flour with inclusion of 20 % lima bean flour showed comparable overall acceptability with the control. Bakare et al. 2016 Bread The sensorial qualities of the 5 % blend were not significantly different from the wheat flour. 27 Source Product Findings Bates et al. 1991 Fried chips Partial air drying prior to frying reduced oil absorption from 42 % to 26 %. Clark et al. 2018 Gluten-Free Bread Breadfruit flour can be used at <= 20 %, when leavened with yeast, to produce quality gluten-free bread Daley et al. 2016 Pulp Identified a range of skin, pulp colour and skin textures among the breadfruit cultivars Esuoso and Bamiro, 1995 Bread No significant difference at p<0.05 between the control (100 % WF) and the composite bread samples up to 30 % level of breadfruit flour. Gao et al. 2019 Probiotic Beverage A novel fermented breadfruit-based beverage with acceptable sensory characteristics and cell viability using a mixture strain of L. acidophilus and L. plantarum DPC 206. Haydersah et al. 2012 Fermented New functional fermented beverages can be produced from breadfruit and sweet potato Levai et al. 2024 Biscuits Breadfruit can be successfully processed into acceptable cookies. Ma et al. 2005 Extruded The best range of crispness of the extrudate was from 2.0 to 3.5 N/s, hardness from 60 to 75 N and bulk density from 0.3 to 0.38 g/cm. Mayaki et al. 2003 Stiff porridge Concentrations of the chemical components of pre-cooked pulp flour were higher than that of the raw pulp flour, with the exception of starch, while flour from the mixture of pulp and core contains higher percentages of ash, fat, protein, and fibre compared with either the raw or pre-cooked pulp flours. Narasimham and John, 1995 Pulp Controlled low temperature vacuum drying gave a dehydrated product. sensory quality equivalent to conventionally freeze-dried products. Nelson-Quartey et al. 2007 Infant food The incorporation of breadfruit and breadnut into locally produced infant foods can provide a nutritious and palatable alternative. Nochera and Ragone, 2016 Bar Evaluation indicates that 81 % of the panelists found the bar acceptable from breadfruit flour developed from the Micronesian variety, Meinpadahk. Nochera and Ragone, 2019 Pasta 80.3 % of the panelists (N=71) found the pasta acceptable while 18.3 % disliked the pasta. Olaoye and Onilude, 2008 Bread Composite breads were not significantly different (p<0.05) from the whole wheat bread with respect to internal texture, taste, appearance, and general acceptability, up to 15 % BF levels. Roberts et al. 2007. Fried chips Pre-drying affected sensory characteristics of fried breadfruit chips negatively. 28 Source Product Findings Ragone and Cavaletto, 2006 Pulp Significant differences exist in the taste and flavour of breadfruit cultivars. Souza et al. 2012 Instant flour The breadfruit puree was well-accepted by consumers, based on the sensory attributes evaluated Worreell et al. 1998 Pulp Sensory tests of cooked fruit revealed that only fruit 15 - 19 weeks old was acceptable 1.9. Volatile Compounds Discovered in Breadfruit Research works have reported that breadfruit cultivars differ in nutritional composition, functional properties, and morphology (Adebowale et al., 2005; Jones et al., 2011a, 2011b, 2013). However, these previous studies have not demonstrated what could be responsible for this flavour formation, especially the intense flavour perceived by consumers from some cultivars. Additionally, in all these studies, the cultivar name is difficult to know, therefore, creating confusion as to which of the A. altilis cultivar has the intense flavour. From an understanding of flavour, it is formed by the integration of volatile and non-volatile compounds. The reported studies in the literature on volatile compounds in breadfruit were conducted by Iwaoka et al., (1994) (Table 1-6). Iwaoka et al. (1994) work was focused on one cultivar; however, this has not been extended across the cultivars to ascertain what could be responsible for the differences in their flavour intensity. Based on the available literature, the non-volatile compounds that could be responsible for this flavour have not been revealed. It is imperative to adopt a systematic approach in solving this problem. Moreover, the information that would be derived from this study will not only be useful for the flavour formation in breadfruit, but this approach can also be extended to other fruits that face similar challenges. Table 1-6. Volatile compounds found in a single breadfruit (data from Iwaoka et al., 1994) Compound Known flavour Other fruits References Methyl acetate Fruity Apple, cherry, tomato (FooDB, 2024) Ethyl acetate Ethereal, sweet, or fruity Pineapple (FooDB, 2024) 2-butanone/methyl ethyl ketone Pleasant Pungent Paints, coatings (PubChem, 2024) Ethanol Sweet Orange, apple, grape (GSC, 2024) 29 Compound Known flavour Other fruits References 2-pentanone Sharp ethereal banana (PubChem, 2024) Chloroform Pleasant Apple, corn, berries (GSC, 2024) Toluene Paint Pears, citrus, apples (GSC, 2024) Ethyl butyrate Fruity Apple, strawberry (GSC, 2009) 1-methybutyl acetate Fruity Banana (PubChem, 2024) Hexanal Beany, grassy, fatty Faba beans (Akkad et al., 2022) Trans-2-pentenal and 2-pentanol Fruity Apple, tomato (GSC, 2024) 2-methyl-4-pentanal Cheesy Cheese (PubChem, 2024) Butanol Harsh fuel Banana (GSC, 2024) l-penten-3-ol Pungent Grape, tomatoes (Mall et al., 2018) 2-heptanone Fruity Banana, coconut (GSC, 2024) cis-2-hexenal Fresh fruity-green Tomatoes (GSC, 2024) isoamyl alcohol Fruity Banana (GSC, 2024) trans-2-hexenal Green-vegetable-like Tomatoes, black tea (GSC, 2024) amyl alcohol Balsamic, sweet Apricot, banana (GSC, 2024) hexyl acetate Fruity Apple, pear (GSC, 2024) 3-hydroxy-2-butanone Woody, bland, yogurt Yogurt (PubChem, 2024) 2-ethenyl-2-butenal (tentative) ----- ----- ----- cis-3-hexenyl acetate Sweet Apple, banana (FooDB, 2024) cyclopentanol Fragrance Cashew, cauliflower (GSC, 2024) 2-heptanol Fresh lemon grass Lemon (GSC, 2024) Hexanol Fatty-fruity Coconut berry (GSC, 2024) trans-3-hexenol Grassy, green aroma Beer (PubChem, 2024) cis-3-hexenol Grassy-green odor Banana, green tea (GSC, 2024) trans-2-hexenol Fruity, fatty grassy, green Apple, banana (GSC, 2024) 2-cyclohexenone ----- ----- ----- l-octen-3-ol ----- ----- ----- 2-cyclohexenol ----- ----- ----- trans, trans-2,4-heptadienal Fatty green Melon (GSC, 2024) 3-cyclohexenol ----- ----- ----- benzaldehyde Bitter almond-like Almond, apricots (PubChem, 2024) ethyl 3-hydroxybutyrate ----- ----- ----- 2,3-butanediol Buttery, creamy, fruity Beer, coconut (FooDB, 2024) γ-valerolactone ----- ----- ----- 30 Compound Known flavour Other fruits References diethylene glycol monoethyl ether ----- ----- ----- butyric acid Buttery with a fruity Garlic, artichokes (GSC, 2024) trans-2(or 4)-chlorocyclohexanol ----- ----- ----- ethyl benzoate Floral, fruity Apple, banana (GSC, 2024) cyclohexyl benzene Sweet fruity Banana (PubChem, 2024) 2-methylbutyric acid Fruity Cheese (GSC, 2024) γ-hexalactone Coumarin-like Tobacco, coconut (GSC, 2024) benzyl acetate Fruity Cranberry, peach (PubChem, 2024) 5-ethyl-2(5H)-furanone ----- ----- ----- hexanoic acid Sweaty Apple, wine grapes (GSC, 2024) benzyl alcohol Phenolic, balsamic (GSC, 2024) 3-hexene-2,5-diol (tentative) ----- ----- ----- trans-3-hexenoic acid Cheesy, green, fruity Apple, strawberry (GSC, 2024) 1,2-cyclohexanediol ----- ----- ----- 3-hexene-2,5-diol (tentative) ----- ----- ----- phenylpropyl alcohol Balsamic, fruity Guava (GSC, 2024) Octanoic acid fatty waxy rancid Apple, banana, cheese (GSC, 2024) ethyl palmitate Waxy, fruity creamy Apricot, cherry, guava (GSC, 2024) β-β-dimethylbenzenepropionic acid ----- ----- ----- cinnamic alcohol Balsamic, fruity Cocoa (GSC, 2024) Vanillin Vanilla Vanilla (GSC, 2024) 1.10. Chromatographic Separation and Techniques The goal of chromatography is to separate two or more solutes. Chromatography relies on the approach of separating molecules in mixture, which are applied to a surface and fluid stationary phase, separating each other while in the motion through the mobile phase. The mobile phase is either a liquid or a gas that passes through the column, while the other phase called stationary, is either a liquid chemically attached to the inside of a capillary tube, or solid particles packed in a 31 column. Due to the variation in the properties of the compounds, their motion across the chromatography system vary, making some pass slower or faster than the others. A stronger interaction between the solute and column shows a slower motion, and vice versa. Hence, the adsorbed solutes are released at different rates, giving rise to separation factors (Eqn 1.1). This arises from the differences in their equilibration between the mobile and stationary phases. The chromatogram illustrate how the detector respond as a function of time. Where the retention time is the period of injection onto the column to the arrival at the detector. The difference in elution time and how broad the peaks are factors that affect how well compounds are separated. The narrower the column, the better the resolution (Eqn.1.2) (Coskun, 2016; Harris and Lucy, 2019). ð¼ =ð¡â²ð 2ð¡â²ð 1= ð¾2ð¾1 (Eqn. 1.1) ð ðð ððð¢ð¡ððð =âð4 (ð¼â1)ð¼ ð¾1+ð¾ (Eqn. 1.2) Liquid chromatography - The separation is based on differences in properties such as polarity, size, charge, or affinity between the components and the stationary phase. In this technique, the mixture is dissolved in a solvent (the mobile phase) and passed through a column packed with a stationary phase. The stationary phase can be a solid or a liquid supported on a solid. As the mixture travels through the column, the components interact differently with the stationary phase, causing them to elute (come out) of the column at different times (retention time). The mobile is always a mixture of solvents. This can be subjected to continuous changes to increase the mobile strength (gradient) or at constant depending on the nature of analyte. Reducing the particle size in liquid chromatography will improve resolution or separation occurs in a shorter time, chromatography with 1.5 to 2 µm now used for Ultra-high-performance-liquid-chromatography for improved resolution (UHPLC) (Harris and Lucy, 2019). Gas chromatography - It is thus used to separate and detect compounds in the gas phase. The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert. The sample is either a gas or a liquid that is vaporized in the injection port. The mixture of components to be separated is converted to vapor and mixed with the gaseous mobile phase. Component with more interactions with the column travel slower and eluted later. The column must be hot enough to provide sufficient vapor pressure for analytes to 32 elute in reasonable time. The detector is maintained at a higher temperature than the column so analyte will remain gaseous. Largely, Gas chromatography (GC) is run with temperature programming and pressure programming where temperate is ramped continuously either increased or decreased at a predetermined rate throughout the chromatographic run. Pressure of gas increased to increase the flow, decrease retention time, and peaks are sharpening. Through the kovats retention index (I), the retention time can be interpolated with alkane standards (Eqn.1.3) (Harris and Lucy, 2019). ð° = (n + (N â n) =log ð¡ð (ð¢ððððð¤ðâðððð¡ð (ð))log ð¡ð (ð)âlog ð¡ð (ð)) (Eqn. 1.3) n = number of carbons with smaller alkane; N = number of carbons with larger alkane; tR = adjusted retention time 1.11. Mass Spectrometry Mass and electric charge, which are expressed as m/z is used in mass spectrometer (MS) to characterize different atomic or molecular ions. According to classical electrodynamics, particles with the same m/z ratio would move in an equivalent path in a vacuum provided they are subjected to an equal electromagnetic field. MS ionizes the chemical compounds into ions (charged molecules), which are then separated by application of electrostatic or magnetic fields or by a combination of both, fragments and measures their m/z as âchemical fingerprint.â A mass spectrum displays the different ions detected at each unique m/z and their relative abundance. The mass of the molecular ion is equal to the molecular weight of the compound. Since the charge of ion formed in the mass spectrometer is usually +/ or -1, the m/z is usually equal to the mass of the ion. Thus, the m/z of the molecular ion is equal to the molecular weight of the compound (Busch, 2011). The mass of a molecular ion is an important parameter in the identification of the compound. For example, an ion of 1000 amu or Da with 2-charges will generate m/z of 500. The Dalton is defined as one-twelfth of the mass of an unbound neutral atom of carbon-12 at rest, in its electronic ground state. The unit dalton (Da) has been accepted as an alternate name for the unified atomic mass unit and is commonly used by biochemists. Thus, one 12C atom has a mass of 12.0000 Da (Ho et al., 2003; Harris and Lucy, 2019). Mass accuracy is the closeness of measurement of mass to the exact or theoretical mass of an object or material in question. It also indicates the accuracy of the mass information provided by the mass spectrometer. It is usually expressed in parts per million (ppm) 33 (Eqn. 1.4). The resolution of the mass analyzer, the quality, and the stability of calibration of the m/z axis determine the achievable mass accuracy in practice. In particular, the resolution of the MS helps to eliminates possible contaminating peaks at the same nominal mass. (Arrebola-Lie´banas et al., 2017). Greater resolution corresponds to increased ability to separate/differentiate ions (Eqn. 1.5). Mass accuracy (ppm) = (m/z taken - m/z Theoretical)/m/z Theoretical Ã 106 (Eqn. 1.4) m/z taken = m/z value for the unknown; m/z Theoretical = m/z value the true value Mass resolution = ðð¥ð (Eqn. 1.5) m = m/z; Îm = full width at half maximum Electrospray Ionization (ESI) is moving thermally stable and charged nonvolatile ions from outlet of liquid chromatography into the inlet of MS. ESI has the capacity to generate multiple charged ions and so it expands the analyzerâs mass range. The ESI procedure can be split into 3 important steps: formation of droplets, desolvation, and ion formation. At a high voltage, the sample in solution is passed through a metal capillary. As the sample leaves the capillary, it is nebulized by a flow of nitrogen gas and generates fine aerosol solely containing charged dropletsâdroplet formation. To attain desolvation, gentle heating of the droplets with the flow of nitrogen gas should be ensured. Upon reaching the Rayleigh limit, droplets reduce in size, and the forces between sample ions in the charged droplet are fused together. and stabilize with the surface tension of the sample ions droplet resulting in small, charged droplets. This causes the ions to turn into gas phase ions and be removed from the surfaceâgaseous formation. Evaporation happens rapidly in organic components in organic/aqueous mixtures causing a gradual increase in water percentage. The ions are then pulled via a tiny orifice to the MS vacuum (Figure 1-9). The technique provides a chance to ionize polar metabolites and heat labile molecules like proteins in multiple ionization modes. Depending on the operating polarity, either [M+H]+ or [MâH]â is produced in most instances. Mass spectra obtained through ESI are generally simple because it is a soft ionization technique, producing little fragmentation compared to electron ionization. Overall, ESI is applicable to polar metabolites (Ho et al., 2003; Kebarle, 2009). 34 Figure 1-9. Electrospray Ionization in Positive Mode (Figure created on Biorender). Electron Ionization â The electrons migrate from a filament of rhenium or tungsten to a positively charged metallic conductor. Electrons are released from the filament heated to ~2000 ºC through a potential difference of 70 electron volts (eV). Molecules are always stable with even a number of electrons. Gaseous molecules (M) with the heated beam of electrons at a pressure of 10-3 Torr by raising elevating the energy to form charged molecules, molecular ions, with a lower energy state, that will still retain the elemental composition of the original molecule. The ionization is maintained at ~300 ºC, to mitigate the condensation of the analyte and solvent. This ionization technique can be applicable to many MSs operations such as quadrupole MS, quadrupole ion trap etc. However, EI causes a lot of fragmentation, causing molecular ion not to be observed for many compounds (Medhe, 2018; Harris and Lucy, 2019). 1.11.1. Mass Spectrometry Methods The following are some of the common MS instruments and its methods used in metabolomics: Quadrupole MS (Q-MS) is also a relatively simple detector suitable for gas and liquid chromatography (Figure 1-10). This consist of four parallel metal rods having both constant voltage and radio- frequency oscillatory voltage. Ions are deflected by the electric field, while allowing ion with particular mass to charge ratio (m/z) to reach the detector. However, other ions lose their charge by colliding with the metal rods. Through the optimization of the voltages, this 35 allows the detection of different ions with their unique m/z. The attractive feature of Q-MS is that it is suitable for mass filtering in getting the desired mass by alternating the RF voltage. Furthermore, Time of Flight (ToF-MS) is another mass analyzer. ToF-MS performs well in terms of fast scanning capability, wide mass ranges from small to large complex molecules, such as proteins, with high resolution and quadrupole as mass filter (Liang et al., 2015; Harris and Lucy, 2019; Guo et al., 2023). The principle follows that in a constant kinetic energy where a beam of ions moving in a similar direction, and having a distribution of masses, will have a variation of velocities. Where velocity is in reverse to the square root of m/z. Their arrival times at a target plane in parallel to the origin will be distributed according to the square root of m/z. Due to its principle of operation, ToF MS have always been used with a pulsed ion source. Also, it is for this reason that they are frequently coupled to MALDI sources as lasers which frequently operate in pulsed mode. The upper mass range of a ToF instrument has no limit, which makes it fit for soft ionization techniques, and could even be up to 300 kDa to be examined by MALDI-ToF. They also have higher mass resolution, particularly when reflectrons are integrated into its operation to compensate for different kinetic energies of the analyte ions. ToF instruments are capable of high sensitivity because they record all the ions in a single ionization pulse. However, the ToF has a limitation in profiling compounds at ultra-trace levels. Figure 1-10: Quadrupole Mass spectrometer (Figure created on Biorender). Tandem mass spectrometry involves the connection of two mass analyzers separated by a collision cell. Tandem MS can also be referred to as the hybrid approach of involving more than one type of MS to increase specificity and/or mass resolving capability through fragmentation and 36 mass analysis of fragment ions. Tandem MS is of many diverse types, each has comparative advantages, drawbacks, and applications. All consist of four major sections linked together inletâionization sourceâanalyzerâdetector. They are commonly referred to as MS/MS or (MS)n techniques in literature with two mass analyzers separated by a collision cell filled with a gas. The molecular ions are sorted and weighed in the first MS, then fragmented into smaller masses in the collision cell, and the pieces are sorted and weighed in the second MS. Time of Flight, (ToF) can be hybrid with quadrupole or with additional double ToF (Q-ToF/ToFqToF), Orbitrap with quadrupole, triple quadrupole (QqQ) etc. Through this approach, it measures masses correspond to molecular structure and atomic composition of parent molecules, hence, it allows determination and elucidation of molecular structure and chemical pathways (Harris and Lucy, 2019). Quadrupole Time of Flight (Q-ToF)-MS is one of the most used instruments in metabolomics research. Despite its moderate mass resolution and mass precision as compared to Orbitrap MS, it can be compensated by the very fast scan rate; thus, it is better to define the chromatographic peak (Zhang et al., 2014). Q-ToF has the capacity to acquire MS1 data over a wide m/z range during scanning (Farre et al., 2012; Schwaiger-Haber et al., 2021). Triple Quadrupole MS (QqQ) -- measures masses corresponding to molecular structure and atomic composition of parent molecules, hence, suitable in quantitative analysis of known metabolites (Jeevan, 2012; Roberts et al., 2012). QqQ includes a wide range of capabilities beyond the standard product ion scanning operation, including SRM, MRM, precursor scan, neutral loss, and others (Rubino, 2020). It is particularly suitable for combination with LC and is now widely used for the screening and quantification of metabolites (Guo et al., 2023). QqQ is regarded as the gold standard for targeted approach, especially small molecules with specific transitions. QqQ excels in performance with its coordinated trio of quads and the ability to utilize multiple reaction monitoring (MRM), making it a preferred choice for advanced and precise analytical applications because of its high signal/noise ratio in comparison to other mass analyzers by achieving subfemtomole detection levels. As a result, a routine quantification analysis of biomolecules using validated assay is hard to beat. Additionally, QqQ are not designed to acquire MS1 data over a wide range m/z where they only provide nominal mass measurements. So, it might be difficult to acquire unexpected compounds. Therefore, it could be regarded as the best choice for qualifications but not untargeted (Miller and Denton, 1986; Herrero et al., 2014). 37 Orbitrap MS -- This MS has been built for metabolites structural identification and is an excellent choice for providing MS/MS sensitivity with improved certainty when coupled with another mass analyzer. The first high-performance MS was introduced in early 2000, which uses electrostatic fields to capture ions. It is another mass analyzer of the Fourier transform series and are used in combination with other mass analyzers (Zubarev and Makarov, 2013). The ion source is linked to the curved linear trap (C-trap). The heart of orbitrap mass analyzer is an axially symmetrical mass analyzer, which consists of a spindle-shape central electrode (~5 v) surrounded by a pair of bell-shaped outer electrodes (ground potential) devoid of magnetic and radiofrequency field (Harris and Lucy, 2019). Orbitrap-based MS system joined to LC allows a possible resolution of generating incredibly high-resolution measurements of up to 500,000 resolution capabilities (Pan et al., 2020). An orbitrap MS has high mass resolution and is suitable for target, post target, and nontarget analysis of small molecules with no specific SRM transitions, thereby revealing unexpectant compounds in a sample (Farre et al., 2012). This property enables an accurate mass analysis with a mass accuracy ~3 ppm (Niessen and Falck, 2015). Under the name of Q-Exactive, a combination of orbitrap with a quadrupole mass analyzer has been introduced and became popular for proteomics and high throughput screening. Even though detached orbitrap systems have been produced, the full power of the orbitrap mass analyzer is maximized by its use in an MS/MS setting (Figure 1-11). Usually, samples can be introduced into the ion source by various methods either ESI or matrix-assisted laser desorption (MALDI). The injection flat pole passes ions in the source to the quadrupole. The quadrupole rod assembly acts as an ion transmission device, filters mass range and transmits ions according to its m/z values. Then, the ions are moved into the C-Trap and injected into the orbitrap mass analyzer to generate mass spectra report. Also, ions are momentarily pushed via the C-Trap into the high energy collisional induced cell (HCD) for the fragmentation analysis in combination with a quadrupole mass analyzer (Zubarev and Makarov, 2013). Fragmentation techniques such as HCD as well as a newly developed electron transfer dissociation (ETD) reagent ion source required are used to obtain structural information that is not possible by low-energy CID fragmentation (McAlister, 2007). Specifically, HCD in Orbitrap offers beam-type collisional dissociation like that obtainable in triple quadrupole instruments, as well as in quadrupole time-of-flight (Q-ToF) MSs that were previously the most widely used accurate mass instruments (Morris et al., 1996). One of many different operating modes for this 38 instrumental setup is all-ion fragmentation (AIF) technology, which applies HCD fragmentation to all ionized molecules without mass filtering (quadrupole not engaged) (Sentandreu et al., 2018). The fragment spectra generated in the HCD cell and detected in the Orbitrap show a fragmentation pattern comparable to the pattern of typical triple quadrupole spectra. Among the clear advantages of implementation of the HCD cell on the ion trap/Orbitrap hybrid instrument includes facilitating the production of de novo sequencing of peptides and building highly informative fragmentation libraries (Eliuk and Makarov, 2015). Orbitrap-based (MS/MS) system is capable and efficient in providing the resolution, mass accuracy, and speed anticipated by the analysts while eradicating the huge regular operational maintenance (as well as wide-ranging laboratory space and site preparation) essential for FT-ICR structures. The available mode features on Q-Exactive can perform all the functions on QqQ and Q-ToF, even with better sensitivity and selectivity. Consequently, in solving research questions about the qualitative nature, high resolution mass spectrometry (HRMS) such as an orbitrap, suits this challenge and applications (Eliuk and Makarov, 2015). Figure 1-11: Schematic for Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer There are several mass spectrometers commonly used in metabolomics analysis. Although some factors could affect the choice, which includes a mass range, availability, selectivity, 39 specificity, resources etc. In summary the strengths and weaknesses of some mass spectrometers are highlighted below (Table 1.7). Table 1-7. Strengths and Weaknesses of Orbitrap and Triple Quad Mass Spectrometric Methods Methods Strengths Weakness References Orbitrap Possess high mass resolution and resolving power with mass accuracy suitable for untargeted analysis. An important feature of the instrument is the automatic gain control (AGC) procedure, which uses a brief pre-scan in the linear ion trap to measure the ion current within the specified mass range. C-trap helps in subjecting ions into multiple injections and fragmented or selected under varying conditions can be accumulated and analyzed within a single Orbitrap spectrum One of the most expensive spectrometers. Analytes present in high-background matrices frequently exhibit reduced signal intensity compared to those in clean standard solutions. This effect arises from AGC-regulated reduction of ion injection time, implemented to control the total ion current. Farre et al., 2012. Hecht et al., 2025 Kaufmann & Teale, 2016 Triple Quad Reproducibility, and better limits of detection and quantification, compared with a single quadrupole mass spectrometer. High sensitivity make it suitable for trace analysis Low mass resolution. Best choice for quantification and not for untargeted. Herrero et al., 2014 TOF High scan speed. Resolving power for large m/z ions. Relatively high baselines noise than orbitrap. Kaufmann & Teale, 2016 1.11.2. The Application of Metabolomics The term âmetabolomicsâ was first used in 2000 by Oliver Fiehn to describe the phytochemistry of metabolites in any biological sample to create an image that would depict a metabolome in a complex system and understand the impacts of genetics, environment, or stressors (Turi et al., 2015b; Fiehn et al., 2000). Large datasets are generated from untargeted analysis of sample extracts using mass spectrometry or NMR (Mudge et al., 2019; Turi et al., 2014, 2015b; Turi & Murch, 2013). Statistical tools to analyze features in the complex untargeted datasets reveal previously unknown metabolites and relationships between metabolites for the discovery of new 40 biochemical pathways, bioactive compounds activated and deactivated forms of metabolites, precursors, and products of catabolism (Turi et al., 2015b; Erland et al., 2020b; Ye et al., 2021). Due to this approach, significant advances have been achieved in agriculture and food research to characterize food related material by achieving relevant information through statistical tools known as âchemometricsâ (Fernie and Schauer, 2008). The application of metabolomics has been used to generate new hypotheses for some pertinent issues or give clarity about the nature of biological systems. Metabolomics and HormonomicsDB approach were able to suggest new avenues of research about the mechanism of plant growth and regeneration under the influence of Thidiazuron application, of which the action has not been properly explained despite its use for over 40 years (Erland et al., 2020a). In another study, the approach was able to generate a model iron-mediated metabolism via shikimic acid pathway in a complex physiological phenomenon, plant morphogenesis (Erland et al., 2020b). Similarly, there has been inconsistent data on how probiotics promote health outcomes (Ye et al., 2021). Then, metabolomics technique was used to better understand the colonization and persistence of biotherapeutic products. Untargeted metabolomics data were able to unravel the processes involve in the modulation of microbiome health by live biotherapeutic products (Ye et al., 2021). In another study, targeted and untargeted metabolomics was adopted to assist in better knowing the aroma characteristics in chemovars during cannabis breeding, high potency chemovars are believed to selected by the cannabis breeder due to their scent notes. But these chemovars are known to have several phytochemicals that influenced their aroma. The approach was able to detect some terpenes which are compounds influencing the clandestine breeding programs (Mudge et al., 2019). Volatile metabolitesâExploratory GC-MS of hydro-distillate in the determination revealed the volatile for mosquito deterrence from dried male inflorescence (Jones et al., 2012). The detector was set in low and high mass ranges of 40 - 550 with MS quad temp of 150 ºC, and match spectra matching NIST chemical balance. The putative identification produced 30 peaks which revealed the presence of terpenes, aldehydes, fatty acids, and aromatic compounds within the mixture. Noestheden et al. (2017) examined the volatile phenols (VPs) in berries during the analysis of QqQ MS method. Initially, single quads have always been used for volatile compounds. Mudge et al. (2019) applied GC-MS with electron impact and mass spectra from 50 â 500 m/z to studying the terpenes in cannabis chemovars. The injector was in a split ratio of 10:1 to avoid overload and ensure adequate detection in the MS. In many instances, the purification of solid 41 phase extraction is required to avoid ion suppression in the MS, which can significantly impact the sensitivity and reliability of the results (Liu et al., 2020). Interestingly, Noestheden et al. (2017) has demonstrated the quantification, qualifying transitions, and MS voltages were optimized in similar manner to a single quad (Singh et al., 2012). The broad range of VP-glycosides created isobars resolution; however, it was proved that the tandem GC-MS/MS is appropriate in quantifying VPs even at low (2 n/g) and high (100 ng/g) standard recoveries. As a result, this approach has enabled the accurate quantification of VPs prior to fermentation of perceptible smoke-taint in the berries (Noestheden et al., 2017). Non-volatile metabolitesâIn studying plant morphogenesis, Erland et al. (2020a) used a ToF-MS metabolomic profiling to examine the physiological cascade that affects the metabolic resources and growth in filbert. This technique generated novel hypotheses for the morphoregulatory role of ferric ethylenediamine-N,Nâ-di-(ortho-hydroxyphenyl) acetic acid (Fe-EDDHA) in filbert shoot organogenesis in vitro. The MS was coupled to ESI at cone voltage of 60 ºC and desolvation temperature of 250 ºC in positive mode with mass range of 100 â 1000 amu. The ToF-MS resolved and generated the datasets of axenic filbert shoot at varying levels Fe-EDDHA within the specified mass range where they had different retention time in the drift region. This method profiled the ions from polar metabolites extracted with ethanol in an untargeted manner. In particular, the method identified phenolic conjugates and catabolites. A similar protocol has earlier been applied in Brown et al. (2012) to profile anthocyanin to explain the phytochemical diversity of five cranberry cultivars. Recently, the protocol with ToF-MS generated global fingerprints which led to the generation hypotheses that explain the plant growth regulation in Wollemi Pine (WP) and Norfolk Island Pine (NIP) about brassinosteroids and indolamine metabolism (Giebelhaus et al., 2023). Furthermore, Giebelhaus et al. also demonstrated the use of Xevo TQ-S QqQ in single ion reaction (SIR) mode, which detected, and had quantifiable levels of melatonin in samples of WP and NIP within the concentration of 300.4 ± 30.0 ng/g and brassinolide with 84.7 ± 44.2 ng/g for WP and NIP had 217.4 ± 139.6 ng/g respectively, above the LOQ standards. Metabolites derived from colon using Live Biotherapeutic Products (LBPs) were examined via orbitrap MS with heated ESI in positive mode at a full MS range between 100 - 1200 m/z. The metabolites ion was mapped into databases and showed differences in serotonin, antioxidant production and bile acids accumulation between the LBPs (Ye et al., 2021). The addition of MS has proven to be a great technique in generating fingerprints of molecules in a complex biological sample due to its relative resolution and mass 42 accuracy of the monoisotopic masses in a molecule/analyte of interest. HRMS (Orbitrap and ToF/Q-ToF) have proven to be the best choice for untargeted metabolomics while QqQ is regarded as the gold standard for targeted approach, especially small molecules with specific transitions. However, the resources for HRMS are always twice that of QqQ. 1.12. Mass Spectrometry Data Processing through Software Proprietary/commercial software programs available data processing LC-MS and GC-MS data such as: MassHunter Profinder by Agilent Ltd; Compound Discovererâ¢ by ThermoFisher Inc.; Progenesis QI by Waters Ltd (Agilent Technologies, 2019; Peralbo-Molina, 2023). But they are prohibitively expensive. On the other hand, open sources are free to use and versatile in their applicability, in data processing (Fernández-Ochoa et al., 2020), for example, MZmine, MS-DIAL and XC-MS (Pluskal et al., 2010; Tsugawa et al., 2015; Smith et al., 2006). However, features quality and detection of true positive features has been the challenge in the workflow of XC-MS, while the performance of MZmine and MS-DIAL are close (Cambiaghi et al., 2017). Particularly, MZmine has been one of the most widely used software in the data processing, since its official release in 2004. The latest version have new functionalities like a highly interactive graphical user interface (GUIs) and improved detection workflow, which allow users to make better informed decisions (Schmid et al., 2023; Chang et al., 2021). The processing of the software is generally done by using the following key steps, such as peak picking, resolving, smoothing, alignment, and gap-filling (Hendriks, 2011). Peak picking is defined as the conversion of raw data acquired by instruments like a mass spectrometer into a list of peaks (Rafiei and Soleno, 2014). The chromatograms are always built by the ADAP chromatogram builder module over a minimum number of consecutive scans to derive the extracted ion chromatogram (EIC). The EIC if jagged, which can lead to inaccurate peak integration can be smoothen. In addition, the algorithm further separate partially co-eluting peaks into individual features through local minimum resolver. Untargeted mass spectrometry (MS) experiments conducted on multiple samples aim to qualitatively or quantitatively compare the analytes detected across the analyzed set. However, chromatography-MS experiments are subject to instrumental drift, which leads to fluctuations in retention time (RT), ion mobility, and mass accuracy throughout the analysis. As a result, the same analyte is rarely detected with identical RT, and m/z. Feature alignment addresses these variations by aligning features corresponding to the same molecular entity across different instrument runs. 43 This process allows for the merging of feature lists from multiple samples into a unified, aligned feature table. Through peak finder module, the MZmine offer the chance to re-introduce low-intensity signal that might have been omitted or filtered (Smith et al., 2015; Heuckeroth et al., 2024). MS-DIAL is also an open-source software for identification of data-independent acquisition (DIA) coupled to the tandem MS/MS. DIA-type mass fragmentation are common in hard ionization in GC-MS as against the LC-MS/MS approach. Therefore, the development of MS-DIAL introduces the deconvolution algorithm for DIA datasets. Firstly, the data is converted from mzML to Analysis Base File (ABF) for rapid retrieval to spot the precursor ion using retention time and accurate mass, then, deconvolute according to their precursor range. Additionally, MS2 Dec algorithm the extract the product spectra from each precursor ion to achieve the alignment between the precursor ion and product ion, without background noise or co-eluting metabolites in the MS/MS spectrum (Tsugawa et al., 2015). 1.13. Statistical Modeling for Metabolomics Data In providing comprehensive support for complex data generated from metabolomics studies, Metaboanalyst serves as friendly web-based platform that processes the data for univariate, multivariate, enrichment and functional analyses (Xia et al., 2009, 2012, 2015). Earlier versions (1.0 to 3.0) of MetaboAnalyst were built primarily for statistical and functional analysis of targeted metabolomics data. Recent versions (4.0 to 6.0) have incorporated a new module to support functional analysis from LC-MS peaks, meta-analysis, and integration with other omics data (Pang et al., 2024). In functional analysis to detect the underlying metabolomic patterns, this involves the identification of statistically significant associations with the metabolites and pathways (Garcia-Campos et al., 2015; Nguyen et al., 2019). As such, metabolomics provides deeper understanding regarding complex biochemical relationships and incorporate orthogonal information about the features under study (Picart-Armada et al., 2017). Through the use of either m/z or m/z and retention time, mummichog algorithms are widely used for the functional metabolomic analyses. The mummichog algorithm involve the use of univariate statistical models for example t-test or ANOVA for the identification of significant features through the differences in the distribution of concentration or their intensities while not accounting for covariance and correlation in the data (Xia et al., 2010). One of the advantages of the univariate analysis is their easy interpretation (Alonso et al., 2015). Moreover, the assumption is that metabolites are 44 independent of each other. However, this may not be absolutely right since the metabolites are linked to each other through complex networks. Unlike multivariate analysis, it considers simultaneously all the metabolic features necessitating the need for data reduction to reduce data dimensionality. Hence, the use of multivariate methods, principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) with increased sensitivity to detect thread in the data. Consequently, Kuligowski et al. (2024) identified the contributions of functional analysis and multi-variate discriminant methods, although they have been reviewed independently. The study recommended the possibility of integrating results from multivariate models with functional metabolic analysis through orthogonal PLS-DA (OPLS-DA). Therefore, univariate and multivariate analysis are complementary approaches, both are essential in metabolomics analysis. (Peralbo-Molina et al., 2023). The strengths and weaknesses of the highlighted analyses and others are summarized below (Table 1-8). Table 1-8. Strengths and Weaknesses of the Statistical Concepts Model Strengths Weakness References PCA Reduce dimensionality in large datasets and identify variance regarded as principal components which are orthogonal. PCA may not prioritize class separation (focuses on explaining the overall variance in the data, which may or may not correspond to class separation. Ruiz-Perez et al., (2020) PLS-DA Full awareness of class labels. Maximize the covariance of the input variable while detecting the latent variables in the labels. It is prone to overfitting. Cross validation is required. It is prone to noise. Mishra et al., (2017) OPLS-DA Predictive scores are less subject to orthogonal variation. Easy illustration of the predictive information in the components. Ability to differentiate between predictive and non-predictive. Computational analysis is complex. Cross validation is required. Trygg & Wold, (2002). Bylesjö et al., (2006) Student t-test Simple interpretation between two groups. Size of sample when small. means are affected by outliers. Mishra et al., (2019) 45 Model Strengths Weakness References ANOVA Suitable for comparing groups of three or more. Post hoc tests determine the significant pairs. Post hoc tests required for pair-wise multiple comparison Mishra et al., (2019) Volcano plot Convenient for quick deductions that are involved in the class model of interest. It entails the use of preliminary statistical and mathematical knowledge. Santello, (2021) SAM It uses a non-parametric approach to identify significant features while providing the estimate of the false discovery rate. The cut-off might get rid of features that are relevant. Tusher et al., (2001) Pattern Hunter Metabolites identified based on how well their behavior follows a defined trend. The output depends on the chosen patterns. Ma et al., (2002) *PCA - principal component analysis PLS-DA â partial least square-discriminant analysis OPLS-DA â orthogonal partial least square-discriminant analysis 1.14. Study Rationale It is estimated that by the year 2050, there will be >9 billion people to feed, and an increase of >60 % of the current global food production is required to meet this reality (FAO, 2022). Underutilized crops are important sources of macro- and micronutrients that contribute to improved food and nutritional security (Liu et al., 2020; Erland et al., 2022). Breadfruit (Artocarpus altilis Parkinson (Fosberg)) and the hybrids (Artocarpus altilis Ã Artocarpus mariannensis) is one such important underutilized crop with the potential to improve food security listed in Annex 1 of the International Treaty on Plant Genetic Resources for Food and Agriculture (Liu et al., 2020). With >3,000 breadfruit varieties selected across the Pacific and the Caribbean Islands, breadfruit shows a wide range in phenotypic and morphological properties, nutritional compositions, taste, and flavour (Jones et al., 2011a; Ragone et al., 2006). Selection of elite varieties with pleasing flavour profiles and widespread use requires an understanding of the taste chemistry. A sensory evaluation panel compared the flavours of 20 breadfruit cultivars and identified cultivars with intense flavour (Ragone and Cavaletto, 2006). Flavour is the human experience modulated by the interaction of food chemistry (Chen et al., 2022) with taste and odor receptors and is perceived differently between people (Kader, 2008). The specific compounds 46 responsible for the taste of breadfruit are not known, and to fully understand the flavour diversity of breadfruit varieties, there is a need to use a technique that would simplify this complex system. Metabolomics is the use of high-resolution analytical instruments and statistical tools to discover and characterize all the small metabolites in a biological sample (Fiehn et al., 2000; Turi et al., 2015b). A metabolomics approach was recently used to characterize flavour and to identify key chemical components of unpalatable taste in the tropical fruit tree, Baccaurea ramiflora (Chen et al., 2022). The application of metabolomics in fruit quality studies has revealed changes in the metabolic profiles of sugars and organic acids closely related to sugar and acid metabolism, as well as other important nutrients, including anthocyanins, L-amino acids, free fatty acids, and phenolic acids (Xu et al., 2018; Zhu et al., 2018). Therefore, metabolomic changes can be considered a biomarker of fruit quality (Monti et al., 2016). The current study seeks to understand the physicochemical properties of fresh and cooked breadfruit by using untargeted metabolomics to discover the phytochemistry responsible for flavour intensity. I hypothesized that unknown specific metabolites in breadfruit varieties are responsible for the flavour intensity. To test this hypothesis, I compared 3 varieties previously characterized as having intense flavours (Porohiti, Meiarephe, Meinpadahk) with 3 varieties identified as having a mild flavour profile (Ulu fiti, Yap, Maâfala). Here, I describe the key components of breadfruit flavour which can help inform variety choice to improve acceptability of breadfruit-based products and uptake of breadfruit, leading to improved food security globally 1.14.1. Thesis Objectives The overall objective of my thesis is to identify and quantify specific phytochemicals responsible for the taste of breadfruit. To achieve this target, my specific objectives were: 1. To determine the broad physicochemical properties and phytochemical composition of a subset of breadfruit varieties identified as having a mild or intense flavour profile, including pH, titratable acidity, sugars, phenolics, tannins, proteins, vitamins, and so on. 2. To discover differences in metabolic profiles of breadfruit varieties identified as having mild or intense flavour by untargeted metabolomics and phytochemical discovery methods. 3. To characterize the flavour profile of a set of commercially prepared breadfruit flours from different geographies as either mild or intense based on the taste metabolites present. 47 4. To make recommendations for breadfruit variety selection, methods of preparation, and use for future producers of breadfruit food products. 48 Chapter 2: Understanding the Flavour Chemistry of Breadfruit Varieties through Phytochemical Assays 2.1. Synopsis of Chapter The selection of the six cultivars from the 20 examined by Ragone and Cavaletto et al. (2006) was based on their lower or higher in flavour intensity, compared to the other cultivars in the study. While the remaining cultivars scored within the moderate range for aroma and flavour in the sensory evaluation, the breadfruit flavour largely mirrored the aroma characteristics. These six stood out for their differencesâeither mild or intense flavour characteristics. Porohiti, Meiaraepe, and Meinpadahk exhibited the most intense flavour while Yap, Maâfala, and Ulu fiti are mild in their flavour characteristics. The breadfruit underwent moderate processing, resulting in a form similar to that presented to the panelists during the sensory evaluation by Ragone and Cavaletto (2006). Several differences have been reported among different breadfruit cultivars, particularly in terms of their morphology traits, physicochemical properties, and phytochemical composition (Jones et al., 2011a, 2011b). Breadfruit cultivars vary in their attributes, which can impact the acceptability and application of the crops in processing of foods and food products. However, information is still limited in the morphological traits, physicochemical properties, and phytochemical composition of breadfruit that would align with our anticipated phenotype trait of interest, which is the âflavour intensity.â For instance, proteins have been shown to vary in breadfruit cultivars, containing all the essential L-amino acids (Liu et al., 2015; Golden and Williams, 2001; Morton, 1987). Therefore, assessing these chemical compounds, along with others, is crucial to uncover the potential links to the intense flavour observed in certain cultivars. 2.1.1. Objectives The objective of this study is to scientifically investigate and evaluate if the differences in the morphological traits, physicochemical properties, and phytochemical composition correlate with the flavour profiles. The specific objectives are: 1. To determine differences in growing regions, island of origin, morphology, yield, and other phenotypic characteristics among the breadfruit cultivars. 2. To determine differences in acid levels, titratable acidity, total protein, amino acids, sugars, phenols, vitamins, antioxidant potential, and other chemical parameters. 49 2.2. Materials and Methods 2.2.1. Breadfruit sample collection and preparation The breadfruit varieties used in the study were selected from Ë300 accessions of breadfruit (Aa and Aa Ã Am), breadnut (A. camansi Blanco), and dugdug (A. mariannensis Trecul.) domiciled at the Kahanu Garden of National Tropical Botanical Garden (NTBG) in Maui, Hawaii (20°47â²57.07â³N, 156°02â²18.42â³W). The germplasm collection was established by NTBG botanists and represents 34 Pacific Islands, the Philippines, Indonesia, and Seychelles. The micro-climate in the breadfruit collection has an average minimum temperature of â19 °C, average maximum temperature of â27 °C, and a mean annual precipitation of â2050 mm (Western Regional Climate Centre; www.wrcc.dri.edu/) at 15 m elevation (Jones et al., 2011a, 2011b). Three mature breadfruits were collected from each individual tree of each of the breadfruit varieties. The fruit was weighed, peeled, quartered, cored, seeds removed, and weighed again to determine the edible weight. One-half of each breadfruit was sliced, dried, and ground into flour, and the remaining half of each breadfruit was frozen before shipping to UBC for analysis (Jones et al., 2011a, 2011b; Liu et al., 2015, 2020). Samples for this study were stored frozen following harvest in vacuum-sealed bags at â 80 °C. Previous studies on the sample set have characterized and found differences in minerals, proteins, L-amino acids, vitamins, and starches composition (Figure 2-1) (Jones et al., 2011a, 2011b;; Liu et al., 2015; Kehinde et al., 2022). Specific varieties for the current study were selected to represent those identified as having more or less intense flavour profiles in a prev1ious taste panel (Ragone and Cavaletto, 2006; Table 2-1). 50 Figure 2-1. The workflow for the Mild and Intense flavour varieties of breadfruit (Picture created on Biorender). (a) the removal of peels and seeds, dicing the pulp, and washing. (b) increasing the surface area through milling (c) Packed in a container free of air and transporting the flour/samples through air travels (d) pretreatment and extraction (e) separation and detection (f) chemometrics and metabolomics analysis. Table 2-1. Breadfruit (Artocarpus altilis and A. altilis Ã A. mariannesis) selected for flavour intensity (Ragone and Cavaletto, 2006) Variety Name Species/Hybrid Ploidy Island of Origin Latitude coordinate Equator distance Flavour Intensity Meiaraepe Hybrid Diploid Pohnpei, FSM 6°57â²07â³N 479 Intense Meinpadahk Hybrid Triploid Pohnpei, FSM 6°57â²07â³N 479 Intense Porohiti A. altilis Triploid Moorea, S. Islands 17°32â²00â³S 1209 Intense Maâafala A. altilis Diploid Upulo, Samoa 13°55â²15â³S 960 Mild Ulu fiti A. altilis Diploid Fiji Island 17°59â²59â³S 1241 Mild Yap Hybrid Triploid Koror, Palau 7°14Ì57â³N 500 Mild 51 2.2.2. Reagents D-Glucose Assay Kit (GOPOD Format) (Wicklow, Ireland). AccQFluor kits, comprising borate buffer (AccQFluor 1), reagent powder (AccQFluor 2A), and reagent diluent (AccQFluor 2B), and AccQ Tag (6-aminoquinolylN-hydroxysuccinimidyl carbamate, AQC) were purchased from Waters (Milford, USA). Norleucine (Sigma Chemical Company, Louis, USA) stock solution (2.5 mM) was made in 0.1N HCl. DPPH (2,2-diphenyl-1- picrylhydrazyl; free radical; Sigma, USA), methanol (HPLC grade; Fisher Scientific, USA), Trolox ((Â±)-6-hydroxy-2, 5, 7, 8-tetramethylchromane-2-carboxylic acid; 97 %; Sigma), BCA (Bicinchoninic Acid) protein assay kits (Thermo Fisher Scientific, Manassas, USA), 4-(2-Hydroxyethyl)-piperazine ethane sulfonic acid (HEPES) (fine white crystals, bioreagents; Fisher Scientific), Tween 20 (PhytoTechnology Laboratories®, Shawnee Mission, USA), Hemoccult Sensa (Beckman Coulter, USA). 2.3. Chemical Analyses 2.3.1. Acidity Determination Measurements of titratable acidity (TA) followed standard protocol (Adebowale et al., 2020). In brief, 10 g of each of the breadfruit flour was suspended in 100 mL of MilliQ water for about 30 min to form a slurry solution. The slurry was centrifuged (Eppendorf Centrifuge 5810R) at 1800 Ã g for 10 min. Potentiometric method was adopted for this determination, the pH meter (VWR Symphony Portable Bench Top with Thermo Scientific Orion pH Electrode) was calibrated with buffer 4, 7 and 10 respectively at 25 °C. The measurements of the hydronium ions were first obtained for each sample in room temp in triplicates. The solutions were titrated with 0.1 N NaOH solution on a Fisher Scientific Isotemp Stirrer till the reaction reaches an equivalent point at pH 8.2. The volume (v) of the titrant was used to get the percentage of the TA in the samples. The milli equivalent weight of malic acid (67.05) was used in the calculation (Eqn. 2.1). % TA = (N of titrant Ã v of titrant Ã milli. equivalent of acid/weight of the sample) Ã 10 (Eqn. 2.1) N = normality, v = volume 52 2.3.2. Brix and D-Glucose analysis The protocol for determining Brix levels followed a method previously established by Adebowale et al. (2020). Calibration of the Hanna Instruments Portable Refractometer H196801 was conducted according to the manufacturer's instructions. This calibration involved the use of a plastic pipette to apply MilliQ water as a blank, ensuring it was shielded from light to minimize or eliminate any light-induced interference, until the instrument registered zero. For the analysis, 200 mg of the ground breadfruit samples were dissolved in 1 mL of MilliQ water, and centrifuged at 1800 Ã g for 10 min (Eppendorf Centrifuge 5810R). The zero-water standard on the prism was gently absorbed with Kimwipe to avoid scratching the prism surface, and left until dried. A plastic pipette was used to transfer the drip sample and completely covered the prism surface before taking the measurements. The determination of D-Glucose concentration in breadfruit flour was performed according to the Megazyme D-Glucose Assay Procedure (Megazyme Co. Wicklow, Ireland). Glucose oxidase/peroxidase (GOPOD) Buffer containing 50 mL of p-hydroxybenzoic acid and sodium azide (0.095 % w/v) with pH 7.4 was diluted to 1L with MilliQ water to obtain Solution I. The GOPOD Reagent Enzymes were dissolved in 20 mL of Solution I to derive the GOPOD Determination Reagent. Briefly, 100 mg of the flour was weighed and dissolved in 10 mL MilliQ water. 3.0 mL of GOPOD Reagent was added to 0.1 mL of the sampleâs solution containing D-glucose in triplicate and incubated in a water bath (Rose Scientific Limited, Edmonton, Canada) at 45 °C for 20 min. Absorbance reading was taken at 510 nm on Thermo Electron Mass Spectronic 20D+ with cuvette of light path length 1 cm and compared against the reagent blank (MilliQ water and GOPOD Reagent) to obtain the absorbance for samples and glucose standard (mg/ml). The amount of D-glucose in the sample was calculated based on the equation below: D-glucose (µg/mg) = (Sample absorbance/D-glucose standard (100 µg)) Ã 100 (Eqn. 2) 2.3.3. Reducing Sugars Determination The determination of reducing sugars was conducted using the Schoorl Method (AACC, 2000). Briefly, 5 g of breadfruit samples were dissolved in MilliQ water to a final volume of 250 mL. To neutralize the solution, 1.5 g of calcium carbonate (CaCOâ) was added, followed by the addition of alcohol to reach half the total volume. This mixture was then boiled for 2 hours and 53 allowed to cool overnight. The resulting solution was diluted to the mark with 95 % methanol. A 150 mL portion of the supernatant was transferred to a 400 mL beaker and evaporated until approximately 20 - 30 mL remained. The residue was then quantitatively transferred into a 100 mL volumetric flask, and 1 g of neutral lead acetate was added. The mixture was shaken and allowed to stand for 15 min. Filtration was carried out into a 150 mL flask, and the filtrate was precipitated with sodium carbonate (NaâCOâ). To the filtered solution, 10 mL each of Fehling solutions A and B were added, and the volume was adjusted to 50 mL using an appropriate aliquot of the breadfruit solution and water. The mixture was then boiled for 2 min and cooled under running water. Finally, 10 mL of 30 % potassium iodide (KI) solution was added, followed by 10 mL of 25 % sulfuric acid (HâSOâ), and the solution was titrated immediately using 0.1 N sodium thiosulfate (NaâSOâ). 2.3.4. Total Phenol and Flavonoid Content Methods for detection of total phenolics were modified from previously published works by Adebowale et al. (2020) and Badejo et al. (2016). To prepare the sample, 1 g of the breadfruit was dissolved in 10 mL of methanol (1g/10mL). Then, an aliquot of 0.2 mL of the mild and intense dried breadfruit sample extracted in methanol was mixed with 2.5 mL of 10 % Folin-Ciocalteau reagent (Sigma-Aldrich, Canada) and 2 mL of 7.5 % sodium carbonate. Gallic acid (Sigma-Aldrich, Canada) was used as the standard and expressed as milligram gallic acid equivalent (GAE) per g of sample at a concentration range of 0 to 200 mg/L. The mixture was incubated in the dark for 30 min at 45 °C and the absorbance reading was taken at 750 nm on a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Vermont, USA) using Gen5: Microplate Data Collection and Analysis Software (BioTek). The flavonoid content was determined using the aluminum chloride colorimetric method described by Adebowale et al. (2020) and Badejo et al. (2016). An aliquot of 0.2 mL of the extract was added to 0.3 mL of 5 % NaNO3. 0.6 mL of 10 % aluminum chloride and 2 mL of 1 M NaOH were added to the mixture after 5 and 6 min respectively. An additional 2.1 mL of distilled water was introduced to the mixture. The absorbance reading was taken at 510 nm on a Synergy HT Multi-Mode Microplate Reader (BioTek) against the reagent blank data expressed as mg rutin equivalent per g of the sample. 54 2.3.5. 1,1-diphenyl-2-picrylhydrazyl (DPPH) Determination for Antioxidant Potential The determination of antioxidant potential were modified from previously published analytical methods according to Adebowale et al. (2020) and Turi and Murch, (2013) to test for radical-scavenging ability in breadfruit. In brief, 200 mg of the breadfruit flour was dissolved in 10 mL methanol to create a 3:2 (v/v) dilution and this was prepared by dissolving the stock solution in 800 µL of methanol. A serial dilution series in methanol within the working range of 0.6 μL to 0.079 μL was made. Trolox was used as the standard, prepared serially from 10 â 50 μM Trolox solution. 0.039 mg/mL DPPH (Sigma-Aldrich, Canada) in methanol was prepared. The wells were filled with 100 μL of either the prepared sample or Trolox solution. Then, 100 μL of DPPH was added to these wells. To prepare the blank wells, 100 µL of methanol was added to wells separately containing 100 μL of sample, DPPH, and Trolox. The absorbance was read by a Synergy HT Multi-Mode Microplate Reader (BioTek) using Gen5: Microplate Data Collection and Analysis Software (BioTek). The antioxidant potential was assessed against DPPH scavenging ability as the change in absorbance at 562 nm for 20 min and the reaction blanks to determine the 50 % reduction in radical production. The equation below was used for the calculation: % DPPH = (Sample corrected absorbance)/(Corrected DPPH) Ã 100 (Eqn. 3) 2.3.6. Vitamin C Determination The determination of vitamin C content was done using a modified version of the method described by Zhang et al. (2013). Briefly, 500 mg of ground breadfruit samples were dissolved in 250 mL of MilliQ water. A 25 mL aliquot of the resulting solution was then transferred to another 250 mL of distilled water. The iodometric method was employed for vitamin C quantification. Specifically, 25 mL of potassium iodate (KIOâ) was added to 0.2 g of potassium iodide and 1 mL of sulfuric acid. The resulting mixture, containing excess iodide, was then reacted with 10 mL of the prepared vitamin C solution, which was quantitatively transferred into the iodate solution. The mixture was stirred vigorously to allow vitamin C to reduce the iodine. A back-titration method, using sodium thiosulfate (NaâSâOâ) as the titrant, was employed to determine the unreacted iodine. The amount of vitamin C in the sample was calculated by subtracting the moles of unreacted iodine from the total excess iodine, and the corresponding amount of vitamin C was determined in both mild and intense-flavour breadfruit varieties. 55 Vitamin C (mg/g) = No. of moles of vitamin C. Ã molar mass Ã 1000 (Eqn. 4) 2.3.7. Total Protein Analysis The total protein content of the breadfruit flour was determined using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific, Montreal, Canada) as previously described in Jones et al. (2011a); Kehinde et al., (2022) and following manufacturer instructions. Briefly, 10 mg of the flour was mixed with 1 mL of protein extraction buffer (20 mM HEPES, Fisher Scientific, Mississauga, ON; 150 mM NaCl, Fisher Scientific, Mississauga, ON; 0.3 % Tween 20, MP Biomedicals, California, USA), and then sonicated for 20 min at room temperature. 25 μL of protein extract and bovine serum albumin standards (BSA) with a working range of 125 - 2000 μg/mL) was pipetted into a 96-well plate in triplicate (Bio-Rad Laboratories, Ontario, Canada) with 200 μL protein working solution, then heated for 30 min at 35 °C. The absorbance was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek) using Gen5: Microplate Data Collection and Analysis Software (BioTek) at 562 nm. The protein content of the ground flour was calculated from the linear equation in standard linear curve with varying concentrations of BSA standard solutions. The protein working solution was used as a blank to determine any contribution from the reagents. 2.3.8. Tannin Determination The tannins concentration in breadfruit was determined according to AOAC (1995) using the Folin-Denis reagent with minor modifications. In brief, 1 g breadfruit flour was extracted in 25 mL of deionized water at room temperature for 24 hours with continuous agitation. The mixture was then centrifuged at 4000 g for 5 min. Sodium carbonate (Na2CO3) was prepared by dissolving 35 g anhydrous in 100 mL. The solution was heated to 70 â 80 °C until complete dissolution. Then, the solution was allowed to cool overnight. The crystallization process was performed through filtering through glass wool. Aliquots of 0 to 1 mL from the standard tannic acid solution into 15 mL centrifuge tubes containing 7.5 mL H2O. Subsequently, 0.5 mL of Folin - Denis reagent and 1 mL Na2CO3 solution were added. The final volume was diluted with deionized up to 10 mL. Finally, the mixture was homogenized, and absorbance A was taken after 30 min at 760 nm. A calibration curve was generated by plotting A against the concentration of tannic acid (mg/100 mL). Calculations: 56 Grams tannic acid/10 mL of aliquot = (mg tannic acid/10 mL)/1000 Ã Reciprocal of dilutions. 2.3.9. L-amino acids Determination The L-amino acids were determined as described in Liu et al. (2015) via AccQFluor kits, comprising borate buffer (AccQFluor 1), reagent powder (AccQFluor 2A) and reagent diluent (AccQFluor 2B), and AccQ Tag (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, AQC) purchased from Waters (Mississauga ON). In brief, 200 mg of each frozen sample (6 varieties Ã 3 replications) was weighed and transferred to a 1.5 mL MCT graduated natural tube (Fisherbrand, Mississauga, ON). In a CentriVap DNA Vacuum Concentrator (Labconco, Kansas City, USA) the sample was placed for an 18 h drying process, at 1725 RPM. The output was weighed to determine the dry matter, reduced, and put to a screw cap Pyrex vial (17 Ã 60 mm) (Fisherbrand). The vial containing aliquot of 20 μL of the 2.5 mM norleucine internal standard was added to 2000 μL 6.0 N HCl was quickly capped after flushing with nitrogen. In a standard heating block (VWR, Radnor, USA), the hydrolysis was performed at 110 °C for 19 h. An aliquot of 1 μL of the hydrolyzed protein sample from the 500 μL hydrolyzed sample filtered through an Ultrafree MC Centrifugation Unit (Fisherbrand), was diluted to a total volume of 40 μL with borate buffer. The sample was derived by reacting a 20 μL diluted sample with 20 μL AccQTag derivatizing reagent in 60 μL borate buffer. The prepared samples for injections were put in 250 μL glass conical insert bottom spring (6 Ã 29 mm) (Canadian Life Science, Peterborough, ON), and held inside a screw cap injection vial. To complete the reaction, the vials were subjected to 55 °C for 10 min in the heat block. The samples were analyzed with Waters 2659 the high-performance liquid chromatography (HPLC) Separations Module comprising of a multi wavelength (λ) fluorescent detector (Waters 2475) and a photodiode array detector controlled by Empower 3 software (Waters) and data system. Then, 10 μL derivatized sample were injected into the HPLC instrument using a 4 μm NavaPak C column (3.9 Ã 300 mm) (Waters). The gradient elution of eluent A was ACCQ Tag/water (10/90, %v/v) and eluent B was acetonitrile/water (50/50, %v/v). The emission wavelength of 395 nm with an excitation wavelength of 250 nm through a period of 75 min. Table 2-2. HPLC gradient elution for the breadfruit amino acid study Time (min) Flow (mL/min) %A %B Gradient Curve 1 100 0 57 Time (min) Flow (mL/min) %A %B Gradient Curve 2 1 93 7 6 4 1 90 10 6 28 1 80 20 3 32 1 75 25 8 38 1 61 33 4 50 1 35 65 6 52 1 0 100 6 55 1 100 0 6 60 1 100 0 2 Eluent A: ACCQÂ·Tag/water (10/90, v/v) and Eluent B: acetonitrile/water (50/50, v/v) Genesis 1.7.5 (Institute for Genomics and Bioinformatics; Graz, Austria) was used to build the essential amino acid profile. All statistical analyses were conducted using RStudio Version 0.98.1062 (RStudio Inc. USA) with a type 1 error rate of 0.05. Essential amino acid data for other staples listed in this article are reported by the Food and Agriculture Organization of the United Nations (FAO, 1981). 2.3.10. Correlation-Based Network The correlation calculator was used to arrange the features and metabolites of interest. After which, the uploaded data were normalized and autoscale, annotated using the build-network option on Metascape plugin in Cystoscape 3.10.1. The Pearsonâs correlation coefficient (Pearsonâs r) parameter was set between -1 to 1 and threshold value at p<0.05 (Basu et al., 2017). 2.4. Results 2.4.1. Preliminary Data Analysis of Breadfruit Cultivars Using the data from Ragone and Cavaletto (2006), as the independent variables âmildâ and âintenseâ flavours varieties, I conducted a re-analysis of existing datasets within the Murch Lab for the specific selected cultivars. Data for 3 mild and 3 intense flavour cultivars was extracted from the large datasets for protein, minerals, vitamins, , starches, and fibers previously published by Murch lab students Jones, Liu, and Kehinde. Analysis of these data discovered increased magnesium, phosphorus, and calcium in the intense cultivars and a significant difference in the phosphorus (p-value, 0.05). Among all the vitamins, vitamin C appeared to be evident in the 58 intense cultivars. Interestingly, the total dietary fiber in the intense flavour varieties exhibited more percentage composition (5.72 %) than the mild cultivars (3.45 %), equally, the carbohydrates is marginally higher in the intense flavour cultivars. To put together, these data provide a foundation for my studies to understand nutritional profiles in breadfruit (Figure 2-2a-d). Figure 2-2a: Figure 2-2. Nutritional Composition of Breadfruit (A. altilis, A. altilis x A. mariannensis). (a) sodium, (b) phosphorus (c) potassium, (d) vitamin C (e) Carbohydrates (CHO) (f) total dietary fibre. Blue bars represent the means for the mild cultivars and green bars represent means for Intense cultivars. Error bars represent the standard error of the means. Significance difference (p<0.05) was observed using t-test with between the means (N=3). 2.4.2. Breadfruit Morphological Attributes The development of plants in the tropics is believed to be influenced by environmental signals, such as shifts in light intensity and quality, sun declination, and variations in precipitation (Borchert et al., 2005; Yeang 2007). The distance from the equator of both mild and intense breadfruit varieties shows no significant difference (Table 2-1). Our data indicates that intense 59 varieties appeared to have a slightly longer fruiting season as compared to mild varieties (Figure 2-2a). The climatic conditions of the islands of origin for these breadfruit varieties indicate significant differences in parameters such as temperature, precipitation, humidity, and number of rainy days. However, the average number of sunny days remained consistent across these regions and was not significantly different (Figure 2-3b). The difference in the climatic parameters is possibly a contributing factor to the morphological differences (Jones et al., 2013). The results for both external and internal morphological attributes of the varieties are presented in Table 3-3. The breadfruit size, fruit weight, peeled weight, and total fruit flesh are economic traits contributing to the productivity of a breadfruit variety. The mild flavour varieties were significantly larger, longer, and wider than intense varieties. Interestingly, the percentage of the eaten portion is marginally higher in the intense varieties compared to the mild (Table 2-3). External attributes (skin texture, skin color, and peel weight) exhibit no significant difference in the varieties. The breadfruit is monoecious, the male inflorescence in the axils of breadfruit varieties leaves, data summary from January 1996 to January 2006 compiled from Jones et al. (2011a), showed that between April and September months over the decade exhibited the peak season in the breadfruit varieties (Figure 2-2a). From the data, the intense appeared to have higher marginally presence of the male inflorescence than the mild during this period which would drive the development of the flowers into fruitlets. The climatic condition of the island of origin for the breadfruit varieties demonstrated that these regions are different in parameters, for example, temperature, precipitation, humidity, and rainy days except average of sunny days. Comparatively, differences in climatic parameters are likely among the environmental factors that influenced variations in the morphological properties of breadfruit cultivars, as reported by Jones et al. (2013) (Table 2-3). Table 2-3: Breadfruit morphological quality properties Mild Intense Fruit Weight (g)* 1722.89 ± 227.12 977.89 ± 322.44 Length (mm)* 22.08 ± 1.38 14.28 ± 0.36 Shape 3.7 ± 0.26 3.83 ± 0.37 Width at the middle (mm)* 13.45 ± 0.41 11.84 ± 0.26 Width at the top (mm)* 7.42 ± 0.48 8.94 ± 0.37 Width at the base (mm)* 7.13 ± 0.28 7.97 ± 0.20 60 Mild Intense Skin Texture 4.73 ± 0.27 4.33 ± 0.23 Color 2.4 ± 0.26 1.93 ± 0.10 Others Peel weight (g) 118.56 ± 17.29 88.78 ± 9.92 Peeled weight (g)* 1604.33 ± 210.52 889.11 ± 106.38 Seed weight (g)* 19.22 ± 8.66 n.a. Total fruit flesh (g)* 1418.33 ± 205.85 820 ± 98.70 % Edible portion 81.52 ± 1.43 83.7 2 ± 0.42 *n.a. â not applicable (seedless) Figure 2-3a: 61 Figure 2-3b: Figure 2-3. Growth of Different Varieties of Breadfruit (Artocarpus altilis) and Its Hybrids (Artocarpus altilis Ã Artocarpus mariannensis) determined by geographical origin and season (Jones et al. 2010). Season, climate and origin did not differ for the mild and intense flavour varieties chosen for this study. (a) Male inflorescence of selected mild and Intense breadfruit varieties, (b) Climatic conditions of the genetic origins of selected breadfruit varieties with different in flavour intensities i- temperature, ii- rainfall, iii- sunlight, iv-humidity. Bar represents the standard error of 24 replicates within each treatment. 2.4.3. Physicochemical Properties The intense flavour varieties were significantly less acidic (pH = 6.15 ± 0.07) than the mild varieties (pH = 5.59 ± 0.27) (Figure 2-4a; p<0.05). There was no significant difference observed in the titratable acidity between mild and intense flavour varieties with an average value of 0.18 %TA (Figure 2-4b). There was no significant difference detected in the D-glucose levels (Figure 62 2-4c; p>0.05). The soluble solids (Brix) were marginally higher in intense varieties 2.35 °B ±0.45; Figure 2-4d). No significant difference was observed between mild and intense flavour varieties in the measurements of total polyphenols (Figure 2-4e; p>0.05). Significant differences were observed in the total flavonoids (Figure 2-4f; p<0.05)). Mild varieties had significantly more protein (32.66 ± 2.73 mg/100mg), than the intense flavour varieties (21.12 ±2.43 mg/100mg) (Figure 2-4g; p<0.05). Our analysis showed that the intense flavour varieties had significantly higher ascorbic acid than the mild varieties (16.778 ± 0.0002 mg/100g Figure 2-4h; p<0.05). The measure of reactive oxygen species (ROS) scavenging ability demonstrated that the intense flavour varieties have a higher significant antioxidant potential than mild varieties and significantly less amount of sample from intense varieties is required to detoxify 50 % of the reactive oxygen species (ROS) (0. 0303 ± 0.002 ppm) as compared to mild varieties 3.17 ± 0.183 ppm) (Figure 2-4i; p<0.05). The intense flavour varieties had marginal amount of reducing sugars (Figure 2-4j). There was no significant difference between the tannins, although marginally higher in the mild (Figure 2-5; p>0.05). Consistent with the observed protein levels, the L-amino acids analysis showed marginally higher levels of 15 out of 19 L-amino acids in the mild varieties which includes leucine, phenylalanine, alanine, serine, histidine, glycine, arginine, threonine, valine, methionine, isoleucine, tryptophan, lysine, and cysteine (Figure 2-6a). The correlation analysis identified links between the samples and the L-amino acids. From the data, aspartic acid and tyrosine appeared to have stronger correlation with the mild varieties than with the intense ones (Figure 2-6b). 63 64 Figure 2-4. Physicochemical analyses of the Mild and Intense flavour breadfruit varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The spectrophotometric analyses were performed on Microplate Reader. The flavonoids, proteins, soluble solids and DPPH exhibit significant differences between the groups. (a) pH, (b) Titrable Acidity (potentiometric method, %malic acid), (c) d-glucose (Megazyme assay), (d) Brix - soluble solids, (e) Total Phenols (gallic acid equivalent), (f) Total Flavonoids (rutin equivalent), (g) Total Protein content (Bicinchoninic acid assay), (h) Vitamin C - Iodine Titration, (i) DPPH (2,2-diphenyl-1-picrylhydrazyl). Plot represents mean±standard error of mean calculated from the nine replicates in each variety (N=9). Yellow denotes increased in mild and blue denotes increased in intense 65 Figure 2-5b: Figure 2-5. L-amino acids Determination in the Mild and Intense Breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) through high performance liquid chromatography (HPLC) method. (a) quantity of each of the amino acids in each variety group. Yellow denotes mild and yellow denotes intense. (b) Correlation of the L-amino acids. Blue shape indicated a known compound. Pink indicated positive correlation. Thickness signifies the strength of the correlation. Figure from Metascape on Cystoscape 3.10.1. 66 67 Figure 2-6. (a) Total Tannins in Mild and Intense Flavour Varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). (b) Sodium Bisulfite Treatment (0.03 %) to the Mild and Intense Breadfruit (c) Sodium Bisulfite Treatment (0.3 %) to the Mild and Intense Breadfruit. Plot represents mean±standard error of mean calculated from the nine replicates in each variety (N=9). Yellow denotes increased in mild and blue denotes increased in intense. analyses were performed on Microplate Reader. 68 2.5. Discussion In this study, the intense breadfruit varieties have demonstrated desirable qualities in its morphological, physiological, and phytochemical compositions. In addition to breadfruit been resilient crop to changing climatic conditions (Erland et al., 2022). Relativity, the intense flavour varieties appeared to have a higher percentage edible portion. Even during the busiest months of its availability, the edible portion further gives the economic reason for its propagation. Thus, edible portion likely corresponds to the motive of the Lapila people that prioritize the clonal propagation of seedless, and starchy breadfruit across the Pacific Islands, and not flavour or other organoleptic properties (DeLoughrey, 2007; Jones et al., 2011a, 2011b, 2013). Phenotypic and metabolic diversity in the fruits and vegetables from wild relatives, could be natural or human breeding mediated (Zhu et al., 2018). The diversity of breadfruit is manifested as variation in not only its flavour but nutritional composition, leaves, fruit colour, texture etc. (Jones et al., 2011a) and this has been demonstrated in Table 2-3. In particular, flavour is an elusive trait and could determine the acceptability and applications of foods (Knee, 2001; Roberts-Nkrumah and Badrie, 2005). According to the findings of Bakare et al. (2020) that studied the acceptability of cookies made from one cultivar of breadfruit flour, which has been reported in literature in relation to the acceptability of breadfruit products in a certain region, however, I noticed that there are some gaps in their findings. Specifically, the addition of an edible fish meal could possibly have affected or masked the flavour and taste of the breadfruit variety used. In fact, the identity of breadfruit varieties was not stated that could help in differentiating the breadfruit from many other varieties. In addition, the high temperature used could have affected the sensorial properties of the flour; as a result, the information provided is still sparse. Then, it is imperative to understand the compounds responsible for this trait, using an unbiased and comprehensive technique. To start with, I commence the findings using Ragone and Cavaletto (2006) as a basis which evaluated 20 breadfruit varieties for the understanding that represented the vast diversity in breadfruit metabolites, the study discovered no significant difference in the sweetness between the mild and intense flavour varieties. Sweetness is most defined as the difference in sugar content of the foods consumed. Considering physiological perspective, one could consider sweetness to be the generally appetitive sensation, that arises when sugars or other sweet compounds stimulate specialized receptor proteins expressed in a subset of cells in taste bud (Trumbo et al., 2020). The human soft palate contains taste buds, the main organ of taste is classically considered the tongue, 69 and the primary structure that house the sensory endings are the papilla (Azzali et al., 1996). Taste buds are the primary sensory unit of the taste system and are imbedded under the keratinous layer of the papillae with a taste pore exposed to the external milieu. At least 5 types of cells make up a taste bud: type 1, 2, 3 cells, basal cells, and neuronal processes. Evidence now suggests that each taste modality is mutually exclusive to a subset of individual taste cells or 1 taste modality for 1 taste cell. This varies from individual to individual. Also, ASTM international defined as perception resulting from stimulating a combination of the taste buds or combined effect of taste sensations, aromatics, and chemical feeling factors evoked by a substance in the oral cavity (ASTM, 2009). Consequently, sensory processing at the level of the taste bud is complex (Zhang et al., 2003; Gravina et al., 2013). In understanding taste metabolism, it is practical to approach from the angle of the interaction of these metabolites with the taste buds, and the significant intensity that would provide beyond threshold. To this effect, the Association of scientists recommended the adoption of methods that rely on partitioning details of the flavour into portions, that can be evaluated, understood, and acted upon. The intensity or amount of a particular attribute is only one part of flavour; the length of time it lasts, which attributes are dominant at any given point, how the attributes blend together, and how they compare to our mental picture of the product, or similar products all impact the actual âflavourâ of a product (Caul, 1957). The correlation between ºBrix and sweetness is low in many instances because the soluble solids determined may include sugars, organic acids, and polyphenols. Kehinde et al. (2022) noted that despite hybridization having effects on starch quality in breadfruit varieties, there was no significant difference (p<0.05) in starch yield between A. altilis and A. mariannesis. This trend is like the D-glucose and reducing sugars discovered in this study. But the marginal difference recorded could signify impart to the sweetness and health benefits. Mulberry fruit is rich in glucose, sucrose, and fructose (Przeor et al., 2020). Adewusi et al. (1995) also explained that sucrose, D-glucose, and D-fructose followed a similar pattern as the reducing sugars in a variety of breadfruit. These might not be significant difference due to their family trait, but the marginal differential levels in these sugars might be a pointer to their uniqueness (Sahu et al., 2019). L-amino acids and proteins contribute umami taste to foods (He, 2021). Proteins containing L-glutamic acids and aspartic acids can influence the perception of taste as umami; also known as the âsavoryâ or meaty taste (Yamamoto and Inui-Yamamoto, 2023). Jones et al. (2011ab) reported that Maâafala is an elite mild variety that is higher 70 in protein than other breadfruit varieties. Specifically, proteins could influence the perception of flavour and taste like other macro ingredients via van der Waals interaction, and covalent or electrostatic linkages (Kinsella, 1989). OâNeil (1996) reported that chemical modification of β-lactoglobulin by ethyl esterification of a proteinâs carboxyl side chains, and reduction of disulfide bonds with sodium sulfite lowered the apparent binding constant to 2-nonanone, while forming a large number of binding sites. Suppavorasatit and Cadwallade (2012) successfully demonstrated that enzymatic deamidation by glutaminase decreased the overall binding affinity of vanillin, and maltol with soy proteins. Since the ultimate goal is to recover bound volatile compounds and restore a complete aroma profile during food consumption, methods that can destabilize the hydrophobic interior regions of proteinsâby removing or substituting reactive functional groups (e.g., free âNHâ or âSH groups)âwarrant further attention. Based on these findings, I presumed that there is influence from protein concentration, acidity, and other chemicals on flavour perception. Interestingly, my data showed that the mild exhibits significantly higher protein level than the intense (Figure 2-4g; p<0.05). Built on this finding, there is a possibility of protein and flavour interaction more in the mild which could lead to reduced intensity, with strong binding in the mild varieties due to high concentration of amino acids such as cysteine, and lysine (Figure 2-5ab). Comparatively, Land et al. (1996) discovered that a substantial decrease in the flavour intensity occurs when the concentration of protein is improved regardless of the type of protein involved. In addition, the interaction mechanism of protein and flavour are influenced by the intrinsic and extrinsic factors such as pH, with binding with protein decrease from pH 5 to 7 and denatured at extreme pH of 3 and 11 (Wang and Arntfield, 2015; 2017). My data showed that the level of pH in the varieties is also significant (p<0.05) (Figure 2-4a). Recent findings proposed covalent bonding and soluble complexes as possible result of interactions between tannins and proteins (Hagerman et al., 2012), which was demonstrated in Figure 2-6abc, with sodium bisulfite concentration leads to the release of the tannins. Although diverse food chemical composition may affect the perception of flavour, flavour and protein has been the most diverse due to varying L-amino acids chains, terminal ends, and hydrophobic pockets (Reineccius, 2006). Polyphenolic compounds such as tannins form complexes with either precipitating them or causing sufficient conformational changes that they lose their lubricating power, thus, making the mouth feel rough, puckery, and dry (Lawless and Corrigan, 1994). Proline provides a flat, rigid, open, hydrophobic surface favorable for association 71 with other flat, rigid, hydrophobic structures such as the aromatic rings of gallic acid in galloyl-glucose or other hydrolysable types of tannins (Lawless et al., 1996). Despite the relatively marginally higher levels of tannins in the mild flavour varieties, however, with covalent bonding the lower perception of the bitterness from the tannins will be perceived (Figure 2-6a). Even though tannins can still aggregate with protein far from the iso-electric point. But at a pH level around the isoelectric point, proteins aggregate more readily due to no net electrical charge (Hagerman and Butler, 1978). An alternate route for formation of covalent tanninâprotein complexes involve oxidized protein. Under oxidizing conditions, phenolics react covalently with nucleophilic amino acid side chains for example lysine or cysteine (Pierpoint, 1969). Protein oxidation products may react with phenolics to yield new products, not obtained with native proteins (Figure 2-7). Reactive oxygen species (ROS) or high-energy radiation promote protein oxidation (Davies et al., 1995). Scavenging of the ROS was found to be lower in the mild flavour varieties (Figure 2-4i). In addition, I observed that the intense flavour varieties scavenge free radicals by IC50 in 10 min at lesser amount than the mild varieties which explains the potential to scavenge free radical generation at a relatively reduced quantity (Figure 2-4i) and richer in vitamin C (Figure 2-4h) content even as non-enzymatic antioxidant that could cause off-flavour (Gordon et al., 2020). As many natural compounds play key roles in taste properties of food, vitamins have varying taste properties according to their structure, which can influence the sensorial quality of foods. In a study carried out by Schiffman and Dackis (1975) on vitamin C supplements evaluation, they described the taste as sour and fruity, with a pleasant taste noted by half of the judges, and an unpleasant taste for the other half. Also, Delompré et al. (2019) affirmed that vitamin C possesses sour taste with antioxidant properties. This suggests their enhanced potential for scavenging free radicals at reduced quantities, and being richer in vitamin C content, even as an antioxidant that might cause off-flavour (Gordon et al., 2020). Badejo et al. (2012) explained that the change in ascorbate levels during fruit maturation is a trait dependent on the species, hence, the possible difference between the mild and intense flavour varieties. The difference in pH between the varieties may affect the action of the enzymes for instance ascorbic acid oxidase, and ascorbic acid peroxidase as well as its degradation (Aloni et al., 2008; Chen and Asada, 1989). Different groups are known to contribute to different tastes (Robinson, 2015). Phenols and flavonoids; organic 72 acids; and total soluble solids have been linked to bitter and astringency flavours in many foods (Drewnowski and Gomez-Carneros, 2000). 2.6. Conclusion and Further Analysis This study highlights the importance of addressing the noticeable intense flavour from the breadfruit, and the economic benefits to their region; through the yield indicating higher percentage eaten portion, towards ensuring food and nutrition security. In addition, its health benefits through attributes to antioxidant properties, which enhance the ability to scavenge free radicals, better with the intense breadfruit varieties that could induce oxidative stress, and other inflammations potentially leading to various diseases. Based on the data generated in this study, there is a likelihood of a complex interaction between the chemical compounds, which appear to be affecting the chemistry of flavour formation. In particular, proteins and phenols through Schiff base, and Michael addition (Figure 2-7). Given the nutritional benefits of these varieties, and their dominance as source of local food crops in some places, the need to determine the metabolites, and biochemical pathways synthesizing the compound through global profiling is imperative. To deepen the understanding of the morphological, physiochemical properties, and phytochemical profiles, it is advisable to further look at this interaction using metabolomics technology. Figure 2-7. Possible Proteins and Phenols Structural Changes + pH Oxidation 73 Chapter 3: Metabolomics-guided Discovery of Flavour Chemistry in Breadfruit Varieties using Ultra-High Pressure Liquid Chromatography â Mass Spectrometry 3.1. Synopsis Despite its promising potential to enhance food security, breadfruit remains an underutilized species in the global markets (Ragone, 1997; Jones et al., 2011a, 2011b). Although, Omobuwajo (2003) earlier revealed that biscuits from 100 % biscuit were scored low in overall quality. Similarly, Oke et al. (2005) observed that the breadfruit flour in doughnut lowers the âlikenessâ in a taste panel. Taste is a primary determinant of consumer preference for breadfruit (Roberts-Nkrumah and Badre, 2005). Then, a sensorial evaluation organized at NTBG, and University of Hawaii was able to discover some differences in flavour intensity among some breadfruit species and hybrids (Ragone and Cavaletto, 2006). Based on the outcome of this sensorial study, the perceived attribute discovered could be as a difference in the breadfruit chemical composition of the cultivars, potentially driven by underlying biochemical pathways. This mechanism remains unclear, I hypothesize that unknown metabolites in âPorohitiâ, âMeinpadahkâ, and âMeiarepheâ are responsible for its high flavour intensity and are either absent or at low levels in less intense flavour cultivars (âMaâafalaâ, âUlu fitiâ, and âYapâ). Hence, it is important to determine the polar metabolites underlying the formation of the taste compounds in the breadfruit varieties, through untargeted LC â high resolution mass spectrometry (HRMS) approach. 3.2. Objectives The specific objective of this study was to discover the unknown metabolites responsible for flavour intensity in the breadfruit through untargeted metabolomics, and to generate novel hypotheses about the metabolic processes that modulate the flavour intensity perceived in the breadfruit. 3.3. Materials and Methods 3.3.1. Breadfruit Sample Collection and Preparation The sample collection used for the study has been described in Chapter 2, section 2.2.1. The workflow for untargeted metabolomics analysis is described in Figure 2-1. 74 3.3.2. Reagents Acetonitrile, methanol, and acetic acid, OptimaTM LC-MS grade (Fisher Scientific, Ottawa, ON, Canada) were used as solvents. Water was deionized, and purified to 18.2 MΩ Ã cm MilliQ water system (ThermoFisher Scientific). A standard mixture at three dilution levels containing 50 selected metabolites, representing a range of polarities, was used for instrument calibration, and retention time prediction as previously described (Giebelhaus et al., 2022). 3.3.3. Liquid Chromatography â High Resolution Mass Spectrometry Breadfruit tissues (~100 mg) were sampled while frozen directly into pre-weighed 1.5 mL Eppendorf microcentrifuge tubes. Samples were suspended in 1mL of MilliQ water. The tissues were boiled for 15 min in a water bath (Rose Scientific Limited, Edmonton, AB) at 100 °C to simulate cooking. The cooked samples were centrifuged at 13000 Ã g for 3 min (VWR Galaxy 16DH centrifuge). The supernatant was decanted and filtered by centrifugation (0.2 μm Ultrafree-MC, Millipore, Mississauga, ON, 13000 Ã g for 1 min). Aliquots of 100 µL of each were diluted in 200 µL of acidified methanol to precipitate any residual protein and 200 µL of the extract was quantitatively transferred into amber glass autosampler sample vials, stored at â 20 °C until use in chemical and metabolomic analyses (Figure 2-1) (Erland et al., 2020; Ye et al., 2021). Aliquots (10 µL) of extracts, blanks, and standards were separated by reverse phase chromatography using a Phenomenex Kinetex EVO C18 column (2.1 Ã 150 mm, 1.7 µm; Torrance, CA, USA) at 30 °C. Metabolites were eluted by a gradient of (a) 0.1 % formic acid in water and (b) acetonitrile with an initial condition of 95:5 (a:b), reaching 5:95 (a:b) at 25 min and 95:5 (a:b) at 30 min using a re-equilibration time of 5 min at initial starting conditions (Thermo Scientific Vanquish UHPLC linear system). Compounds were ionized by electrospray ionization (heated electrospray ionization (HESI) probe and detected with a Q-Exactive Hybrid Quadrupole-Orbitrap (ThermoFisher Scientific). The mass detection settings were as follows: spray voltage:â7 kV; sheath gas pressure: 48; aux gas flow rate: 11 mL/min; sweep gas flow rate:1 mL/min; capillary temperature: 250 °C; auxiliary gas heater temperature: 300 °C, S-lens RF level: 50.0, scan mode - Full MS â Full MS resolution: 70,000; polarity: positive; scan range: 120 â 1800 m/z; AGC target: 1e6; loop count: 5; aux gas heater temp: 300 °C. The data were collected in Data Dependent Acquisition (DDA) mode. The total method duration was 20 min (Ye et al., 2021; Giebelhaus et al., 2023). 75 3.3.4. Data Processing and Exporting Mass Spectrometry data were processed in MZmine 3.2.8 open-source software (Pluskal et al., 2010). Mass detection was performed with baseline peak intensity of 3 Ã 104 to remove noise while retaining lower intensity peaks. Feature detection was performed using ADAP chromatogram builder with a group intensity threshold at 3 Ã 104, and a minimum highest intensity of 7 Ã 104. The generated chromatograms were smoothed using Savitzy-Gozay algorithm after deconvolution through Local Minimum Resolver setting border points bands with a chromatographic threshold of 90 %, minimum absolute height of 3 Ã 104 , and minimum ratio peak of 1.70. C13 isotopes between the 0.0010 m/z tolerance were removed from the features, and alignment was performed using Join Aligner to generate a matrix of features across samples. Gap filling was performed on this feature list with an intensity tolerance of 20 %. The feature list was then exported for downstream analysis including feature-based molecular networking, and functional analysis on MetaboAnalyst Ver. 6. 3.3.5. Detection of Significant Features and Functional Analysis Filtering normalization, transformation, and scaling of data normalization were done on MetaboAnalyst Ver. 6 (www.metaboanalyst.ca/) prior to the chemometric analyses. The volcano plot was used to visualize the level of the statistical significance, and fold change between the groups. Pattern search analysis and significance analysis of microarray (SAM) was used as a tool to determine homologous significant features. Features with both positive and negative correlation (Pearson r) between the samples that showed increase and decrease across the flavour intensity were examined (Erland et al., 2020; Ye et al., 2021). Significant features identified by pattern search hunter were used to generate the pathways (p<0.05) that are upregulated or downregulated in intense flavour varieties. The Pattern Hunter output was formatted as three columns (m/z, p-value, and t.score) with a mass accuracy of 5 ppm, and submitted to mummichog, and gene set enrichment analysis (GSEA) for ranking by t.score. Identified metabolites were matched to Arabidopsis thaliana in Kyoto Encyclopedia Biomolecules of Genes and Genomes (KEGG) database (www.genome.jp/kegg/pathway.html/) (Erland et al., 2020). 76 3.3.6. Synthetic Biotransformation Following previously established protocol, using the algorithms in Microsoft Excel, which simulated the following possible enzymatic reactions +H2, âH2, âCH3, +CH3, +C6H12O6, +OH, +OH (Ã2), +COOH, +NH2, +NH3 (Murch et al., 2004; Turi and Murch, 2013). The monoisotopic mass + H for each biotransformation (±0.02 Da), were mined within our dataset in order to identify potential anabolic or metabolic products, as described above, for significant compounds exhibiting linear trends. 3.3.7. Putative Metabolite Annotation The significant features derived through pattern search hunter were identified in FooDB (M+H, ±0.02, v1.0, https://foodb.ca/) as per published protocol in Sumner et al. (2007), annotated and mapped using the KEGG Compound database (https://www.kegg.jp/kegg/compound/). The authentic standards were not analyzed in the untargeted analysis to make comparison, but the metabolites separated at least two orthogonal informationâmonoisotopic mass and retention timeâand were putatively identified according to Metabolomics Standards Initiative (MSI) Level 2 to have unique metabolites. 3.3.8. Statistical Analysis of Data All analyses in the study were performed in triplicate. For statistical comparisons between treatments, the results were subjected to Studentâs t-tests using Microsoft Excel 2016, with differences considered significant at p<0.05. Data transformation and chemometric analyses were conducted using the MetaboAnalyst ver 6.0 platform. 3.3.9. Correlation-Based Network The Correlation calculator in Cystoscape 3.10.1 was used to organize the features and metabolites of interest. The uploaded data were normalized and autoscaled, then annotated using the built-network option on Metascape plugin. The Pearsonâs correlation coefficient (Pearsonâs r) parameter was set to range from -1 to 1, with a significance threshold of p<0.05 (Basu et al., 2017). 3.4. Results 3.4.1. Data Quality and Characterization 77 Chemical fingerprints associated with mild and intense flavour variation were generated using untargeted metabolomics analysis. Three replicate extracts were prepared from each frozen sample representing pseudoreplication. The extracts contained between 3145 and 5347 distinct features detected in the mass spectrometry analysis. Precision of the extraction, chromatography and feature detection was between 2 and 10 % for all of the extractions and analyses. There were 100 to 233 unique features characteristic of each the varieties. Interestingly, there were ~1900 features found in the mild varieties, Maâafala, and Yap that were not found in Ulu Fiti. According to the data, we observed ~450 features, and below in the intense flavour varieties that were not present in the mild varieties. This information was used in putting together a dataset for thorough and detailed compound identification (Table 3-1). However, the principal component analysis did not find an individual distinguishing feature that accounted for significant variance between the mild and intense treatment groups (Figure S3). The supervised partial least squares discriminant Analysis (PLS-DA) was utilized, as it can effectively mitigate the effects of individual outliers. Features identified by the variable importance in projection (VIP) scores in PLS-DA demonstrated R2, Q2, and an accuracy of ~1 with 5 components (Figure 3-1ab). With positive Q2 the model is not overfitted (SzymaÅska et al., 2012). Through the analysis and mapping to FooDB database, I discovered classes of carboxylic acids and derivatives, fatty acyls, benzenoids, organonitrogen, phenols, pyridines and derivatives, and piperidines. Further putative identification discovered the following pathways, biosynthesis of plant secondary metabolites, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism, biosynthesis of unsaturated fatty acids, lysine biosynthesis, and shikimate pathway (Table 3-2). Table 3-1. Quality Control for the Features in the Mild and Intense Flavour Varieties Variety Maâfala Ulu fiti Yap Porohiti Meinpadahk Meiaraepe Treatment Group Mild Mild Mild Intense Intense Intense Average Features 4850 3145 4995 5347 4285 4997 %RSD 2.5 5.9 1.6 1.5 10.1 2.9 Unique to an Individual Variety 160 206 317 242 129 117 Features Found in 2 of 3 of either Mild or Intense Flavour Varieties Ma'afala and Ulu Fiti 194 Ma'afala and Yap 1890 Ulu Fiti and Yap 206 Porohiti and Meinpadahk 212 78 Variety Maâfala Ulu fiti Yap Porohiti Meinpadahk Meiaraepe Features Found in 2 of 3 of either Mild or Intense Flavour Varieties Porohiti and Meiaraepe 452 Meinpadahk and Meiaraepe 91 Average Features by Data by Treatment Group Treatment Group Mild Intense Average Features 4330 4876 %RSD 21 11 Unique to each treatment 176 233 Figure 3-1a. Scores plot in PLS-DA between the Mild and Intense Flavour Varieties 79 Figure 3-1b. Significant features in the PLS-DA Figure 3-1c. Model validation Figure 3-1. Significant features for the Mild and Intense flavour varieties through partial least squares-discriminant analysis (PLS-DA) (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The PLS-DA identified features (VIP) scores. This analysis was performed on MetaboAnalyst ver. 6.0. (a) scores plot for the mild and intense, (b) Features identified between the groups, (c) accuracy of the components. 80 Table 3-2. Putatively Identified Top 10 Features identified as significant by the Partial Least Squares-Discriminant Analysis (PLS-DA) for Intense Flavour Cultivars m/z monoisotopic mass Putative ID Class Associated KEGG Pathways 132.1021 131.0946 L-Leucine Carboxylic acids 6-Deoxyfagomine Piperidines Biosynthesis of plant secondary metabolites Aminocaproic acid Fatty Acyls Phenylpropanoid biosynthesis 166.0864 165.0789 (Â±)-Phenylalanine Carboxylic acids Ethyl anthranilate Benzenoids Biosynthesis of unsaturated fatty acids 346.259 345.2668 Eicosapentaenoyl Ethanolamide Organonitrogen compounds 293.2112 292.2038 cis-12-Oxophytodienoic acid Fatty Acyls alpha-Linolenic acid metabolism [7]-Paradol Phenols Shikimate pathway 182.0814 181.0739 L-Tyrosine Carboxylic acids Phenylpropanoid biosynthesis 4-Hydroxy-4-(3-pyridyl)-butanoic acid Pyridines and derivatives Butanoate metabolism 275.2007 274.1933 Apo-13-zeaxanthinone Prenol lipids Biosynthesis of various alkaloids Lysyl-Lysine Carboxylic acids Lysine biosynthesis 3.4.2. Detection of Significant Ions for The Polar Metabolites The volcano analysis shows that the data are normally distributed; 159 were significantly upregulated, and 57 significant features downregulated in intense flavour varieties (Figure 3-2a). Pattern search hunter analysis, which hinges on the homologous features, identified 16 features that are most significantly increased in the intense flavour varieties (Figure. 3-3c). Pathway analysis using an increasing model (mild<intense) discovered three overrepresented biochemical pathways (p<0.05) in the intense flavour varieties. Furthermore, pathway analysis can be used to better understand the trans-formation relationship between various metabolites and substrates, and to identify key metabolic pathways and key metabolites. The significant features demonstrated in the PLS-DA, volcano plot, and pattern hunter were used to build a model for PCA (Figure 3-3ab). The features in the loading plot appeared in all the statistical models used for the PCA. 81 Figure 3-2a. Volcano plot for the significant features Figure 3-2b. Heatmap cluster analysis 82 Figure 3-2c. Pattern Hunter Search for the significant features in Mild and Intense Figure 3-2. Detecting the significant features for the polar metabolites in Mild and Intense Flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) through Chemometrics analyses on Metabonalystver6.0. Filtering, normalization, transformation, and scaling of data normalization were performed prior to the analyses. The significant features correlate across the analyses (a) Volcano plot with the Fold change between the groups showed 159 significant features upregulated and 57 significant features downregulated. (b) Heatmap cluster analysis (c) Pattern hunter analysis, Pearson r as a choice of distance measure. 83 Figure 3-3a. Scores plot analysis for the significant features Figure 3-3b. Loading for the significant features Figure 3-3. Principal Component Analysis for the significant in the PLS-DA, Volcano plot and Pattern Hunter between the Mild and Intense flavour for the polar metabolites. Filtering normalization, transformation, and scaling of data normalization prior to the analyses. The features mapped to FooDB (M+H, ±0.02; v1.0) and classes of the metabolites were studied (a) Scores plot analysis for the significant features, (b) Loading for the significant features in the varieties. 84 The significance analysis of microarray (SAM) indicated 49 significant metabolites between the mild and intense flavour varieties (Figure 3-4ab). The analysis showed that metabolites within the class carboxylic acids and derivatives mutually ~50 percent of the overall metabolites. Particularly, the subclass appeared to be L-amino acids, peptides, and analogues followed by fatty acyls. Figure 3-4a: Significance analysis of microarray Figure 3-4b. Signifiance metabolites proportion Figure 3-4. Summary of Significance Analysis of Microarray (SAM) between the Mild and Intense flavour for the polar metabolites. Filtering normalization, transformation, and scaling of data normalization prior to the analyses. The features mapped to FooDB (M+H,±0.02;v1.0) and classes of the metabolites were studied (a) SAM plot showing the significant features, (b) the proportion of the significant metabolites in the varieties. 85 3.4.3. Functional Analysis MetaboAnalyst Ver. 6.0 analysis of pathway regulation revealed that phenylpropanoid biosynthesis was significantly increased in intense flavour varieties (Figure 3-5a). In addition, the most significant hits from the datasets come from the phenylpropanoid biosynthesis, diterpenoid biosynthesis, alpha-linolenic acid metabolism, and biosynthesis of various plant secondary metabolites (p<0.05) (Figure 3-5ab). The relative intensities of the pathways were identified (S4- S7). Additionally, the dataset was further mined and detected the peaks to pathways analysis which identified 233 features unique to the intense flavour varieties. These features were mapped and annotated by some pathways that are significant to the varieties. The results of the analysis generated cyanoamino acid metabolism, pyrimidine metabolism, flavone and flavonol biosynthesis for Meinpadahk, Porohiti and Meiarephe (1-2-1) (Figure 3-6ab). Additionally, alpha-linolenic acid metabolism, glycerolipid metabolism, n-glycan biosynthesis, caffeine metabolism and pyrimidine metabolism were also discovered for the reverse model (2-1-2) (Figure 3-7ab). The Artocarpus genus is known to be rich in secondary metabolites, phenylpropanoid (Nomura et al., 1998). Interestingly, examination of these distinct features showed that while Meiaraepe and Meinpadahk are hybrid varieties of breadfruit, it is possible that there could be a unique composition of metabolites in Meinpadahk different to the other intense flavour varieties, Porohiti and Meiarephe and can be same to either of these three. Orthogonal partial least squares-discriminant analysis (OPLS-DA) with GSEA was used to detect pathways associated with the models. The pathway revealed alpha-linolenic metabolism as one of the significant pathways (Figure 3-8ab). Additionally, the pattern hunter search with GSEA indicated pathways like arachidonic acid metabolism, glucosinolate biosynthesis and phenylpropanoid biosynthesis (p<0.05) (Figure 3-9ab). 86 Figure 3-5a. Peak to pathways analysis (Mild<Intense) 87 Figure 3-5b. Results for Mummichog and Gene Set Enrichment Analysis (GSEA) Pathway Total_size All_Hits Sig_Hits P_vals α-linolenic acid metabolism 26 12 12 0.005 Diterpenoid biosynthesis 29 16 15 0.006 Biosynthesis of various plants sec. metabolites 29 12 10 0.037 Phenylpropanoid biosynthesis 43 22 18 0.038 Figure 3-5. Untargeted metabolomics profiling for the polar metabolites through LC-MS/MS in the Mild and Intense Flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The significant features from Pattern Hunter were used for the analysis and mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Mummichog and Gene Set Enrichment Analysis (GSEA) as the algorithm for functional analysis and Arabidopsis thaliana as the model plant (a) Peak to pathways analysis (Mild<Intense), (b) Results for mummichog and GSEA. Blue areas are pathways significantly affected based on mummichog analysis, pink by GSEA and the purple were significantly modified in both analyses (p<0.05). âTotal sizeâ indicates total number of compounds in Arabidopsis thaliana KEGG pathways, âAll.Hitsâ the number of matches in the data set (expected and significant) based on m/z value after mapping to KEGG pathways, âSig.Hitsâ the number of significant matches based on the analysis. âPvalsâ are the combined p-values of both tests using Fisherâs method. 88 Figure 3-6a. Peak to pathways analysis (Mild<Intense). 89 Figure 3-6b. Results for Mummichog and Gene Set Enrichment Analysis (GSEA) (1-2-1). Pathway Total_size All_Hits Sig_Hits P_vals Cyanoamino acid metabolism 29 20 11 0.026 Pyrimidine metabolism 41 21 11 0.040 Flavone and flavonol biosynthesis 10 3 2 0.043 Figure 3-6. Untargeted metabolomics analysis for unique pathways for the Intense flavour varieties (Meinpadahk, Porohiti and Meiarephe). The significant features from Pattern Hunter were used for the analysis and mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Mummichog and Gene Set Enrichment Analysis (GSEA) as the algorithm for functional analysis and Arabidopsis thaliana as the model plant. (a) Peak to pathways analysis (Mild<Intense). (b) Results for mummichog and GSEA (1-2-1). Blue areas are pathways significantly affected based on mummichog analysis, pink by GSEA and the purple were significantly modified in both analyses (p<0.05). âTotal sizeâ indicates total number of compounds in Arabidopsis thaliana KEGG pathways, âAll.Hitsâ the number of matches in the data set (expected and significant) based on m/z value after mapping to KEGG pathways, âSig.Hitsâ the number of significant matches based on the analysis. âPvalsâ are the combined p-values of both tests using Fisherâs method. 90 Figure 3-7a. Peak to pathways analysis (Mild<Intense). 91 Figure 3-7b. Results for Mummichog and Gene Set Enrichment Analysis (GSEA) (2-1-2) Pathway Total_size All_Hits Sig_Hits P_vals α-linolenic acid metabolism 26 13 12 0.066 Caffeine metabolism 10 7 7 0.128 Pyrimidine metabolism 41 21 17 0.155 Figure 3-7. Untargeted Metabolomics Analysis for Unique Pathways for The Intense Breadfruit varieties (Meinpadahk, Porohiti and Meiarephe). The significant features from Pattern Hunter were used for the analysis and mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Mummichog and Gene Set Enrichment Analysis (GSEA) as the algorithm for functional analysis and Arabidopsis thaliana as the model plant (a) Peak to pathways analysis (Mild<Intense), (b) Results for mummichog and GSEA) (2-1-2). Blue areas are pathways significantly affected based on mummichog analysis, pink by GSEA and purple were significantly modified in both analyses. p<0.05. âTotal sizeâ indicates total number of compounds in Arabidopsis thaliana KEGG pathways, âAll.Hitsâ the number of matches in the data set (expected and significant) based on m/z value after mapping to KEGG pathways, âSig.Hitsâ the number of significant matches based on the analysis. âPvalsâ are the combined p-values of both tests using Fisherâs method. 92 Figure 3-8a. Peak to pathways analysis (Mild<Intense). Figure 3-8b. Results for Mummichog and Gene Set Enrichment Analysis (GSEA) Pathway name Total Hits Hits (sig) P_value Pyrimidine metabolism 41 21 21 0.01 Tyrosine metabolism 17 14 14 0.04 Arginine and proline metabolism 32 21 20 0.05 α-linolenic metabolism 26 12 12 0.06 Figure 3-8. Functional analysis for the mild and intense breadfruit varieties through orthogonal partial least squares-discriminant analysis (OPLS-DA) for the Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The OPLS-DA model for the discriminant features. Gene Set Enrichment Analysis (GSEA) as the algorithm for functional analysis and Arabidopsis thaliana as the model plant (p<0.05). 93 Figure 3-9a: Peak to pathways analysis (Mild<Intense) Figure 3-9b. Results for Mummichog and Gene Set Enrichment Analysis (GSEA) Pathway name Total Hits P_value Arachidonic acid metabolism 9 2 0.02 Glucosinolate biosynthesis 65 3 0.02 Phenylpropanoid biosynthesis 43 7 0.02 Carotenoid biosynthesis 43 5 0.05 Valine, leucine and isoleucine degradation 37 2 0.05 Valine, leucine and isoleucine biosynthesis 22 2 0.05 Phenylalanine metabolism 12 2 0.05 Biosynthesis of unsaturated fatty acids 22 2 0.05 Figure 3-9. Untargeted metabolomics profiling for the polar metabolites through LC-MS/MS in the Mild and Intense flavour breadfruit varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The significant features from Pattern Hunter were used for the analysis and mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Gene Set Enrichment Analysis (GSEA) as the algorithm for functional analysis and Arabidopsis thaliana as the model plant (a) Peak to pathways analysis (Mild<Intense), (b) Results for GSEA(p<0.05). âTotal sizeâ indicates total number of compounds in Arabidopsis thaliana KEGG pathways, âAll.Hitsâ the number of matches in the data set (expected and significant) based on m/z value after mapping to KEGG pathways, âSig.Hitsâ the number of significant matches based on the analysis. p-values of the test using Fisherâs method. 94 3.4.4. Hormonomics Analysis The features were uploaded and mapped to the HormonomicsDB for the plant hormones identification, following the previously established protocol (Giebelhaus et al., 2022). The dataset showed hormones and indoleamines. The growth hormone relative intensities were selected for the mild and intense flavour varieties. In particular, gibberellins were putatively identified, which includes GA-53, GA-15 and GA1 (Figure S8). GA1 happened to be significant in the intense while other GAs were significant in the intense. 3.4.5. Biotransformation of the Metabolites A biotransformation approach was used for the flavonoids due to their effects, differences and possible role these chemical compounds could play in the flavour formation and binding with other metabolites in the breadfruit varieties. The transformation resulted in a predicted 55 compounds. However, metabolites with fold change>2 in the intense were selected and putatively identified (Figure 3-10). Most of the identified compounds mapped to the FooDB included mahanimbine, coumarinic acid, 5-methyl-tetrahydrofolate, undecanoylcarnitine, lucidenic acid J, [12]-Shogaol, 4-deoxycohumulone, and dihydroxystearic acid. These compounds have been previously detected in foods such as tomatoes, broccoli, dairy, mushroom, ginger, green leafy vegetables, castor oil. According to FooDB, they are associated with bitter, meaty, nutty, and pungent tastes and flavours. Specifically, the aforementioned compounds were found to be significantly (p<0.05) higher in the intense flavour (Table 3-3). Table 3-3. Biotransformed Metabolites in the Mild and Intense from The Flavonoids. Name of Compounds m/z Class Flavour Dietary source Reference Lysyl-Lysine 275.2078 Carboxylic acids Foodb, 2024 4-Methylphenyl dodecanoate 291.232 Phenol esters Sweet Foodb, 2024 Dihydroxystearic acid 317.2883 Fatty Acyls Bitter Castor oil Usuki and Kaneda, 1980 (Â±)-Mahanimbine 332.3106 Indoles Bitter Murraya koenigii Prakash, 1990 Lucidenic acid J 491.2681 Triterpenoids Bitter Ganoderma lucidum Watanabe et al., 2011 95 Name of Compounds m/z Class Flavour Dietary source Reference Coumarinic acid 165.0548 Cinnamic acids Bitter Peanuts, tomatoes Huang and Zayas, 1991 4-Acetyl-2-methylpyrimidine 137.0792 Organooxygen Burnt GSC, 2024 4-Deoxycohumulone 333.2037 Benzopyrans Pungent Hops Foodb, 2024 [12]-Shogaol 361.2781 Phenols Sweet Ginger Jolad, et al., 2004 Figure 3-10. Relative Intensity of Metabolites through the Synthetic Biotransformation in the Mild and Intense flavour varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The metabolites were hits in the pathway mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Yellow denotes mild and blue denotes intense flavour varieties. Bars represents Mean±Standard error calculated from the nine replicates in each variety. 3.5. Discussion Significant Metabolites and Biochemical Pathways There is insufficient information about the metabolites for sensorial qualities of breadfruit varieties. Therefore, the aim of using the mild and intense flavour varieties and subjecting it to UHPLC-MS was to provide comprehensive profiling of the polar metaboliteâs differences in these varieties as reported by Ragone and Cavaletto, (2006). To fully understand this, chemometrics and functional analysis using MetaboAnalyst ver. 6.0 was performed to discover the function of the genes, and biochemical pathway modulated to better understand these complex interactions and complement the data explained above (Figure 3-2abc; 3-3ab) as earlier demonstrated by Li et al. 96 (2013). Carboxylic acids and derivatives happened to be the most significant compounds in the SAM output (Figure 3-4ab), with sub-class of L-amino acids, and this also reflected in the conjugated form with the indolamines in which they could have bitter-sweet taste (Anson, 2002). Particularly, phenylpropanoids (also known as cinnamic acids) biosynthesis is one of the signature pathways discovered between the mild and intense flavour varieties; they are aromatic metabolites synthesized from the L-amino acids phenylalanine or tyrosine through several biological processes, which mostly generate bitter taste and flavour (Deng and Lu, 2017). Flavonoids and phenolic acid are subgroups of phenylpropanoid (Fraser and Chapple, 2011). Previously, Moco et al. (2007) opined those phenolic acids, usually consisting of gallic, chlorogenic, and ferulic acids, give astringent taste to tomato fruits. The predicted pathways assisted in revealing the role of the putatively identified metabolites in flavour intensities. For instance, naringenin, quercetin, rutin, kaempferol, and catechin are mostly found in the skin of tomatoes for aroma and pigmentation (Raiola et al., 2014). Also, a study has previously identified flavonoids metabolites as markers for several foods which includes wheat, and broomcorn. (Tang et al., 2022). However, Aldin et al. (2006) claimed that isoflavonoids have a negative impact on the sensory attributes. Moreover, it have been found in a study on the impact of different withering approaches on the metabolism of flavour compounds in oolong tea leaves, the analysis on pathway enrichment showed that phenylpropanoid, flavonoid metabolisms, and protein processing in endoplasmic reticulum were the major pathways discriminating the different tea samples (Wang et al., 2022). The study further explained that the significant decreases in flavonoids, xanthine alkaloids, and several L-amino acids contributed to the alleviation of bitter or astringent taste of withered leaves, although the decomposition of L-theanine resulted in the loss of umami flavour over the solar-withering step. Additionally, Liu et al. (2024) explained that some bitter metabolites as flavonoids are mostly found in plants and play a major role in the bitter taste metabolites in fruits and vegetables, and are synthesized through specific taxa. Recently, Wang et al. (2023) discovered α-linolenic acid and phenylalanine pathways as the most significant pathway in the oats which have fruity and nutty flavour, as well as the relative proportion of linoleic/α-linolenic acid ratio affects the flavour quality of cucumber (Cucumis sativus L.). Also, I discovered that diterpenoids correlate with stale off-flavour/gamey and loss of roasted beef (Figure 3-5ab). In addition, biosynthesis of various plant secondary metabolites which comprise of the gibberellins are known to regulate the fruit development from bitterness and astringency to maturity (Takahashi 97 et al., 1991). Flavone and flavonol biosynthesis are one of the pathways unique to the intense breadfruit varieties (Figure 3-6ab). Flavones comprise one of the largest sub-groups of flavonoids. Furthermore, flavonols share a similar structure with flavones, except that there is an extra hydroxyl group substituted at C-3. Flavones are found in orange, olive oil, thyme, broccoli, while flavonols are in lemon, hops, red wine etc. (Kayoto et al., 1988; Basli et al., 2012). Drewnowski and Gomez-Carneros (2000) found that tangeretin, nobiletin, and sinensetin of flavone in fruits and quercetin in the sub-class of flavonols possessed the bitter taste quality. Pyrimidine metabolism plays a vital role in the synthesis and metabolism of nucleotides for genetic replication and cell division. Ishimaru et al. (2016) discovered the purine and pyrimidine compounds in marine products that make them to be associated with the umami taste. The synthesis of pyrimidine can occur through de novo pathway from simple such as glutamine. I found a high amount of glutamic acid in the intense (Figure 2-6a). The conversion of glutamic acid to glutamine in plants occurs through the absorption of inorganic nitrogen as ammonium (NH4+) or nitrate (NO3-) from the soil through Glutamine Synthetase/Glutamate Synthase pathway (Lea and Miflin, 2011). This variation may explain the impart of how environment may affect the breadfruit pathway, leading to the metaboliteâs variation. Caffeine metabolism (Figure 3-7ab) have its own bitter flavour for foods and beverages, although, enzymes can be different between plant species which may affect different between the caffeine content in the breadfruit species. Therefore, the amount of the methylated xanthine, for example (theobromine and theophylline) can lead to the differences in its bitter taste, and flavour has been discovered in Arabica and Robusta coffee (Zhang and Chen, 2023). Arginine and proline metabolism putatively in Figure 3-8ab shared the same precursor and pathway from amino acid glutamate. Zhao et al. (2016) explained that peptides containing proline amino residue often give bitter taste experience, while microbial activity of arginine can give distinct flavour compounds, which may contribute to savory, bitter, and pungent notes in the intense breadfruit varieties. Arachidonic acids are also fatty acids in plants, the biosynthesis of unsaturated fatty acids discovered in the breadfruit (Figure 3-9ab) have been found to contribute to the flavour in cucumbers, fresh tomatoes, and soybeans through lipoxygenase (LOX) pathway to volatile aldehydes, ketones, and alcohol which can be heat induced turning to bitter, rancid, or roasted flavour (Matthäus, 2007). As mentioned, linolenic and α-linolenic through LOX have hexanal, (Z)-3-hexenal as breakdown product indicated in Table 1-6. 98 Synthetic Biotransformation of the Putative Metabolites Putting all of these claims together by the previous studies, it is clear that the mapped biochemical pathways have a link with flavour and taste formation in fruits and other food crops. My data has further proved these claims opined by these studies. More importantly, a targeted analysis for the pathway, and metabolites is required to replicate, and validate these results. As explained by Giebelhaus et al. (2022) biotransformation could show flavonoids that can be generated from their compounds, which can be found above the detection limits, hence, the need of applying logical algorithms which would generate compounds that can be derived, and interesting these demonstrated bitterness, and pungent flavour. Remarkably, I adopted the approach for flavonoid compounds as to significant (p<0.05) (Figure 2-4f), and generated biotransformation metabolites which leads to putative identification like cinnamic acids, fatty acyls, prenol lipids with objectionable odors (Table 3-3; Figure 3-10). Murraya koenigii commonly known as âcurry leavesâ is a major source of Mahanimbine which has a slightly bitter taste (Prakash, 1990). Likewise, coumarinic acid as one phenolic acid has been discovered to have astringent notes, and bitter found in vegetable protein (Huang and Zayas, 1991). Jolad (2004) and Bhattarai (2001) found that the fresh ginger rhizome has a pungent taste due to the presence of gingerol which are thermally labile and changes to shogaol when subjected to temperature. Future studies can also look more deeply for a targeted analysis and quantity to detect if the transformation of these compounds can be over the threshold level in intense breadfruit varieties that would lead to sensorial perception. 3.6. Conclusion and Further Analysis My study revealed that phenylpropanoid biosynthesis is a key pathway which is common to the Artocarpus genus and also associated with the flavour formation in the breadfruit varieties. The findings further emphasized the efficacy of this pathway, with the significance of the flavone and flavonol biosynthesis. Similarly, α-linolenic pathway, diterpenoids also demonstrated its importance in the formation of the polar metabolites in the intense flavour varieties. In addition, caffeine metabolism, arginine and proline metabolism, and biosynthesis of unsaturated fatty acids were shown to influence the flavour active metabolites in the intense breadfruit varieties. Consequently, these findings contribute to a broader understanding of flavour chemistry in breadfruit and provide direction for future work to focus on these specific biochemical pathway 99 routes, that affect the taste difference between the mild and intense flavour varieties. Technically, the antioxidative potential of flavonoids in food products cannot be overemphasized, hence, its efficacious levels do not have to result in increased bitterness and affect consumer preference. But, by understanding how the amount and structural forms of flavonoids alter flavour generation, it becomes possible to develop healthier food products that have preferable or mild taste. 100 Chapter 4: Metabolomics-guided Discovery of Flavour Chemistry in Breadfruit Varieties using Gas Chromatography Tandem Mass Spectrometry 4.1. Synopsis of Chapter Flavour compounds have been identified in cooked breadfruit (Iwaoka et al., 1994). With >300 breadfruit varieties selected across the Pacific and the Caribbean Islands, breadfruit shows a wide range in phenotypic and morphological properties, nutritional compositions, taste, and flavour (Jones et al., 2011a; Ragone et al., 2006). Selection of elite varieties with pleasing flavour profiles and widespread use requires an understanding of the taste chemistry. To this end, it is expedient and beneficial to breadfruit flour free of the odor present in some varieties. In mitigating this, it is important to know the causes of the taste formation. As previously defined, flavour is human experience modulated by the interaction of food chemistry with taste and odor receptors, and is perceived differently between people (Chen et al., 2022; Kader, 2008). However, the specific compounds responsible for the taste of breadfruit are not known. Jones et al. (2012) investigated volatiles compounds using smoke extracts, solvent extracts, and a hydro-distillate in finding insect repellent. The study discovered >30 compounds consisting of terpenes, aldehydes, fatty acids, and aromatics. Faria et al. (2020) revealed that aldehyde represented the VOCs in breadfruit, with the (E,E)-deca-2,4-dienal (23 %), and (E,Z) - deca-2,4-dienal (6.3 %) isomers as the most abundant, both having fatty odor. The most abundant isomer shows a lower odor threshold, revealing its important contribution to the aroma of breadfruit. To the best of my knowledge, no study has studied the volatile metabolites in the mild, and intense flavour breadfruit varieties to identify compounds responsible for their differences in aroma/odor. 4.2. Materials and Methods 4.2.1. Reagents Hexane (Fisher Scientific, ON, Canada), methanol (Fisher Scientific, ON, Canada), alkanes standards (Sigma-Adrich, MA, USA). Grob mix (Sigma-Adrich, MA, USA). Water was deionized and purified to 18.2 MΩ Ã cm MilliQ water system (ThermoFisher Scientific, Canada). 4.2.2. Sample preparation 101 The analysis of the volatile metabolites followed an established protocol with slight modifications (Giebelhaus et al., 2022). In brief, ~100 mg of each of the six breadfruit varieties (N=3) were transferred to glass vials (Fisherbrand, 15 Ã 45 MM), and subjected to temp of 100 °C in a water bath for 15 min. Prior to extraction, the samples were ground to increase the surface area, and 1 mL of 95 % ethanol was added to each vial. The samples were further crushed and briefly vortexed in the tubes to homogenize. After which, the tubes were shaken, agitated, and mixed (New Brunswick Scientific, INNOVA 44) using 160 RPM at 45 °C for 48 hours. The supernatant was transferred to glass vials, and dissolved pellets in hexane, and placed on shaker using 160 RPM at 45 °C for another 48 hours. Centrifuged again at 10000 RPM for 5 min. The aliquot of 200 µL of the solution was transferred into the amber autosampler glass vial with conical insert bottom spring (Life Science, Canada). Blanks of ethanol and hexane were also prepared and injected (Figure 4-1). Figure 4-1. Method of preparation of breadfruit (Artocarpus altilis) and its hybrids (Artocarpus altilis Ã Artocarpus mariannensis) samples for the non-polar metabolites untargeted analysis by Gas Chromatography Tandem Mass Spectrometry (GC-MS/MS). 102 4.2.3. Gas Chromatography - Mass Spectrometry Separation and identification were performed on a TRACE 1310 GC-TSQ 9000AEI MS/MS (Thermo Fisher Scientific, Mississauga, ON) equipped with a ZB-5MS GC column (30 m Ã 0.25 mm Ã 0.25 μm) (Phenomenex) capillary column with the following oven parameters: oven conditions, initial temp of 50 °C for 3 min, ramp at 12.5 °C/min to 325 °C, then held the final temp for 10 min. Injection parameters were as follows: initial inlet temp of 250 °C with an injection volume of 1 μL (1:20), column flow of 1.2 mL/min; split flow of 24.0 mL/min, transfer line temp of 250 °C, MS ion source temp at 300 °C. Mass spectrometric detection was carried out using electron ionization (EI) at 70 eV in multiple reaction monitoring (MRM) mode. 4.2.4. Alkanes and Grobâs Mix Test The separation and identification of analytes were performed using GC-MS/MS, where separation was achieved based on volatility and polarity of some standards. The alkanes standard was diluted to make a 6-point dilution series (0.5, 1, 2.5, 5, and 10 ppm). The Grob Mix was made in ethanol into 3-point dilution series (1 to 8 ppm). These standards were injected to determine the separation efficiency of the analytes identified in the samples. Also, both samples and blanks were passed through columns with the same protocol. The average blank concentration of three replicates subtraction to correct matrix effect. Experimental data was presented as a means of independent measurements (3 replicates for each solvent system) (Grob, 1978). 4.2.5. Putative Metabolite Annotation For non-polar metabolites, peak identification was performed using MS-DIAL ver. 4.9.22 by matching mass spectra to known libraries and calibrating retention behavior through the calculation of Kováts Retention Indices (RI) based on a series of injected n-alkanes. Library matching was guided by comparing the calculated RI values with reference values from the Fiehn Lab database. Metabolites with a calculated retention index greater than 900 were considered for identification. To reduce false positives, all features were manually inspected, and in some cases, selected peaks were further curated through comparison with metabolites exhibiting higher retention indices. This approach enabled the derivation of deconvoluted metabolite peaks with better accuracy. 103 4.3. Results 4.3.1. Column Evaluation The chromatographic behavior of the standards provided further evidence of the columnâs performance characteristics. The more theoretical plates contained in any length of column, translates to more plates per meter and higher column efficiency. Theoretical plate number increases with column efficiency, as more plates per meter indicate greater separation capability. As demonstrated in Table 4-1, the number of theoretical plates was 858,780 for decane, along with similarly high values for other compounds in the mix, and n-alkanes, this was automatically calculated from the peak width 50 % height (W1/2), and retention time (tR) using Chromeleon software which relates to the formula (N = 5.54 (tR/W1/2)2). Additionally, the column demonstrated distinct separation of methyl esters, with methyl laurate exhibiting higher retention than methyl decanoate which similarly thing in decane and undecane. (Table 4-1). Nonanal eluted with a sharp, symmetrical peak, higher than that of 1-octanol, indicating that the column provided good retention, and equilibration for saturated aldehydes with the stationary phase. To assess column bleed, multiple blank injections were performed following the same oven temperature program. No significant baseline drift, low and clean background in both blank injections (ethanol and hexane), signifying no or minimal column bleed under the experimental conditions. Overall, the column exhibited high efficiency, strong separation capability, and excellent thermal stability with negligible stationary phase bleeding (Figure S9 - S10ab). These characteristics suggest the columnâs ability to profile volatile and semi-volatile compounds in complex matrices. Table 4-1. Features of Total Ion Chromatogram (TIC) for the Reference Standard Mix Compounds Chemical formula Retention time (min) Relative Area % Width (50 %) min Plates EP Decane CââHââ 9.969 76.29 0.026 858780 Undecane CââHââ 11.146 30.7 0.02 1757226 Methyl decanoate CââHââOâ 11.4 77.64 0.019 1946358 Methyl laurate CââHââOâ 12.483 37.57 0.019 2344046 104 Compounds Chemical formula Retention time (min) Relative Area % Width (50 %) min Plates EP Methyl undecanoate CââHââOâ 13.503 26.52 0.019 2690154 1-Octanol CâHââO 8.358 7.7 0.019 1028294 2,3-Butanediol CâHââOâ 4.706 2.04 0.023 240207 Nonanal CâHââO 8.808 13.35 0.02 1121586 2,6-dimethylphenol CâHââO 8.857 64.99 0.022 981161 2,6-dimethylaniline CâHââN 9.623 44.01 0.016 8287316 2-ethylhexanoic acid CâHââOâ 8.921 3.89 n.a. n.a. dicyclohexylamine CââHââN 12.543 10.36 0.024 1519597 EP- efficiency plates; n-a. â not applicable. 4.3.2. Untargeted Metabolomics for Non-Polar Metabolites The monoisotopic mass indicated nine putatively identified metabolites with marginal difference in their relative peak intensity between the mild and intense varieties (Figure 4-2). In particular, these were benzyl alcohol, isoleucine, dehydroascorbic acid, palmitic acid, linolenic acid, arachidonic, epicatechin and 3-hydrobenzoic acid. The correlation of the metabolites with mild and intense flavour varieties were described in Figure 4-3. From the GC-MS/MS results, epicatechin, dehydroascorbic, linolenic, palmitic, and arachidonic acid showed higher abundance in the intense breadfruit flavour varieties. 105 Figure 4-2. Determining the significant metabolites between the Mild and Intense Flavour Varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) (Mild<Intense). Epicatechin, dehydroascorbic, linolenic, palmitic, and arachidonic acid showed higher abundance in the intense varieties. The data were processed on MS-DIAL ver. 4.9.22. Aligned to a curated database. 106 Figure 4-3. Clustering analysis for the significant metabolites between the Mild and Intense breadfruit varieties (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). Dehydroascorbic, epicatechin, and isoleucine show positive correlation to the varieties. The data were processed on MS-DIAL ver. 4.9.22. Aligned to curated database. 4.4. Discussion As there were no previous studies that focused mainly on the significant non-polar metabolites between the mild and intense breadfruit varieties, recall that the evaluation of the 20 breadfruit cultivars put together by the University of Hawaii and NTBG used the cooked form for detecting the differences in the flavour intensities of the breadfruit cultivars. My study was designed to be close to the samples used in the University of Hawaii and NTBG in order to detect the differences in the sensorial qualities. As earlier reported by Aalbersberg et al. (1988) on the nutrient composition of 10 breadfruit cultivars from the Pacific indicating that fatty acids varies among the cultivars which may be due to their genetic composition. Even though, I found some volatile metabolites that are different between the mild and intense flavour varieties (Figure 4-2). My focus was centered around compounds that were unique to the intense flavour varieties in reds (Figure 4-3). My approach discovered benzyl alcohol which has a sharp burning taste. Specifically, cherry wood smoked cheeses were distinguished by the presence of a fruity flavour 107 and it was discovered that benzyl alcohol is one of the compounds that contributed to the fruity flavour in the smoked cheeses (Rizzo et al., 2022). Hufnagel and Hofmann, (2008) confirmed that the threshold of the astringent and not of the bitterness. Odor is amongst the first quality characteristics shown when opening food package products, therefore, volatile organic compounds (VOCs) have potential as early markers for consumer acceptability (Holm et al., 2013). Iwaoka et al. (1994) worked on a single variety in both raw and cooked collected from Oahu, Hawaii. The study discovered some volatile compounds for example hexanal, alcohol etc. (Table 1-6). Jones et al. (2012) found fatty acids, aldehydes, and alcohol in breadfruit. These may have occurred through lipid peroxidation as reported in Frankel (2005). Another study on fatty acids from Golden and Williams (2007) reported that breadfruit is a good source of fatty acids such as palmitic acid, linolenic acid etc. Palmitic acid possessed slight characteristic odor and taste (Burdock, 2010). Linolenic acid is more susceptible to oxidation and will become rancid more quickly in foods. Inui et al. stated that epicatechin was identified in hop seeds, and it was reported that the antioxidant activity of catechin and epicatechin is significantly higher than that of prenylflavonoids (as measured by the DPPH assay), which are also compounds found in hop (Inui et al., 2017). Oxidative instability of polyunsaturated fatty acids is one reason why producers choose to partially hydrogenate oils containing linolenic acid and α-linolenic acid, such as soybean oil (Kinney, 2006). Yamaguchi et al. (2012) explained that introduction of arachidonic acid enhances the umami taste. The fatty acyls identified are similar to linolenic acid, they are both carboxylic acids that can contain fatty acid or its derivative, which are sub-group of lipids. This explains the importance of esterification of carboxylic acids derivatives in forming esters that are predominantly found in fruits and vegetables which contribute fruity aromas and flavours. In addition, the approach again identify another class, prenol lipids, which signifies the importance of this group and class. Gray et al. (1999) explained that polyunsaturated fatty acids are converted to 9- and 13-hydroperoxides and later appeared to be transformed into volatile aldehydes. LOX enzymes catalyze at pH 6 -7 which is similar to the pH discovered in this thesis (Figure 2-4a), this happens in flour until they are heat-inactivated (Robinson et al., 2019). Frankel (2005) explained that there are several parameters and target compounds that signal rancidity for example, peroxide value for hydroperoxides, hexanal for volatile compounds, this suggest breadfruit to be susceptible to rancidity. In comparing breadfruit flour to other tropical 108 flour blends from plantain and cassava, Turi et al. (2015) demonstrated better oxidative stability than banana flour but less than cassava. Adding 1 % if moringa leaf powder, can mitigate oxidation for 6 months (Thanyapanich et al., 2020). Although, intense flavour varieties exhibited better DPPH scavenging ability (Figure 2-4i), this chapter points to its nature of having oxidation that leads to rancidity as explained above which may be tested by other assay like lipid peroxidation. 4.5. Conclusion and Further Analysis The current data in this study complement the UHPLC-MS untargeted method. Notably, both methods highlight certain metabolites or pathways, signaling their significance, and relevance to flavour intensity. Fatty acids have been identified as one class of compounds that could contribute to the differences in metabolites in the breadfruit varieties, with carboxylic acids also emerging as another important class to consider. These compounds are probable to rancidity through oxidation which can be initiated by several accelerating factors such as heat, metal ions, light exposure, and lipoxygenase enzyme at a particular pH (Figure 4-4). Further examination of the significant fatty acids as important classes for the metabolites between the mild and intense breadfruit flavour varieties is imperative. Future targeted research is needed to validate these findings and establish the precise role of these compounds in the flavour chemistry of the breadfruit varieties. In addition, there should be analysis that would test the efficacy of the scavenging ability of the phenols in breadfruit against the oxidation. Inclusion of spices like moringa, blanching, metal chelators, and use of light and oxygen barriers (Figure 1-5b) could mitigate the rancidity in the intense breadfruit varieties. Figure 4-4. Formation of Off-Flavour in the Mild and Intense Breadfruit Varieties 109 Chapter 5: Understanding the Flavour Chemistry of Commercial Breadfruit Flour from Different Geographical Locations 5.1. Synopsis Breadfruit flour has now been sold in many parts of the world; it has also been granted âGenerally Recognized as Safeâ status by the United States Food and Drug Administration (Liu et al., 2015). The flour has the potential to provide an abundant source of nutrient-based food for the rest of the tropics, which had been recognized by European colonialists as early as the 1700s for this capacity (Banks, 1962; Jones et al., 2011a). Within this crop, great diversity exist (Ragone, 2007; Jones et al., 2013). Hence, there is a possibility of variation in the elite varieties of these commercialized breadfruit flour. Ragone and Cavaletto (2006) demonstrated that variation exists in sensorial attributes of breadfruit varieties. Information is still scarce in the comparison with the mild and intense flavour varieties to the commercial breadfruit flour. In achieving the sustainable development goals (SDG) 2 and 3 which target zero hunger, good health, and well-being. Breadfruit flour abundance in a particular region should be transported or imported to any areas across the world where they are needed to subdue the pandemic. However, this cannot be possible without knowing the flavour intensity of these flours which will ultimately help potential producers to inform their decisions. Alternatively, local producers can rely on available breadfruit cultivars even with intense flavour rather than importing crops which will reflect in the cost of their products, inadvertently improving the access of the poor and vulnerable class to healthy and nutritious foods. To have a holistic view of the market for breadfruit across the world. Ten commercial breadfruit flour samples used in this study were obtained from the following places: Veyangola, Sri Lanka; Hawaii; Pago, AS, and Apia, Samoa; Ile-Ife, Southwest Nigeria; Hartwell Garden, Clarendon, and St. Catherine Jamaica; Gressier, Haiti; Indonesia; and Costa Rica. I hypothesize that commercial breadfruit flours vary in their flavour profiles and can be classified as Mild or Intense flavour breadfruit products. The intense breadfruit varieties to be used for this characterization will be selected from Table 2-1. Therefore, this chapter seek to understudy the flavour profile of commercial breadfruit flour from different geographical locations and determine if the flour either have Mild or Intense profile. To investigate this, I will compare: (1) the intense breadfruit varieties to the commercial breadfruit flour for tannins composition. (2) the intense breadfruit varieties to the commercial breadfruit flour for acidity level. (3) the intense breadfruit varieties to the commercial breadfruit flour for total soluble solids and sugars. (4) the intense 110 breadfruit varieties to the commercial breadfruit flour for total protein. (5) the intense breadfruit varieties to the commercial breadfruit flour for flavonoids. (6) the intense breadfruit varieties to the commercial breadfruit flour for vitamin C and for scavenging ability. 5.2. Materials and Methods 5.2.1. Reagents Tannic acid, A.C.S. Reagent (MilliporeSigma, Oakville, ON), Folin - Denis reagent. âTo 750 mL H2O, add 100 g sodium tungstate dihydrate (Na2WO4â 2H2O) (Sigma-Aldrich, Canada), 20 g phosphomolybdic acid (Sigma-Aldrich, Canada), and 50 mL phosphoric acid (H3PO4) (Sigma-Aldrich, Canada). Reflux 2 hours, cool, and dilute to 1 L of MilliQ water. 5.2.2. Commercial Breadfruit Origins The ten commercial breadfruit flours were purchased from different geographical locations around the world and shipped to the UBC facility and stored in -20 ºC before the analysis. All samples were coded to conceal their identity during analysis used in this study are listed in Table 5-1. Also, to protect proprietary information, and eliminate bias during internal and external evaluations. Table 5-1. List of Commercial Breadfruit Flour and their Origins Breadfruit (codes) Location Latitude Coordinates GM Indonesia 6° 12' 0.00'' S JF Costa Rica 9° 37' 48.68" N CP Veyangola, Sri Lanka 7° 9' 6" N BN Ile-Ife, Nigeria 7° 27' 59.99" N DLP Gressier, Haiti 18° 33' 0.00" N DBIA Pago, Samoa 14° 16' 32.2752'' S ADS Hartwell, Jamaica 18° 00' 0.00" N SIHCL St. Catherine, Jamaica 17° 59â² 46â³ N NFIL Apia, Samoa 13° 50' 1" S NFIL-M Apia, Samoa 13° 50' 1" S 111 5.2.3. Tannin Determination Tannins were determined to use the AOAC method 952.03 as fully described in Section 2.3.8. In summary, 1 g of the commercial breadfruit flour was soaked in 25 mL of water for 24 hours, agitated, and then centrifuged. A sodium carbonate solution (35 g in 100 mL water) was prepared, heated, and filtered after cooling. A fresh standard tannic acid solution (100 mg in 1 L water) was used for each test. Aliquots of the tannic acid solution were mixed with sodium metabisulfite, Folin-Denis reagent, Na2CO3 solution, and water, then incubated for 30 minutes. Absorbance was measured at 760 nm. Figure 5.1. The Workflow for Tannins Determination in Breadfruit Using Spectrophotometric Approach on a Microplate Reader. 5.2.4. Acidity Level The acidity level was determined using a modified method from Adebowale et al. (2020) as described in Chapter 2, section 3.3.1. 5.2.5. Brix, and Sugars Analysis The determination of Brix and D-glucose analysis was carried out following the procedure described in Chapter 2, section 3.3.2. D-fructose and sucrose was according to standard protocol 112 using Megazyme kits K-SUFRG (Bray, Co. Wicklow, Ireland). The kits contains the following reagents: Buffer 1 (25 mL, pH 7.6) plus sodium azide (0.02 % w/v) as a preservative; NADP+ plus ATP; Hexokinase plus glucose-6-phosphate dehydrogenase suspension, (4.1 mL); phosphoglucose isomerase suspension (2.25 mL); D-Glucose plus D-fructose standard solution (5 mL, 0.2 mg/mL of each sugar); and β-Fructosidase (pH 4.6), lyophilized powder at 340 nm and ~25 ºC Where Hexokinase catalyzes the phosphorylation of D-fructose to fructose-6-phosphate (F-6-P) by adenosine-5â-triphosphate (ATP). The sucrose content was calculated from the difference in D-glucose concentrations before and after hydrolysis by β-fructosidase (Outlaw and Mitchell, 1988; Beutler, 1988; Kunst et al., 1988). Determined the absorbance difference (A3 - A2) for both blank and sample (D-glucose/D-fructose sample only). Subtracted the absorbance difference of the blank from the absorbance difference of the sample thereby obtaining ÎAfructose. The values of ÎAD-glucose, ÎAsucrose, and ÎAD-fructose should as a rule be at least 0.100 absorbance units to achieve sufficiently accurate results. The concentration of D-glucose, sucrose, and D-fructose was calculated as follows: c = (V Ã MW)/ ε Ã d Ã v [g/L] where: V = final volume [mL] MW = molecular weight of the substance assayed [g/mol] ε = extinction coefficient of NADPH at 340 nm = 6300 [l Ã mol-1 Ã cm-1] d = light path [cm] v = sample volume [mL] for D-fructose: c = (2.44 Ã 180.16 Ã ÎAD-fructose) /6300 Ã 1.0 Ã 0.1 = 0.6978 Ã ÎAD-fructose for Sucrose: c = (2.42 Ã 342.3 Ã ÎAsucrose) /6300 Ã 1.0 Ã 0.1 = 1.315 Ã ÎAsucrose 5.2.6. BCA Total Protein Determination The determination of total protein in the commercial flour was performed using a Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Scientific, Montreal, Canada). The procedure for BCA protein are fully described in Chapter 2, section 2.3.7. 113 5.2.7. Total Flavonoids Content The determination of total flavonoids was determined using the method of Adebowale et al. (2020); Badejo et al. (2016) as explained in Chapter 2, section 2.3.4. 5.2.8. Determination of Vitamin C The determination of vitamin C using iodometry method according to modified method by Zhang et al. (2013) as described in Chapter 2, section 2.3.6. 5.2.9. Determination of Antioxidant Potential The determination of antioxidant potential using 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to modified method in Adebowale et al. 2020 and Badejo et al. (2016) as described in Chapter 2, section 2.3.5. 5.2.10. Statistical Analysis All statistical analyses were performed using GraphPad Prism version 10.2.2 (GraphPad Software Inc., La Jolla, CA, USA). A series of one-way ANOVA tests were conducted to assess significant differences between treatment groups, with the significance level set at α = 0.05. Graphs were generated using GraphPad Prism 10.2.2 and Microsoft Excel 2013 (Microsoft Corporation, Redmond, WA, USA). The intense breadfruit varieties (three in total), serving as control samples (Table 2-1), were compared against ten commercial flours (Table 5-1). Where significant differences were detected, further pairwise comparisons were conducted to evaluate differences among individual samples. 5.3. Results 5.3.2. Principal Component Analysis Intense breadfruit varieties and ten commercial breadfruit flour PCA results (PC1 vs PC2) in Figure 5-2ab show that SIHCL shares similarities with the intense varieties, while the plot suggests that ADS exhibits variation from both the intense and other commercial breadfruit flours. Tannins, pH, and soluble solids are clustered together, indicating a correlation. D-glucose, vitamin C, and proteins positively influence both components, which together explain ~80% of the 114 variance. In addition, the PCA (PC1 vs PC3), which accounts for ~70% of the variance, demonstrates clustering among the majority of the samples, except ADS and SIHCL. In both PCAs, NFIL, and NFIL-M show no separation, indicating strong similarity Figure 5-2cd. . 115 Figure 5-2. Principal Components Analysis of Intense Breadfruit Varieties with Ten Commercial Breadfruit Flour. (a) PC1 vs PC2 scores plot (b) PC1 vs PC2 loading plot (c) PC1 vs PC3 scores plot (d) PC1 vs PC3 loading plot. 116 5.3.3. Acidity, Sugars, and Total Soluble Solids in Control and Commercial Breadfruit Flour The control group, consisting of the intense flavour breadfruit varieties, was compared to ten commercial breadfruit flour samples to evaluate acidity levels. Results indicated that four commercial floursâBN, DLP, DBIA, and NFIL-Mâexhibited higher significant different pH values compared to the control (p<0.001) (Figure 5-3a). The control samples had an average pH of 6.15â±â0.07, while the significantly different commercial flours had pH values ranging from 6.43 to 6.80. In addition, D-glucose content in the ten commercial breadfruit flour samples was assessed and compared to the control (intense flavour varieties). The analysis specifically targeted D-glucose concentrations. The results revealed that SIHCL, CP, NFIL, and NFIL-M contained significantly levels of D-glucose to the control samples, with statistical significance at p<0.05, p<0.01 and p<0.001 (Figure 5-3b). The ten commercial breadfruit flour samples were subjected to other sugar analysis, with particular emphasis on D-fructose and sucrose content (Figure S11ab). Due to the notably high levels observed in ADS, this sample was not compared against the control. The results revealed that both SIHCL and ADS contained the highest amount of the sugar. Additionally, the sugar profile of the ten commercial breadfruit flours was evaluated for sucrose content. The results indicated that all samples, except JF to DLP and NFIL to NFIL-M, contained significantly higher levels of sucrose (Figure S11b). Total soluble solids in the intense flavour varieties (used as control) were compared to ten commercial breadfruit flour samples. The evaluation was conducted using degrees Brix (°B), which indicate the density of soluble solids. Results showed that six commercial flours had °Brix values significantly different from the control (p<0.01; p<0.001). The control samples had an average °Brix of 2.35â± 0.45, while the significantly different commercial flours ranged from 1.30 to 3.13â°B. These commercial floursâGM, CP, BN, ADS, NFIL, and NFIL-Mâexhibited varying densities of soluble solids. Notably, CP and BN appeared to contain higher concentrations of soluble solids, suggesting greater density (Figure 5-3c). 117 Figure 5-3. Acidity, Sugars, and Total Soluble Solids in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locations. The intense flavour varieties represent the control. (a) acidity levels through pH, (b) D-glucose assay (c) total soluble solids analysis (ºBrix). Plot represents mean±standard error of mean calculated from three replicates (N=3). *significant difference at p<0.05. **represents significant difference at p<0.01. *** and **** indicates significant difference at p<0.001 using ANOVA with post-hoc Dunnettâs test. 5.3.6. BCA Protein in the Control and Commercial Breadfruit Flour The BCA total protein assay revealed a significant difference in protein content between ADS and the other samples (p<0.001), as well as a significant difference between CP and GM flour (p<0.05). No significant differences were observed in BCA protein content between the intense flavour varieties and the remaining commercial breadfruit flours (Figure 5-4). 118 Figure 5-4: Total Protein in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locations. Bovine Albumin serum as the standard (BSA). Plot represents mean±standard error of mean calculated from three replicates (N=3). *Represents significant difference at p<0.05. ***indicates significant difference at p<0.001 using ANOVA with post-hoc Dunnettâs test. 5.3.7. Scavenging Ability and Phenols in the Control and Commercial Breadfruit Flour The assay results revealed that antioxidant scavenging activity differed significantly between the control and the commercial breadfruit flour samples. This variation aligns with the distinction between mild and intense flavour varieties. Notably, the intense flavor varieties exhibited higher DPPH scavenging activity compared to the commercial flours. Among the commercial samples, GM, SIHCL, DBP, NFIL, and NFIL-M showed significantly different scavenging activity compared to the control (Figure 5-5a; p<0.01). In terms of tannin content, no statistically significant difference was observed between the mild and intense flavour varieties (p>0.05). However, among the commercial breadfruit flours, significant differences were found. For example, JF, and BN differed significantly in tannin content, with higher and lower values of 1.107 ± 0.002 mg/100 mg and 0.808 ± 0.02 mg/100 mg, respectively (p<0.05; p<0.001). No significant differences were observed between CP, and DBIA (Figure 5-5b). Regarding vitamin C content, SIHCL contained significantly higher levels compared to the control (intense flavor varieties). In contrast, other commercial flours did not differ significantly. Although not statistically significant, BN (14.27 ± 0.69 mg/100 g) and JF (15.65 ± 1.25 mg/100 g) had slightly 119 lower vitamin C levels than the control (Figure 5-5c). Flavonoid content also varied significantly among the commercial breadfruit flours, correlating with the distinctions observed in both mild and intense flavor varieties. Higher flavonoid levels were observed in the commercial samples compared to the control varieties. Among these, BN contained significantly higher flavonoid content (14.34 ±1.76 mg/100 g RE) compared to GM, CP, DBP, SIHCL, NFIL, and NFIL-M (Figure S12). Figure 5-5. Scavenging Activity in Intense Breadfruit (cont) and Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locations. (a) 2,2-diphenyl-1-picrylhydrazyl (DPPH) as the free radical for assay. Trolox as the standard. (b) tannins assay, tannic acid as standard (c) vitamin C assay. Plot represents mean±standard error of mean calculated from three replicates (N=3). *indicates significant difference at p<0.05. **indicates significant difference at p<0.01 while ***indicates significant difference at p<0.001 using ANOVA with post-hoc Dunnettâs test. 120 5.4. Discussions 5.4.1. Geographical Profiling of Commercial Breadfruit Flour Breadfruit varieties have been found to vary in flavour intensity (Ragone and Cavaletto, 2006). This study has identified some metabolites significant between the mild and intense flavour varieties that appears to have taste and flavour properties. It is interesting to state that flour from some breadfruit varieties have been commercialized. However, information is scarce about the flavour intensity of these commercialized breadfruit flour. Thus, comparing the significant flavour-active metabolites between intense flavour varieties, and commercial flour. This will explain the prevailing phytochemistry in commercial breadfruit flour. Firstly, the pH level plays a pivotal role in determining the overall quality of products. Adjei et al. (2024) explained that the levels of acidity checked by pH in the flour may substantially influence several critical biological processes, including protein denaturation, enzymatic activities, and overall product deterioration. Crucially, the level of acidity can affect the interaction of protein and flavour (Wang and Arntfield, 2015; 2017). They found that flavour binding was strongest at pH 5, followed by pH 7, 9, 11, and weakest at pH 3. Aloni et al. (2008) highlighted that these activities could result in formation or breakdown of chemicals compounds through biochemical pathways. Although the diverse chemical composition of food can influence flavour perception (Reineccius, 2006), this study found a significant difference (p<0.05) in the acidity levels between the control breadfruit flours and some commercial breadfruit floursâspecifically BN, DBP, DBIA, and NFIL-M. This difference may be attributed to variations in the chemical compounds generated during processing. Notably, differences in D-glucose content and °Brix readings were also observed, which are likely to reflect variation in the total soluble solids present in the flour samples (Figure 5-3abc). This aligns with Nguyen et al. (2023), who reported that the correlation between individual sugars and °Brix, although is often weak. The presence of other sugars such as D-fructose or sucrose may have contributed to the observed variation, supporting this interpretation. For instance, SIHCL may contain higher amounts of other sugars aside from D-glucose. Golden and Williams (2001), as well as Wootton and Tumaalii (1984), previously reported that D-fructose, sucrose, and D-glucose are the dominant sugars in certain breadfruit cultivars. Additionally, Wang et al. (2022) demonstrated that differences in D-glucose, D-fructose, and sucrose concentrations influence sweetness and flavor perception in Chinese cabbage, distinguishing white-inner from yellow-inner varieties. 121 Fructose is ~1.74 times sweeter than sucrose and 2.34 times sweeter than glucose (Pangborn, 1963), which further supports the idea that varying sugar profiles can significantly impact flavour characteristics. Total protein content was determined using the Bicinchoninic Acid (BCA) assay. Recall that the principle behind the BCA method involves the reduction of cupric ions (Cu²âº) to cuprous ions (Cuâº), which likely indicates the presence of certain L-amino acidsâsuch as cysteine, cystine, methionine, tyrosine, and tryptophanâthat facilitate this reduction (Wiechelman et al., 1988). This suggests that variations in protein content between the commercial breadfruit flours and the intensely flavored varieties may reflect differing levels of these specific amino acids. The biological significance of L-amino acids in protein structure and their interactions with other metabolites has been previously emphasized (Wang and Arntfield, 2015, 2017). Specifically, commercial samples such as GM, CP, and ADS, which exhibited significantly (p<0.05; p<0.001) higher protein levels, may have facilitated greater proteinâflavour compound interactions (Figure 5-4). These interactions could involve sulfide or disulfide linkages, which are not susceptible to cleavage under processing stressors such as heat, potentially leading to irreversible reactions or the release of flavour compounds. Furthermore, proteins can interact with other plant metabolitesâsuch as sugars and flavonoidsâto form conjugates. Proteinâpolyphenol interactions have been well documented (Arts et al., 2001; Spencer et al., 1988). Among these polyphenols, flavonoids are of particular interest and are broadly categorized into five subclasses (Harborne, 1994). Flavones and flavonols, for instance, are widely distributed in plant-based foods. Natural flavonoids predominantly occur as glycosides, either O-glycosides or C-glycosides, with O-glycosides being the most abundant (Å amec et al., 2021). In O-glycosides, sugar moieties are attached to the hydroxyl group of the flavonoid, while in C-glycosides, sugars form direct carbonâcarbon bonds with the flavonoid backbone. These structural differences significantly affect their physicochemical properties, bioavailability, and organoleptic qualities (Ji et al., 2020). Beyond environmental and species-specific influences, the chemical composition and molecular structure of plant materials also play crucial roles in their nutritional and sensory profiles. Franke et al. (2004) reported considerable variability in phytochemical concentrations across food typesâfor instance, ascorbic acid ranged from 4 to 801 mg/kg, citrus flavanones from 172 to 905 mg/kg, flavones/flavonols up to 259 mg/kg, and anthocyanidins as high as 1168 mg/kg. In essence, even with a particular value for the assay, the subclass is equally important for flavonoids. Although, 122 vitamin C and flavonoids are richer in SIHCL and BN, respectively (Figure 5-5c, FigureS12). Nonetheless, the sugar monoglycosides in these samples, the type, quantity, and site of sugar moiety could also affect the amount, physicochemical properties, and activities of flavonoids. In short, the effect of glycosylation on flavonoids is multifaceted and complex (Xie et al., 2022). And so, the difference in the sugar compositions and attachments is important. In food technology, the removal or deglycosylation of naringin, one of the main contributors to bitterness in orange and grapefruit juices, has been described as a treatment that improves juice quality because the released aglycone naringenin is perceived as less bitter (Purewal et al., 2020). As tannin-protein interactions are reported to be pH dependent (Sarni-Manchado et al., 1999). Also, Williamson, (1994) demonstrated that the influence of amino acid composition and the conformation of the proteins on the binding ability of polyphenols. However, the proline-rich proteins (PRPs) found in saliva, which are reported as strongly associated with tannins, cannot be conformationally compared with bovine serum albumin (BSA). The larger and more hydrophobic polyphenols have been reported to bind more strongly to the proline-rich peptide, and some of the binding interactions for the larger polyphenols may arise from multidentate association (Murray et al., 1994; Baxter et al., 1997). Remarkably, the study has revealed that some commercial flour such as SIHCL, NFIL-M, and ADS are rich in D-glucose, protein, and acid levels (Table 5-2). Considering the variations in phytochemical composition, commercial breadfruit flours possess potential for optimization and application in various food products. For instance, they can serve as functional fillers in baked goods, reducing the quantity of meat or other high-cost ingredients required in formulations. This not only offers economic benefits but also supports the development of more sustainable and health-conscious products. Moreover, Adjei et al. (2024) emphasized that in order to maintain protein integrity and enhance sensory quality when incorporating breadfruit flour into food products, careful formulation is essential. Specifically, appropriate blending with protein-rich ingredients, and the optimization of processing parameters are critical to achieving desirable texture, flavour, and nutritional outcomes. Regarding the identification of the cultivars used for these commercial breadfruit flours, it is imperative to understand the traditional knowledge of the breadfruit in the selected geographical locations. However, there is limited documentation of the traditional or localized knowledge, often times, they are communicated through oral means (Ragone et al., 2004; Lee et al., 2001). Initially, 123 breadfruit was introduced by Captain Bligh to the Islands of St. Vincent and Jamaica, then, the plants were moved to other part of the Caribbean. Roberts-Nkrumah (2008) identified 4 cultivars of breadfruit in Jamaica (Macca, Yellow Heart, St. Kitts, and Cassava); 4 cultivars in Samoa (Maâafala, Momolega, Puou, Ulu tala, and Uluea). Rahmah and Waluyo (2019) examined the genetic distance and diversity of breadfruit (Artocarpus altilis) native Bawean Island which is known as native place for Breadfruit in Indonesia, the study found that 30 accessions showed moderate diversity due to local cuttings during propagation and their seedless as a result of parthenocarpy. Similarly, the same approaches have been utilized in Polynesia than Micronesia and Melanesia. Moreover, Jamaicans have been identified as the people that took the breadfruit from the Caribbean to Africa through the missionaries (Morrissey, 2017). From this observation, it is difficult and complex to identify the breadfruit used in making the commercial flour in these different geographical locations, due to the use of different vernacular names, and asexual mode of propagation. 5.5. Conclusion and Further Analysis Remarkably, this chapter has shown that commercial breadfruit flour produced from different geographical locations varies in the parameters that appear to differentiate the mild and intense flavour varieties. Based on the parameters that have shown the differences between the mild and intense flavour varieties; CP, BN, and ADS appeared to be denser with chemical compounds as intense flavour varieties. Domestication and improvements would have affected the metabolites present in the commercial flour. GM from Indonesia appeared to be from the parentsâ species, and thus demonstrate better scavenging ability and marginal higher vitamin C than the control. GM signaled dense composition as an intense flavour variety. Also, some commercial breadfruit flour shares similar properties based on their locations, for example ADS, and SIHCL. Potentially, the flour might have been produced from the same cultivar but different production techniques. Specifically, ADS seems to have more protein with the possibility of having protein-flavour compounds leading to reduced intensity as mild flavour variety, but SIHCL appeared to be intense with sour taste due to level of vitamin C, and relatively lower protein, and even the L-amino acids can also be a factor. Where some from different cultivar, such as NFIL and NFIL-M from different cultivar may have experienced similar production techniques. Additionally, BN exhibited higher acidity and flavonoids similar to the trend of intense flavour varieties, but lower 124 tannins which may suggest it as sweet cultivar, and CP has higher tannins which are likely to be a bitter cultivar. Strikingly, BN, and other commercial flour such as JF, CP, DBP, DBIA, and ADS demonstrated health promoting properties due to their comparable scavenging of radicals to the intense flavour varieties. The table below gives a breakdown of how the flour appeared to be either mild or intense flavour varieties based on the assay on the flavour-active metabolites (Table 5-2). Table 5-2. Summary of the Flavour Intensity of Breadfruit Commercial Flour with different Geographical Locations Breadfruit (codes) Mild Intense Location GM â â Indonesia JF â â Costa Rica CP â â Veyangola, Sri Lanka BN â â Southwest, Nigeria DLP â â Gressier, Haiti DBIA â â Pago, Samoa ADS â â Hartwell, Jamaica SIHCL â â St. Catherine, Jamaica NFIL â â Apia, Samoa NFIL-M â â Apia, Samoa *Key: â - Decrease â - Increase â - No difference 125 Chapter 6: Strategic Techniques in Mediating Flavour Intensity in Breadfruit 6.1. Synopsis of Chapter The study has demonstrated that breadfruit varieties differ in flavour intensity, with mild and intense breadfruit varieties each exhibiting unique characteristics. To promote the utilization of these breadfruit varieties across different geographical regions, it is crucial to develop techniques that can mediate the flavour intensities across the varieties. Therefore, the primary objective of this chapter is to recommend practical techniques that can be used to address the distinguished phytochemicals identified in this thesis as influencing flavour intensity, and to suggest appropriate breadfruit varieties for future food processors. I propose that the adoption of these techniques will facilitate the global distribution of commercial breadfruit flour, especially in regions where breadfruit is abundant. By adopting this approach, it will contribute to food and nutrition security in areas affected by hunger and malnutrition, supporting the achievement of SDG 2 and SDG 3: a world free of hunger with good health and well-being. 6.2. Methods 6.2.1. Flavour Techniques for Improved Acceptability in Breadfruit The proposed recommendation in improving the taste and flavour experience in breadfruit stemmed from the metabolites and pathways that have shown differences between the Mild and Intense flavour varieties in previous chapters. Specifically, it was stated that breadfruit metabolites such as tannins, flavonoids, and vitamin C are associated with astringency and bitterness; it is worth nothing that the possible nutritional and health benefits posed by these compounds needs to be conserved to maintain the functional food nature of breadfruit. Due to the variation in taste perception among individuals, as noted in the introduction, people may experience different flavoursâthe variation in receptors is particularly relevant when utilizing breadfruit flour sourced from different geographical locations, highlighting the potential role of receptor blockers in standardizing taste experiences; pleasing taste and flavour could possibly be cultural or known to be associated with a particular tribe to clan, hence the need to mask the other undesirable flavour with commonly used local spices. Additionally, optimizing the unit operations for instance, the control of the meshes for sieving and heat treatments can influence levels of the aforementioned metabolites. Furthermore, the actions of enzymes or microbes can change the concentration of 126 polyphenols metabolites in the pathways or sugars when the flour is used for fermented products. Particularly, this study will focus on how to remediate five types of taste: bitter, sweet, sour, salty, and umami, through application of flavour and taste reduction techniques which have been used and found applicable to mediate the flavour in breadfruit varieties. 6.2.1.1. Modification of Polyphenols Secondary metabolites such as polyphenols have been found to form complexes with protein or starch which reduces their digestibility (Olapade and Umeonuorah, 2014; Widaningrum et al., 2020). Some of the tannins are released during processing breadfruit into flour. Infact, during the technological processes, the low tannins in breadfruit can be concentrated and the perception will be intense. Widowati et al. (2023) found that blanching and sulphiting reduced the tannins and bitterness in some breadfruit varieties. It is intriguing to note that the blanching will slow down the biochemical process of enzymes that catalyzes the breakdown of L-amino acids to phenols, and the sodium bisulfite will serve as reduction agent against the action of polyphenol oxidase (PPO). Specifically, sulphite will reduce disulfide bonds in enzymes to prevent enzyme catalyzation and the oxidation of phenol compounds causing browning and forming quinones (Figure 6-1). Additionally, sodium bisulfite is often used in the food industry to prevent enzymatic and non-enzymatic browning by reacting with hydrogen peroxide and turning it into water (Wood et al., 1989; Danilewicz and Standing, 2018). In modifying or mitigating the tannins formation and concentration in the breadfruit, it advisable to use either of blanching for about 10 min to control the enzymes or extract in 0.03 % sodium bisulphite which I demonstrated in Figure 2-5b. In addition, the data in this thesis showed that the effect has in the extraction of breadfruit flour with higher concentration in the intense breadfruit (0.3 % sodium bisulphite) will lead to the increase in the amount of the tannins (Figure 2-5c). Thus, these affirmed the processes noted by Widowati et al. (2023) are strongly recommended for the breadfruit flour with high tannins and flavonoids for example CP, and BN. In Figure 6-1, it is possible that the increased amount of SO2 abolished the precipitation of proteins with tannins (Arapitsas et al., 2018). Naczk et al. (1996) discovered that the optimum pH for the precipitation of proteins was found to be 0.3 - 3.1 pH units below the isoelectric points of the proteins. Also, Deaville et al. (2007) reported that gallotannins bind more strongly to BSA than ellagitannins due to the aromatic rings in the hydroxydiphenyl groups of ellagitannins that are inhibited by intramolecular biphenyl 127 linkages. Binding polyphenols (proanthocyanidins) to protein depends on the number and location of hydroxyl groups on the aromatic ring (monophenols < metadiphenols < ortho-diphenols < vicinal triphenols) and molecular size (Siebert, 1999). Condensed tannins can attach with protein at pH 3.5 â 7.5 to form protein complexes, which dissociate and release protein at pH less than 3.5 (Jones and Mangan, 1977). The soluble and insoluble complexes formed from the proanthocyanidins-protein interaction similar to the antigen-antibody interactions such that the binding agent and antigen are through their multivalent properties (Hagerman and Butler, 1978). Privalov (1979) had discovered that the increased binding of the globular protein like BSA happened to be the increased accessibility of their exposed peptide backbone. The affinities of the protein are more active around pH of 4.9, and at this point the protein â protein repulsion is minimized (Hagerman and Butler, 1978). In a study conducted by Kilpelainen et al. (2022), pH changes as temperature which may be due to the degradation of compounds in the extract. The sodium bisulfite extracts had similar pH values to the hot water extract at pH 3.3 â 3.9. Additionally, Dentinho and Bessa (2016) discovered that at pH 6 to 9 the tannins in rock rose and grape seed have the ability to bind to the soya meal proteins, however, the protein dissociate at pH 2. Consequently, this explains that acidity levels of the extractions, type of proteins, and its structural arrangement can affect the contribution of tannins or other polyphenols flavour or taste in breadfruit and its food products as it has been demonstrated in Figure 2-5bc. Food products naturally have polyphenols and tannins, but they must be within specified amounts to avoid insipid or astringent and bitter taste. Ricci et al. (2017) suggested that food rich phenols requires the technological approach to control the concentration to be within the threshold that would not affect the taste buds. Consequently, the use of 0.03 % sodium bisulfite solution will reduce tannins, but 0.3 % would be effective in reducing the tannins and other phenols in the intense breadfruit flavour flour such as GM (Table 5-2). Figure 6-1. Enzymatic Conversion of Phenols to O-quinone (bitter compound) through Polyphenol oxidase. 128 6.2.1.2. Encapsulation through Cyclodextrins Cyclodextrins are cyclic oligomers and can be used as food additives for encapsulation and retention of water solubility in foods with the target compounds to be stabilized (Gonzalez et al., 2021). This compound is âgenerally recognized as safeâ (GRAS) by the Food and Drug administration (FDA). The inclusion of cyclodextrins (CDs) as a form of complexation are used in the food industry as form of complexation in reducing the unpleasant tastes and smells. They are mostly used as Ã-form in food applications to mask the bitter taste of various dietary compounds such as protein isolates, vitamins, and phenolic compounds (Szejtli et al., 2005; Munin and Edwards-Lévy, 2011). The molecules can be in α-, β-, and γ-cyclodextrins are composed of 6, 7, and 8 units, respectively, consisting of glucose units and bound through α-(1,4) linkages (Szejtli et al., 2005). Through this approach, the bitter taste of certain substances may be totally or partially eliminated since complex compounds cannot react in the oral cavity with the taste buds. This technique has been applied to bitter and harsh metabolites in soy, beverages, and cigarettes. This works by making sure that the compound complexes with CDs, and are not soluble in water and saliva, thereby preventing the bitter sensation in the tongue (Table 6-1; Figure 6-2). The complexes can be released in the digestive system. However, the effectiveness of this strategy relies on the complex association constant, pH (fewer stable complexes with ionized guest molecules), and guest/host ratio (the higher possible molar excess, the better) (Szejtli et al., 2005). Table 6-1. Application of Cyclodextrins for Flavour and Taste Improvements (Modified from Gonzalez et al., 2021). CDs Compounds Class Effect References α Phenylalanine, tryptophane, tyrosine, isoleucine, proline, and histidine L-amino acids/proteins Reduce bitter taste Linde et al., (2009) β Naringin, limonin, hesperidin, Polyphenols/ flavonoids Reduce bitter taste of naringin, limonin, and hesperidin and prevent precipitation Linde et al., (2011) β Catechin Flavonoids The inclusion complexes. masked the bitterness of catechin in yogurt Ho et al., 2019 129 CDs Compounds Class Effect References β Beany flavour Aldehydes Decrease the major beany flavour compounds (1-octen-3- ol, benzaldehyde, hexanal, and 2-heptanone) when CD is added Lee et al., 2020 β, γ Caffeine and bitter natural extracts Polyphenols Bitter-masking properties Binello et al., (2004) Figure 6-2. The encapsulation and stability of targeted compound in the taste bud release in the Intestines. Guest â the targeted compound. Host â cyclodextrins. Figure created on Biorender. Based on the data in this study and as previous studies described in Table 6-1, the α-CD can be used for commercial breadfruit flour that has high protein such as ADS, where Ã and γ-CD can be incorporated in commercial breadfruit such as SIHCL, BN, CP rich in vitamin C, flavonoids, and tannins in reducing the perception of bitter taste in the taste buds as earlier reported by Linde et al. (2011); Ho et al., (2019). One of the major advantages of this approach is that it helps to preserve the bitter compounds that may have antioxidative properties as demonstrated in the intense breadfruit varieties (Figure 2-4i). Thus, there can be healthier products without losing any compounds while designing consumer acceptable products with desirable flavour. 6.2.1.3. Incorporation of Receptor Blockers and Modifiers Several taste metabolites compounds activate the taste receptors (Figure 1-5). One of the techniques used in food is to inhibit the activation of the taste receptor caused by taste compounds. In human taste receptors, bitterness is perceived by hTAS2Rs, TAS2Rs or T2Rs. The small molecule (4-(2,2,3-trimethylcyclopentyl) butanoic acid (also denoted as GIV 3727) has been 130 reported as inhibitor of six bitter taste receptors. Aftertaste caused by sweeteners acesulfame K and saccharin, has been found to be reduced by GIV 3727 or mitigate the activation of hTAS2R31, hTAS2R4, hTAS2R40, and hTAS2R43 in a dose dependent way without changing the sweet taste perception. Hence, the bitter receptor is not activated (Greene et al., 2011). Although, given the genetic diversity in receptors, a blocker will not have the same reducing effect signal in every individual (Because humans have more than 30 bitter-taste receptors, finding a universal blocker is impossible). Databases such as BitterDB are beginning to fill these gaps by collating information on bitter molecules and their receptors. As more knowledge is generated, the use of bitter-blocking compounds can be better directed and make the art of taste-masking more precise (Mennella et al., 2015; Dagan-Wiener et al., 2019). Ishii et al. (2012) suggested that capsaicin can be exploited as an inhibitor of PKD2L1, and PKD1L3, which signifies that in a medical condition, pre-treatment with capsaicin will probably lessen sour taste sensitivity. Polyols have been used as food additives that affect the hydrogenation of reducing sugars which are found naturally in vegetables and fruits. Many of the polyols including sorbitol, maltitol, mannitol, isomaltose, lactitol, erythritol, and xylitol function as effective sweet modifiers. Consequently, this can allow the masking of sweetness, sourness, and bitterness in breadfruit. In particular, it will be suitable for ADS, SIHCL, BN, and CP with relatively high sugars, vitamin C, flavonoids, and tannins. 6.2.1.4. Enzymatic Modifications and Fermentation Enzymatic treatment and fermentation are possible methods to alter the sensory profiles of food samples. Lactic acid fermentation (LAB) of pea protein preparations leads to a favorable alteration of the volatile profile due to a reduction of off-flavour molecules, undesirable green, and beany flavour which limits its applications in foods (Schindler et al., 2012). Similar results were recorded by Shi et al. (2021) leading to an appreciable reduction in the number of aldehydes and ketones by 64 %. It has been recommended that using LAB increase the productsâ safety, nutritional composition, functional properties, and pleasant sensorial qualities (Kingston et al., 2010; Rodríguez et al., 2021; Shi et al., 2021). The undesirable aroma is removed through the production of VOCs. Although wine produced from a particular variety of grapes often demonstrates its unique attributes, the active-flavour compounds might not be significant at pre-fermentative stages. The enzymes form LAB such as glycosidases, esterases, and proteases can improve the flavour, aroma, and structure of the wine (Liu, 2002; Swiegers et al., 2005; Lonvaud-131 Funel A, 1999). Therefore, the commercial breadfruit flour can be fermented into wine. Through malolactic fermentation, the malic acid potentially present in BN, DLP, DBIA, and NFIL-M will be converted to monocarboxylic lactic acid and carbon dioxide with no free intermediary products achieving flavourful wine with low tartness (Figure 5-2). Still, it is advisable to control the esterase and glycosidase activity of LAB through the pH, and temperature to experience the good aroma from the major contributors which are glycosides and esters of organic acids and fatty acids. Grimaldi et al. (2000) and Mathews et al. (2007) reported β-glucosidase activity was optimal at a pH of 5.5, and esterase activity was at peak in pH 6.0. Thus, this pH range should be considered during the utilization of the breadfruit flour in making acceptable products as in Figure 1-4. 6.2.1.5. Size Reduction To improve the application of the breadfruit flour with the intense flavour and to achieve a minimum level of undesirable volatiles. It is advisable to properly configure the milling or griding of the breadfruit into flour. Gu et al. (2021) have demonstrated that the use of ultra-centrifugal milling configuration with screen aperture size of 500 µm slows down the release of volatiles than 250 µm aperture due to the reduced surface thereby narrowing their escape. This can be applicable to commercial flour that are richer in tannins and flavonoids for example CP, BN, and SIHCL. 6.2.1.6. Use of Spices Flavour of spices have made them to be important food items for culinary and medicinal applications (Parthasarathy et al., 2008). Depending on the producerâs choices, commercial breadfruit flour can optimize to meet consumer favorite using the following: mint or rosemary flowers as clove, bulbs as garlic or onion. These applications of the species can also be to regional or cultural preference. For instance, the British have preference for cloves, coriander, mustard seeds, and ginger, while the Mexicans are known for cinnamon, vanilla, and dried chilies. In addition, nutmeg, garlic, and mustard seeds are preferred by Canadians (Exploratorium, 2013). 6.2.1.7. Adsorbent Resins The debittering compounds are always trapped by polymers and joined to resins. After which, they are precipitated, extracted with solvent, and later turned into less-bitter compounds (Drewnowski and Gomez-Carneros, 2000). Amberlite XAD-16HP as an adsorbent have been 132 found in reducing the bitterness in an orange juice without altering the quality attributes. Tannins adsorbed using polyvinyl polypyrrolidone (PVPP) matrices, which interact with phenolic compounds through hydrogen bonding, and hydrophobic interactions (Kola et al., 2000). This can be incorporated into the breadfruit turning into a beverage that are richer in polyphenols. 6.2.1.8. Extraction of Sub-Critical Water Among other known phenols with bitter or astringent taste properties in tea is catechin. In the removal or reduction of the catechins from green tea, the sub-critical water extraction (SWE) have been found to be suitable (Miyashita and Etoh, 2013). It is possible that some of the breadfruit flours are richer in catechins since they are flavonoids (Figure 5-8). About 300 mg in 100 mL of water of green tea extract was derived through hydrothermal, and pressurized extracting techniques with SWE, where the original flavour extract was retained. 6.3. Industrial Application Technique for Intense Breadfruit The recommended techniques can be adopted as described in Figure 6-3 in making beverages, and confectionaries from the intense flavour commercial breadfruit noted in Table 5-2. The highlighted steps are key processing workflow in making flour from the intense breadfruit or tissue turning to beverage like the mutiny vodka (Figure 1-4) or bread. While peeling the PPO are activated that can cause browning, these enzymatic activities can be inactivated with the use of 0.3 % sodium bisulfite. Through milling, the release of the significant metabolites can be reduced by using 500 µm mesh. Choice of enzymes (LAB) or other available enzymes that allows specificity in the breakdown of metabolites from the phenylpropanoid pathway in oxidizing to phenolate ions that can bind with proteins through Michael addition or Schiff base (Figure 2-7). 133 Figure 6-3. Proposed Schematic Workflow For Reducing The Intense Flavour In Breadfruit. SBS â sodium bisulfite, SCW- subcritical water. 134 Chapter 7: Summary The motivation was to unravel the flavour chemistry of breadfruit varieties. Then, I hypothesized that varieties with more intense flavours contain compounds that are not present in mild-flavoured breadfruit. Understanding the breadfruit flavour profiles will enable development of methods to optimally explore the quality attributes of breadfruit in a wide variety of food products. Interestingly, knowing flavour profiles also requires understanding the complex nature of the five chemical determinants of taste â sweetness, sourness, bitterness, umami, and saltiness. In particular, breadfruit does not contain enough sodium or calcium to be perceived as a salty food (Jones et al., 2011b), and consequently, the flavour characteristics to understudy was modelled around the other 4 tastes. To achieve this goal of understanding the flavour chemistry in breadfruit, I established these specific objectives necessary to investigate this hypothesis. 7.1. Objective 1: . The overall aim of this objective is to investigate the differences in composition of the breadfruit varieties using physiochemical and phytochemical analyses. I used standardized protocols to study the food properties. The assays implied statistically significant differences (p<0.05) in BSA total protein content, flavonoid levels, and vitamin C concentrations between the mild and intense breadfruit flavour varieties. These compounds have been reported to be associated with taste classes such as sourness, bitterness, and umami taste characteristics in foods and their intensity through their interactions. Additionally, the L-amino acids data provided some understanding about the possible reversible and irreversible protein-flavour compound interactions. Furthermore, I examined the tannins too in the samples which happened to be polymers of flavonoids, and the assay revealed that the tannins are marginally higher in the intense flavour varieties at a particular pH. Further examination uncovered tannins precipitate the proteins and they are pH dependent depending on their structure. The BSA protein happened to be significantly in the mild, suggesting the tendency to complexes with the tannins more than the intense flavour variety. Recall, some L-amino acids such as proline which binds to tannins are higher in intense, cysteine that form covalent bonding are higher in the mild flavour varieties 7.2. Objective 2: To use untargeted metabolomics to discover the known unknown metabolites responsible for flavour intensity in the breadfruit, and to generate novel hypotheses about the biochemical pathways that cause the flavour intensity perceived in the breadfruit varieties. I 135 hypothesize that the unknown polar metabolites in âPorohitiâ, âMeinpadahkâ, and âMeiarepheâ are responsible for the high flavour intensity and are either absent or at low levels in less intense flavour cultivars (âMaâafalaâ, âUlu fitiâ, and âYapâ). From the metabolomic study, I discovered that phenylpropanoid biosynthesis which are common to the Artocarpus species was significant in the intense varieties. Furthermore, I applied synthetic biotransformation, which showed the possible transformation for the flavonoids with the putative classes such as fatty acyls and cinnamic acids possessing bitter taste. Based on the outcome of this approach, I proposed that the pathway generates the metabolites that contributes to generating the significant metabolites in the Artocarpus species that react with the taste receptors. Specifically, bitterness and astringency through food perception have been linked to phenylpropanoid pathways. Where flavonoids contain many sub-classes such as flavan-3-ols, flavones, flavonols, flavanones, isoflavones, and anthocyanins. I recommend further targeted studies on flavonoids will reveal the particular compound in the classes influencing the difference in the intensity. 7.3. Objective 3: To use liquid-liquid extraction, separate and identify these volatile compounds present in the selected varieties using the GC-MS/MS. Although not validated, the pipeline method was put through alkanes standards and Grobâs mix. I matched to a curated database to identify the non-polar metabolomics coverage. Therefore, the mass spectra were subjected to metabolic profiling to characterize the known volatile compounds. I hypothesize that non-polar metabolites in âPorohitiâ, âMeinpadahkâ, and âMeiarepheâ are responsible for its high flavour intensity than the other less intense flavour cultivars (âMaâafalaâ, âUlu fitiâ, and âYapâ). I found linolenic, palmitic, and arachidonic acid associated with odor which could form rancid, fruity, and nutty flavour. Strikingly, fatty acids are among the chemical compounds common to the genus Artocarpus. 7.4. Objective 4: To determine if commercial breadfruit flour from different geographical locations is either mild or intense. I collected 10 commercial breadfruit flour from around the world. I hypothesize that the significant metabolites define the flavour intensity in the commercial breadfruit flour. The samples were subjected to phytochemicals assay significant between mild and intense flavour varieties. Using the established protocols, I discovered that some commercial breadfruit flour from the same geographical location shared similar metabolites profiles. Also, I proposed that production techniques may have affected the metabolites profiles of the flour for example ADS and SIHCL, even NFIL and NFIL-M. 136 7.5. Objective 5: To recommend production techniques for future food producers. I proffer some strategic approaches that would subdue the flavour intensity of the commercial breadfruit flour, conserve the sensorial qualities while maintaining the nutritional and health benefits of the breadfruit flour. I discovered that tannins is pH dependent and precipitate proteins. Processing techniques such as polyphenols modification and reduction, encapsulation with cyclodextrins, fermentation, bitter blockers, and local spices with pleasant flavour will preserve the nutritional composition and reduce the intense flavour to be within satisfactory threshold levels, which were proposed for the producer who intend to use the commercial breadfruit flour in their geographical location or elsewhere. 7.6. Conclusion and Recommendation In general, this research addresses the flavour intensities of breadfruit varieties which was investigated via physicochemical properties, phytochemical assays, and untargeted metabolomic methods. The intense flavour arises from the interactions of breadfruit metabolites with specific taste receptors. This thesis uncovered that the flavour intensity could emanate from astringent, bitter, sour, and odor metabolites through the phenylpropanoid biosynthesis, vitamin C, and α-linolenic acid pathways. I recommend that the tastes of phenols, and tannins can be mediated by several techniques such as; the use of sodium bisulfite, blanching, receptor blockers, size reduction, the inclusion of LAB and yeast extract capable of initiating the desire taste through the breakdown of starch granule to sugars, and development of spices which includes: the use of flavouring agents with pleasing flavour such as (vanilla, strawberry, banana flavours etc.) for masking the intense taste; the application of salts to adjust the acidity levels changing the charges on protein functional groups; and the introduction of egg as a protein concentrate to bind the taste of the compounds. To know more about the flavour formation in breadfruit. Future work should focus on the types of polyphenols and tannins, the structure of proteins, effects of heat treatments on these metabolites, and their interactions in mild and intense breadfruit varieties. Consequently, the following are the hypotheses that are generated for further studies: i. Breadfruit flavour improvement may lead to reduction in its functional food properties. ii. Composition and amount of proteins in breadfruit may affect the flavour intensity. iii. Changes in tannins concentration in breadfruit impact its intense flavour. iv. Variations in acidity levels may affect phenolsâ interactions with other metabolites. 137 v. Heat treatment disintegrate more flavour metabolites in intense flavour breadfruit varieties. 138 Bibliography AACC, American Association of Cereal Chemists International (2000). Approved methods of analysis (11th ed.). Method 80-68. St. Paul, MN: AACC International. https://doi.org/10.1094/AACCIntMethod-80-68.01 Aalbersberg, W. G., Lovelace, C. E., Madhoji, K., & Parkinson, S. V. (1988). Nutrient composition of breadfruit cultivars from the Pacific. Journal of Food Science, 53(3), 804â806. https://doi.org/10.1111/j.1365-2621.1988.tb08910.x Abrajano, M., Casillano, M. G. P., Padel, J., Ranido, J., Ronquillo, M., & Trinchera, A. J. D. (2021). Sensory Evaluation of Breadfruit (Artocarpos Altilis) Polvoron. Available at SSRN 3856829. https://doi.org/10.2139/ssrn.3856829 Abbas, F., H.M. Hammad, S. Fahad, A. Cerda, M. Rizwan, W. Farhad, S. Ehsan, and H.F. Bakhat. (2017). Agroforestry: a sustainable environmental practice for carbon sequestration under the climate change scenariosâa review. Environmental Science and Pollution Research, 24(12), 11177-11191. https://doi.org/10.1007/s11356-017-8687-0 Adebowale, A. P., Adeboyejo, F. O. & Adegoke, G. O. (2020). Chemical, Microbiological and Sensory Profiles of Mixed Fruit Wine from Banana (Musa acuminata), Watermelon (Citrullus vulgaris L.), Pineapple (Ananas comoscus L.) and Cucumber (Cucumis sativus). Acta Scientific Nutritional Health. 4. 01 - 07. https://actascientific.com/ASNH/pdf/ASNH-04-0637.pdf Adebayo, A., Brown-Myrie, E., & Itiola, O. (2008). Comparative disintegrant activities of breadfruit starch and official corn starch. Powder Technology, 181, 98 â 103. https://doi.org/10.1016/j.powtec.2006.12.013 Adebowale, A. A., Sanni, S. A., & Oladapo, F. O. (2008). Chemical, functional and sensory properties of instant yam-breadfruit flour. Nigerian Food Journal, 26(1) 2-12. https://doi.org/10.4314/nifoj.v26i1.47417 Adegunwa, M.O., Adebowale, A.A., Bakare, H.A., & Ovie, S.G. (2014). Compositional characteristics and functional properties of instant plantain-breadfruit flour. International Journal of Food Research, 1-7. Adewusi, S. R., Udio, J., & Osuntogun, B. A. (1995). Studies on the Carbohydrate Content of Breadfruit (Artocarpus communis Forst) From SouthâWestern Nigeria. Starch-starke, 47, 289-294. https://doi.org/10.1002/star.19950470802. Adjei, O., Sasu, P., Baidoo, J., Adjei-Mensah, B., Bredu, G., Ansah, T., Attoh-Kotoku, V., Appiah, F., & Donkoh, A. (2024). Breadfruit (Artocarpus altilis) pulp flour as a non-conventional filler in comminuted pork: Effect on physicochemical, sensory attributes, and cost of meatloaves. European Journal of Agriculture and Food Sciences, 6(2), 33-38. https://doi.org/10.24018/ejfood.2024.6.2.778 139 Agilent Technologies. (2019). MassHunter Profinder software quick start guide (Version 10.0). Agilent Technologies. https://www.agilent.com/en/product/mass-spectrometry/software/masshunter Ajifolokun, M. & Adeniran, O. A. (2015). Microbiological studies and sensory evaluation of breadfruit and cassava into gari analogue. Elsevier - Food Webs Journal. https://doi.org/10. 10.1016/j.nifoj.2015.04.003 Akkad, R., Kharraz, E., Han, J., House, J. D., & Curtis, J. M. (2022). The effect of short-term storage temperature on the key headspace volatile compounds observed in Canadian faba bean flour. Food Science and Technology International, 28(2), 135 â 143. https://doi.org/10.1177/1082013221998843 Akouègninou, A., Van Der Burg, W.J., Van Der Maesen, L. J. G., Adjakidjè, V., Essou, J. P., Sinsin, B., & Yèdomonhan, H. (2006). Flore analytique du Bénin. Cotonou & Wageningen, Backuys Publishers 1034 p. Aldin, E., Reitmeier, H., & Murphy, P. (2006). Bitterness of soy extracts containing isoflavones and saponins. Journal of Food Science, 71(S211-S215). https://doi.org/10.1111/j.1365-2621.2006.tb15643.x Aloni, B., Karni, L., Deventurero, G., Turhan, E., & Aktas, H. (2008) Changes in ascorbic acid concentration, ascorbate oxidase activity, and apoplastic pH in relation to fruit development in pepper (Capsicum annuum L.) and the occurrence of blossom-end rot. The Journal of Horticultural Science and Biotechnology, 83 (1), 100-105 https://doi.org/10.1080/14620316.2008.11512353 Alonso, A., Marsal, S., & Julia, A. (2015) Analytical methods in untargeted metabolomics: state of the art in 2015. Front Bioeng Biotechnol, 3 https://doi.org/10.3389/fbioe.2015.00023 Amusa, N. A., Kehinde, L. A., & Ashaye, O. A. (2002). Biodeterioration of Breadfruit (Artocarpus communis) in storage and its effect on the nutrient composition. African Journal of Biotechnology, 1(2), 57 â 60. https://doi.org/10.5897/AJB2002.000-010 Andrade, L., Apolinário, A., Rangel-Yagui, C., Stephano, M., & Tavares, L. (2019). Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus. Journal of Drug Delivery Science and Technology, 55, 101363. https://doi.org/10.1016/j.jddst.2019.101363 Andrews, L., & Mason, Jr. G. (1991). Production of breadfruit (Artocarpus altilis) in the Eastern Caribbean. edited by D. Walmskey. In: Post-harvest management of tropical fruits and ornamentals in the Caribbean region. Trinidad and Tobago: The Technical Center for Agriculture and Rural Cooperation (CTA) and The Caribbean Agricultural Research and Development Institute (CARDI); 1991. 140 Anson, L. (2002). Neurobiology: The bitter-sweet taste of L-amino acids. Nature, 416, 136-136. https://doi.org/10.1038/416136b Anwar, S. H., Hasni, D., Rohaya, S., Antasari, M., & Winarti, C. (2020). The role of breadfruit OSA starch and surfactant in stabilizing high-oil-load emulsions using high-pressure homogenization and low-frequency ultrasonication. Heliyon, 6(7). https://doi.org/10.1016/j.heliyon.2020.e04341 AOAC. (1995). Method of Analysis. Association of Official Analytical Chemistry, Washington, DC. (accessed 15 December 2024). Appiah, F., Oduro, I. & Ellis, W. O. (2011). The proximate and mineral composition of Artocarpus altilis pulp flour as affected by fermentation. Pakistan Journal of Nutrition, 10 (7)653 â 657. https://doi.org/10.3923/pjn.2011.653.657 Arapitsas, P., Guella, G., & Mattivi, F. (2018). The Impact of SO2 on Wine Flavanols and Indoles in Relation to Wine Style and Age. Scientific Reports, 8. https://doi.org/10.1038/s41598-018-19185-5 Arinola, S. (2023). Quality Evaluation of Flour Blends and Biscuits Produced from Breadfruit and Lima Bean Flours. The Annals of the University Dunarea De Jos of Galati. Fascicle VI - Food Technology, 47(2), 77-94. https://doi.org/10.35219/foodtechnology.2023.2.05 Arrebola-Lie´banas, F. J., Romero-Gonza´lez, R., & Frenich, A. G. (2017). HRMS: Fundamentals and Basic Concepts. Applications in High Resolution Mass Spectrometry. Elsevier Inc. https://doi.org/10.1016/B978-0-12-809464-8.00001-4 Assogba, K., Bokossa, Y., Innocent, T., Celestin, B., Roseline, S., Pivot, D., Anayce, B., & Jultesse, P. (2018). Uses of the fruit of Breadfruit tree (Artocarpus altilis) in the Republic of Benin: Bibliographic Synthesis. ASTM, American Society for Testing and Materials. (2009). Standard Terminology Relating to Sensory Evaluations of Materials and Products, E253-09a. ASTM International, West Conshohocken, PA. E253-09a. Arts, M. J. T. J., Haenen, G. R. M. M., Voss, H. P., & Bast, A. (2001). Masking of antioxidant capacity by the interaction of flavonoids with protein. Food Chem. Toxicol, 39, 43-47. https://doi.org/10.1016/S0278-6915(01)00020-5 Audi, L., Shallow, G., Robertson, E., Bobo, D., Ragone, D., Gardner, E., Jhurree-Dussoruth, B., Wajer, J., & Zerega, N. (2023). Linking breadfruit cultivar names across the globe connects histories after 230 years of separation. Current Biology, 33. https://doi.org/10.1016/j.cub.2022.12.001 141 Azzali, G., Gennari, P. U., Maffei, G., & Ferri, T. (1996). Vallate, foliate and fungiform human papillae gustatory cells. An immunocytochemical and ultrastructural study. Minerva stomatologica, 45(9), 363â379. Baba, S., Chan, H., Kezuka, M., Inoue, T., & Chan, E. (2016). Artocarpus altilis and Pandanus tectorius: Two Important Fruits of Oceania with Medicinal Values. Emirates Journal of Food and Agriculture, 28, 1. https://doi.org/10.9755/ejfa.2016-02-207 Badejo, A. A., Wada, K., Gao, Y., Maruta, T., Sawa, Y., Shigeoka, S., & Ishikawa, T. (2012). Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway. Journal of experimental botany, 63(1), 229â239. https://doi.org/10.1093/jxb/err275 Badejo, A. A., Adebowale, A. P., & Enujiugha, V. N. (2016). Changes in Nutrient Composition, Antioxidant Properties, and Enzymes Activities of Snake Tomato (Trichosanthes cucumerina) during Ripening (2016). Prev Nutr Food Sci., 21(2), 90-6. https://doi.org/10.3746/pnf.2016.21.2.90 Bakare, A. H., Osundahunsi, O. F. & Olusanya, J. O. (2016), Rheological, baking, and sensory properties of composite bread dough with breadfruit (Artocarpus communis Forst) and wheat flours. Food Sci Nutr, 4, 573-587. https://doi.org/10.1002/fsn3.321 Bakare, A. H., Adeola, A. A., Otesile, I., Obadina, A. O., Afolabi, W. A., Adegunwa, M. O., Akerele, R. A., Bamgbose, O. O., & Alamu, E. O. (2020). Nutritional, Texture, and Sensory Properties of composite biscuits produced from breadfruit and wheat flours enriched with edible fish meal. Food Science & Nutrition, 8(11), 6226â6246. https://doi.org/10.1002/fsn3.1919 Balick, M. J. (2009). Ethnobotany of Pohnpei, University of Hawai'i Press. Basli, A., Soulet, S., Chaher, N., Mérillon, J. M., & Others. (2012). Wine polyphenols: Potential agents in neuroprotection. Oxidative Medicine and Cellular Longevity, 2012, Article 805762. https://doi.org/10.1155/2012/805762 Basu, S., Duren, W., Evans, C. R., Burant, C. F., Michailidis, G., & Karnovsky, A. (2017). Sparse network modeling and Metascape-based visualization methods for the analysis of large-scale metabolomics data. Bioinformatics, 33, 1545â1553. https://doi.org/10.1093/bioinformatics/btx012 Bates, R. P., Graham, H. D., Matthews, R. F. & Clos, L. R. (1991). Breadfruit Chips: Preparation, Stability and Acceptability. Journal of Food Science, 56, 1608-1610. https://doi.org/10.1111/J.1365-2621.1991.TB08652.X Baxter, N. J., Lilley, T. H., Haslam, E. & Williamson, M. P. (1997). Multiple interactions between polyphenols and a salivary proline-rich protein repeat result in complexation and precipitation. Biochemistry, 36, 5566-5577. https://doi.org/10.1021/bi9700328. 142 Betiku, E., & Taiwo, A. E. (2015). Modeling and optimization of bioethanol production from breadfruit starch hydrolyzate vis-Ã -vis response surface methodology and artificial neural network. Renewable Energy, 74, 87â94. https://doi.org/10.1016/j.renene.2014.07.054 Beutler, H. O. (1988). D-Fructose. âMethods of Enzymatic Analysisâ (Bergmeyer, H. U., ed.), 3rd ed., Vol. VI, pp. 321-327, VCH Publishers (UK) Ltd., Cambridge, UK. Bezerra, E., Santos, E., S. Farias, N., & Cavalcanti, M. (2019). Coating based on breadfruit starch (Artocarpus altilis) for fruit conservation: influence of glycerol, sorbitol, and mannitol as plasticizers. Food Science and Technology, 39. https://doi.org/10.1590/fst.17518 Bhattarai, S., Tran, V.H., Duke, C.C. 2001. The stability of gingerol and shogaol in aqueous solutions, Journal of Pharmaceutical Sciences, 90(10), 1658-1664. https://doi.org/10.1002/jps.1116 Binello, A., Cravotto, G., Nano G. M., & Spagliardi, P. (2004). Synthesis of chitosan-cyclodextrin adducts and evaluation of their bitter-masking properties. Flavour Fragr. J., 19, 394â400. https://doi.org/10.1002/ffj.1434 Bligh, W. (1792). A Voyage to the South Sea, Undertaken by Command of His Majesty, for the Purpose of Conveying the Bread-Fruit Tree to the West Indies, in His Majestyâs Ship the Bounty, commanded by Lieutenant William Bligh . George Nicol, London, UK. Boonyaketgoson, S., Du, Y,. Valenciano Murillo, A. L., Cassera, M. B., Kingston, D. G. I., & Trisuwan K. (2020). Flavanones from the twigs and barks of Artocarpus lakoocha having antiplasmodial and anti-TB activities. Chem Pharm Bull, 68(7), 671â674 https://doi.org/10.1248/cpb.c20-00080 Borchert, R., Renner, S. S., Calle, Z., Navarrete, D. A., Tye, L., Gautier, R., Spichiger, and von Hildebrand, P. (2005). Photoperiodic induction of synchronous flowering near the equator. Nature, 433(7026), 627 â 629. https://doi.org/10.1038/nature03259 Brown, P. N., Murch, S. J., & Shipley, P. (2012). Phytochemical diversity of cranberry (Vaccinium macrocarpon Aiton) cultivars by anthocyanin determination and metabolomic profiling with chemometric analysis. Journal of Agricultural and Food Chemistry, 60, 261 â 271. https://doi.org/10.1021/jf2033335. Buck, P. (1959). Vikings of the Pacific. Chicago, Univ. of Chicago Press. Burdock, G. A. (ed.) (2010). Fenaroli's Handbook of Flavour Ingredients. 6th ed.Boca Raton, FL, p. 1584. https://doi.org/10.1201/9781439847503 Busch, K. L. (2001). Units in mass spectrometry. 28 Spectroscopy 16(11). 143 Bylesjö, M., Rantalainen, M., Cloarec, O., Nicholson, J., Holmes, E., & Trygg, J. (2006). OPLS discriminant analysis: Combining the strengths of PLS-DA and SIMCA classification. Journal of Chemometrics, 20(8â10), 341â351. https://doi.org/10.1002/cem.1006 Calpe, C. (2007). Review of the rice market situation in 2007. Food and Agriculture Organization of the United Nations. https://www.fao.org/4/a1410t/a1410t.pdf Cambiaghi, A., Ferrario, M., & Masseroli, M. (2017). Analysis of metabolomic data: Tools, current strategies and future challenges for omics data integration. Briefings in Bioinformatics, 18(3), 498â510. https://doi.org/10.1093/bib/bbw050 Caul, J. F. (1957). The profile method of flavour analysis. In Advanced in Food Research, Vol. 7, (E.M. Mrak and G. F. Steward, eds.) p.1, Academic Press. New York, USA. https://doi.org/10.1016/S0065-2628(08)60245-1 Chang, H. Y., Colby, S. M., Du, X., Gomez, J. D., Helf, M. J., Kechris, K., Kirkpatrick, C. R., Li, S., Patti, G. J., Renslow, R. S., Subramaniam, S., Verma, M., Xia, J., & Young, J. D. (2021). A Practical Guide to Metabolomics Software Development. Analytical chemistry, 93(4), 1912â1923. https://doi.org/10.1021/acs.analchem.0c03581 Chaurasia, S., & Pandey, A. (2023). Phytochemistry and Pharmacology of Genus Artocarpus: A Review on Current Status of Knowledge. Russ J Bioorg Chem, 49, 481â514. https://doi.org/10.1134/S1068162023030081 Chen, G. X. & Asada, K. (1989). Plant Cell Physiol. 30, 987-998. https://doi.org/10.1093/oxfordjournals.pcp.a077347 Chen, C., Zhang, B., Fu, X., You, L. J., Abbasi, A. M., & Liu, R. H. (2016) The digestibility of mulberry fruit polysaccharides and its impact on lipolysis under simulated saliva, gastric and intestinal conditions. Food Hydrocoll, 58, 71â178. https://doi.org/10.1016/j.foodhyd.2016.03.024 Chen, J., Wu, F., Wang, H., Guo, C., Zhang, W., Luo, P., Zhou, J., Hao, W., Yang, G., Huang, J. (2022). Identification of key taste components in Baccaurea ramiflora L our fruit using non-targeted metabolomics. Food Science and Human Wellness, 12(1), 94â101. https://doi.org/10.1016/j.fshw.2022.07.027 Clark, E. A., & Fadi, M. Aramouni, (2018). "Evaluation of Quality Parameters in Gluten-Free Bread Formulated with Breadfruit (Artocarpus altilis) Flour, Journal of Food Quality, 12, 2018. https://doi.org/10.1155/2018/1063502 Coronel, R. E., (1983). Rimas and kamansi, Promising fruits of the Philippines. pp: 379-396, College of Agriculture, University of the Philippines at Los Banos. Coskun O. (2016). Separation techniques: Chromatography. Northern clinics of Istanbul, 3(2), 156â160. https://doi.org/10.14744/nci.2016.32757 144 Czerny, M., Brueckner, R., Kirchhoff, E., Schmitt, R., & Buettner, A. (2011). The influence of molecular structure on odor qualities and odor detection thresholds of volatile alkylated phenols. Chemical Senses, 36 6, 539-553. https://doi.org/10.1093/chemse/bjr009 Dagan-Wiener, A., Di Pizio, A., Nissim, I., Bahia, M. S., Dubovski, N., Margulis, E., Niv, M. Y. BitterDB: Taste Ligands and Receptors Database (2019). Nucleic Acids Res, 47(1), 1179â1185. https://doi.org/10.1093/nar/gky974 Daley, O. O., Roberts-Nkrumah, L. B., Alleyne, A. T., & Gloster, M. C. (2022). Folk nomenclature and traditional knowledge of breadfruit (Artocarpus altilis (Parkinson) Fosberg)) diversity in four Anglophone Caribbean countries. Journal of ethnobiology and ethnomedicine, 18(1), 65. https://doi.org/10.1186/s13002-022-00562-4 Daley, Oral O., Laura B. Roberts-Nkrumah, & Angela T. Alleyne. (2016). Sensory and instrument assessment of colour and texture among breadfruit (Artocarpus altilis (Parkinson) Fosberg)) cultivars. Tropical Agriculture, 93. Daley, O., Roberts-Nkrumah, L., & Alleyne A. (2020). Morphological diversity of breadfruit (Artocarpus altilis (Parkinson) Fosberg) in the Caribbean. Sci Hortic. 266, 109278. https://doi.org/10.1016/j.scienta.2020.109278 Danilewicz, J. C., & Standing, M. J. (2018). Reaction Mechanisms of Oxygen and Sulfite in Red Wine. Am. J. Enol. Vitic, 69, 189â195. https://doi.org/10.5344/ajev.2018.17095 Davies, M. J., Fu, S. & Dean, R. R. (1995). Protein hydroperoxides can give rise to reactive free radicals. Biochemical Journal, 305, 643â649. https://doi.org/10.1042/bj3050643 Deaville, E., Green, R., Mueller-Harvey, I., Willoughby, I., & Frazier, R., (2007). Hydrolysable tannin structures influence relative globular and random coil protein binding strengths. J. Agric. Food Chem., 55, 4554-4561. https://doi.org/10.1021/jf063770o Deewa, B., Banerjee, M., & Chowdhury, S. K. (2023). Pharmacological Treasures of the Moraceae Family: Bioactive Compounds and Therapeutic Potential. Plant Science Today. https://doi.org/10.14719/pst.2385 Defilippi, B., Dandekar, A., & Kader A. (2005). Relationship of ethylene biosynthesis to volatile production, related enzymes and precursor availability in apple peel and flesh tissues. Journal of Agricultural and Food Chemistry, 53, 3133-3141. https://doi.org/10.1021/jf047892x Delompré, T., Guichard, E., Briand, L., & Salles C. (2019). Taste Perception of Nutrients Found in Nutritional Supplements: A Review. Nutrients, 11, 2050. https://doi.org/10.3390/nu11092050 DeLoughrey, E. M. (2007). âGlobalizing the routes of breadfruit and other bounties. Journal of Colonialism and Colonial History, 8(3). https://doi.org/10.1353/cch.2008.0003 145 Deng, Y., & Lu, S. (2017). Biosynthesis and regulation of phenylpropanoids in plants. Critical Reviews in Plant Sciences, 36, 1-34. https://doi.org/10.1080/07352689.2017.1402852 Dentinho, M. & Bessa, R. (2016). Effect of tannin source and pH on stability of tannin-protein complexes. Revista de Ciências Agrárias, 39. https://doi.org/10.19084/RCA15062 Devkota, K.P., Beebout, S.E., Bunquin, M.A., (2022). Setting sustainability targets for irrigated rice production and application of the sustainable Rice platform performance indicators. Environ. Impact Assess. Rev. 92, 106697. https://doi.org/10.1016/j.eiar.2021.106697 Domínguez, R., Pateiro, M., Gagaoua, M., Barba, F. J, Zhang, W., & Lorenzo, J. M. (2019). A comprehensive review on lipid oxidation in meat and meat products. Antioxidants, 8, 429. https://doi.org/10.3390/antiox8100429 Drewnowski, A., & Gomez-Carneros, C. (2000). Bitter taste, phytonutrients, and the consumer: A review. The American Journal of Clinical Nutrition, 72(6), 1424â1435. https://doi.org/10.1093/ajcn/72.6.1424. Elevitch, C., Ragone D., & Cole I. (2014). Breadfruit Production Guide: Recommended practices for growing, harvesting, and handling (2nd Edition). Breadfruit Institute of the National Tropical Botanical Garden, Kalaheo, HI and Hawaii Homegrown Food Network, Holualoa, HI. www.breadfruit.org and www.breadfruit.info. https://ntbg.org/wp-content/uploads/2020/02/breadfruit_production_guide.pdf Eliuk, S., & Makarov, A. (2015). Evolution of Orbitrap Mass Spectrometry Instrumentation. Annu. Rev. Anal. Chem. 8, 61â80. https://doi.org/10.1146/annurev-anchem-071114-040325 Englberger, L., Lorens, A., Alfred, J., & Iuta, T. (2007). Screening of selected breadfruit cultivars for carotenoids and related health benefits in Micronesia. Acta Horticulturae, 757, 193â200. https://doi.org/10.17660/ActaHortic.2007.757.26 Englberger, L., Lorennij, R., Taylor, M., Tuia, V., Aalbersberg, W., Dolodolotawake, U., Tibon, L., Tibon, J., & Alfred, J. (2012). Carotenoid content and traditional knowledge of breadfruit cultivars of the Republic of the Marshall Islands. Journal of Food Composition and Analysis, 34. https://doi.org/10.1016/j.jfca.2014.04.003 Erland, L., Turi, C. E., Saxena, P. K., & Murch, S.J. (2020a). Metabolomics and hormonomics to crack the code of filbert growth. Metabolomics, 16, 62. https://doi.org/10.1007/s11306-020-01684-0 Erland, L. A. E., Giebelhaus, R. T., Victor, J. M. R., Murch, S. J., & Saxena, P. K. (2020b). The morphoregulatory role of thidiazuron: Metabolomics-guided hypothesis generation for mechanisms of activity. Biomolecules, 10(9), 1â34. https://doi.org/10.3390/biom10091253 Erland, L. A. E., Needham, A. M. W., Kehinde, A. Z., Adebowale, A. P., Lincoln, N. K., Ragone, D., & Murch, S. J. (2022). Impact of microclimate on Artocarpus altilis (Parkinson) Fosberg 146 var Maâafala fruit and nutritional quality, Journal of Food Composition and Analysis, 115. https://doi.org/10.1016/j.jfca.2022.104983 Esuoso, K. O., & Bamiro, F. O. (1995). Studies on the baking properties of non-wheat flours â I. Breadfruit (Artocarpus altilis). International Journal of Food Sciences and Nutrition, 46(3), 267â273. https://doi.org/10.3109/09637489509012558 Exploratorium, (2013). Spice blends In: Spice blends of the World Science cooking California USA available online http://www.exploratorium.edu/cooking/seasoning/map/spicemap.html Famurewa, J., Pele, G., Esan, Y., & Jeremiah B. (2016). Influence of maturity and drying methods on the chemical, functional and antioxidant properties of breadfruit (Artocarpus altilis). Biotechnol J Int. 16:1â9. https://doi.org/10.9734/BBJ/2016/25535 FAO, (2008). The State of Food Insecurity in the World 2008. Food and Agricultural Organization, Rome. https://www.fao.org/4/i0291e/i0291e00.htm FAO, (2009a). 1.02 Billion People Hungry: One sixth of humanity undernourishedâMore than ever before. Food and Agriculture Organization, Rome. https://www.fao.org/newsroom/detail/1-02-billion-people-hungry/en FAO, (2009b). The International Treaty on Plant Genetic Resources for Food and Agriculture. Food and Agricultural Organization, Rome. https://www.fao.org/3/i0510e/i0510e00.htm FAO, (2009c). Yield Per Hectare of World Maize Crops. Food and Agricultural Organization, Rome. www.fao.org/es/ess/chartroom/chart.asp?image=img/charts/42.gif. FAO, (2022). The State of Food Security and Nutrition in the World 2022: Repurposing food and agricultural policies to make healthy diets more affordable. FAO. https://doi.org/10.4060/cc0639en Farag, M. A., Abdelfattah, M. S., Badr, S. E. A., & Wessjohann, L. A. (2014). Profiling the chemical content of Ficus lyrata extracts via UPLC-PDAqTOF-MS and chemometrics. Nat Prod Res, 28(19):1549â56. https://doi.org/10.1080/14786419.2014.926353 Faria, J. V., Valido, I. H., Paz, W. H. P., da Silva, F. M. A., de Souza, A. D. L., Acho, L. R. D., Lima, E. S., Boleti, A. P. A., Marinho, J. V. N., Salvador, M. J., Dos Santos, E. L., Soares, P. K., López-Mesas, M., Maia, J. M. F., Koolen, H. H. F., & Bataglion, G. A. (2021). Comparative evaluation of chemical composition and biological activities of tropical fruits consumed in Manaus, central Amazonia, Brazil. Food research international (Ottawa, Ont.), 139, 109836. https://doi.org/10.1016/j.foodres.2020.109836 147 Farré, M., Kantiani, L., Petrovic, M., Pérez, S. & Barceló, D. (2012). Achievements and future trends in the analysis of emerging organic contaminants in environmental samples by mass spectrometry and bioanalytical techniques. J. Chromatogr. A, 1259, 86. https://doi.org/10.1016/j.chroma.2012.07.024 FDA Agency Response Letter GRAS Notice No. GRN 000596. (2016). Available online: https://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm495765.htm Fernández-Ochoa, Ã., Quirantes-Piné, R., Borrás, I., Cádiz-Gurrea, M., Alarcón-Riquelme, M., Brunius, C., & Segura-Carretero, A. (2020). A case report of switching from specific vendor-based to R-based pipelines for untargeted LC-MS metabolomics. Metabolites, 10(1), 28. https://doi.org/10.3390/metabo10010028 Fernie, A. R., & Schauer, N. (2008). Metabolomics-assisted breeding: a viable option for crop improvement? Trends Genet. 25, 39-48. https://doi.org/10.1016/j.tig.2008.10.010 Fiehn, O., Kopka, J., Dörmann, P., Altmann, T., Trethewey, R. N., & Willmitzer, L. (2000). Metabolite profling for plant functional genomics. Nature Biotechnology, 18, 1157â1161. https://doi.org/10.1038/79800 Fielding, R. (2024). Voltaireâs Breadfruit: Thoughts on the Inspiration for an Eighteenth-Century Colonial Botanical Transfer. Plant Perspectives. 1. https://doi.org/10.35074/PP.2024.1.1.0001 Franke, A., Custer, L., Arakaki, C., & Murphy, S. (2004). Vitamin C and flavonoid levels of fruits and vegetables consumed in Hawaii. Journal of Food Composition and Analysis, 17, 1-35. https://doi.org/10.1016/S0889-1575(03)00066-8 Frankel, E. N. (2005). Lipid oxidation (2nd ed.). The Oily Press. https://doi.org/10.1533/9780857097927 Fraser, C. M., & Chapple, C. (2011). The phenylpropanoid pathway in Arabidopsis. The Arabidopsis Book, 9. https://doi.org/10.1199/tab.0152 FooDB, (2024). https://foodb.ca/compounds/FDB098215 (assessed 8 February 2024). Fosberg, F. R. (1946). Economic survey of Micronesia. Vol. 11: Botany. Part 1: Botanical economics. Honolulu: U.S. Commercial Company. Fosberg, F. R. (1960). Introgression in Artocarpus (Moraceae) In Micronesia. https://doi.org/10.2307/2805210 148 Fownes, J. H., & Raynor, W. C. (1993). Seasonality and yield of breadfruit cultivars in the indigenous agroforestry system of Pohnpei, Federated States of Micronesia. Tropical Agriculture, 70(2), 103-109. Gao, Y., Hamid, N., Gutierrez-Maddox, N., Kantono, K., Kitundu, E. (2019). Development of a Probiotic Beverage Using Breadfruit Flour as a Substrate. Foods, 8, 214. https://doi.org/10.3390/foods8060214 García-Campos, M. A., J. Espinal-Enríquez, E. Hernandez-Lemus, (2015). Pathway analysis: state of the art, Front. Physiol. 6383. https://doi.org/10.3389/fphys.2015.00383 Giebelhaus, R. T., Erland, L. A. E., & Murch, S.J. (2022). HormonomicsDB: a novel workflow for the untargeted analysis of plant growth regulators and hormones. F1000Research, 11, 1191. https://doi.org/10.12688/f1000research.118019.1 Giebelhaus, R. T., Sorochan Armstrong, M. D., de la Mata, A. P., & Harynuk, J. J. (2022). Untargeted region of interest selection for gas chromatography - mass spectrometry data using a pseudo-F-ratio moving window. Journal of chromatography. A, 1682, 463499. https://doi.org/10.1016/j.chroma.2022.463499 Giebelhaus, R. T., Laura Biggs, Murch, S. J. & Erland, L. A. E (2023). Untargeted and targeted metabolomics to understand plant growth regulation and evolution in Wollemi pine (Wollemia nobilis). Botany. 101(9), 377 - 390. https://doi.org/10.1139/cjb-2023-0017 Goff, S. A., & Klee, H. J. (2006). Plant volatile compounds: Sensory cues for health and nutritional value? In Science, Vol. 311, Issue 5762, pp. 815â819). https://doi.org/10.1126/science.1112614 Golden, K. D., & Williams, O. J. (2001). Amino acid, fatty acid, and carbohydrate content of Artocarpus altilis (breadfruit). Journal of Chromatographic Science, 39(6), 243â250. https://doi.org/10.1093/chromsci/39.6.243 Golden, K. D., & Williams, O. J. (2007). The amino acid, fatty acid and carbohydrate content of Artocarpus altilis (breadfruit): the white cultivar from the West Indies. Acta Hort. (ISHS) 757, 201â208. https://doi.org/10.17660/ActaHortic.2007.757.28 Gonda I, Bar E, Portnoy V, Lev S, Burger J, Schaffer A, Tadmor Y, Gepstein S, Giovannoni J, Katzir N, & Lewinsohn E. (2010). Branched chain, and aromatic amino acid catabolism into aroma volatiles in Cucumis melo L. fruit. Journal of Experimental Botany, 6, 1111-1123. https://doi.org/10.1093/jxb/erp390 Gordon, D. S., Rudinsky, A. J., Guillaumin, J., Parker, V. J., Creighton, K. J. (2020). Vitamin C in Health and Disease: A Companion Animal Focus. In Topics in Companion Animal Medicine (Vol. 39). W.B. Saunders. https://doi.org/10.1016/j.tcam.2020.100432 Gosse, P. (1952). Dr. Viper: The Querulous Life of Philip Thicknesse. London: Cassell. 149 Gonzalez, P. A., Carpena, M., García Oliveira P, Mejuto, J. C., Prieto, M. A., & Simal, G. J. (2021). Main Applications of Cyclodextrins in the Food Industry as the Compounds of Choice to Form Host-Guest Complexes. Int J Mol Sci, 22(3), 1339. https://doi.org/10.3390/ijms22031339 Gray, D. A., Prestage, S., Linforth, R. S., & Taylor, A. M. (1999). Fresh tomato specific fluctuations in the composition of lipoxygenase-generated C6 aldehydes. Food Chemistry, 64, 149-155. https://doi.org/10.1016/S0308-8146(98)00163-0 Gravina, S. A., Yep, G. L., & Khan, M. (2013). Human biology of taste. Ann Saudi Med., 33(3), 217-22. https://doi.org/10.5144/0256-4947.2013.217 Greene, T. A., Alarcon, S., Thomas, A., Berdougo, E., Doranz, B. J., Breslin, P. A., & Rucker, J. B. (2011). Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin. PloS one, 6(5), e20123. https://doi.org/10.1371/journal.pone.0020123 Grimaldi, A., McLean, H., and Jiranek, V. (2020). Identification and partial characterization of glycosidic activities of commercial strains of the lactic acid bacterium, Oenococcus oeni. American Journal of Enology and Viticulture, 51(4), 362-369 https://doi.org/10.5344/ajev.2000.51.4.362 Grob, K., Grob, G., & Grob, K. (1978). Comprehensive, standardized quality test for glass capillary columns, Journal of Chromatography, 156(1), 1-20. https://doi.org/10.1016/S0021-9673(00)83120-9 GSC (2024). https://www.thegoodscentscompany.com/. (Assessed 8 February 2024). Gu, Z., Jiang, H., Zha, F., Manthey, F., Rao, J., & Chen, B. (2021). Toward a comprehensive understanding of ultracentrifugal milling on the physicochemical properties and aromatic profile of yellow pea flour. Food Chemistry, 345, 128760. https://doi.org/10.1016/j.foodchem.2020.128760. Guo, S., Qiu, S., Cai, Y., Wang, Z., Yang, Q., Tang, S., Xie, Y., & Zhang, A. (2023). Mass spectrometry-based metabolomics for discovering active ingredients and exploring action mechanism of herbal medicine. Front Chem. 11. https://doi.org/10.3389/fchem.2023.1142287 Hakim, E. H., Achmad, S. A., Juliawaty, L. D., Makmur, L., Syah, Y. M., Aimi, N., Kitajima, M., Takayama, H., & Ghisalberti, E. L. (2006) J. Nat. Med, 60, 161â18. https://doi.org/10.1007/s11418-006-0048-0 Hagerman, A. E. & Butler, L. G. (1978). The specificity of proanthocyanidins-protein interactions. Journal of Biological and Chemistry. 9, 4494-4497. https://doi.org/10.1016/S0021-9258(19)69462-7 Hagerman, A. (2012). Fifty Years of Polyphenol-Protein Complexes. Recent Advances in Polyphenol Research, 3(3), 71-97. https://doi.org/10.1002/9781118299753.ch3 150 Handa, S. S., Khanuja, S., Longo, G., & Rakesh, D. D. (2008). Extraction technologies for medicinal and aromatic plants. International Centre for Science and High Technology, 21â25. https://doi.org/10.1016/S0021-9258(19)69462-7 Harborne, J. B. (1994). The Flavonoids: Advances in research since 1986. London, U.K.: Chapman & Hall. Harris, D. C. & Lucy, C. A. (2019). Quantitative Chemical Analysis. 10th Edition. 22, 583 â 611. Hawkesworth, J. (1773). An Account of the Voyages Undertaken by the Order of His Present Majesty for Making Discoveries in the Southern Hemisphere. London: Strahan and Cadell. Haydersah, J., Chevallier, I., Rochette, I., Mouquet-Rivier, C., Picq, C., Marianne-Pépin, T., Icard-Vernière, C. & Guyot, J. P. (2012), Fermentation by Amylolytic Lactic Acid Bacteria and Consequences for Starch Digestibility of Plantain, Breadfruit, and Sweet Potato Flours. Journal of Food Science, 77, 466-472. https://doi.org/10.1111/j.1750-3841.2012.02811.x He, H. (2021). Ultimate Molecular Theory of Bitter Taste. Hecht, E. S., Scigelova, M., Eliuk, S., & Makarov, A. (2025). Fundamentals and advances of Orbitrap mass spectrometry. In R. A. Meyers (Ed.), Encyclopedia of analytical chemistry. https://doi.org/10.1002/9780470027318.a9309.pub2 Hendriks, M. M. W. B, van Eeuwijk F. A., & Jellema, R. H. (2011). Data-processing strategies for metabolomics studies. Trends Anal Chem, 30, 1685â1698. https://doi.org/10.1016/j.trac.2011.04.019 Heng, L. (2005). Flavour Aspects of Pea and its Protein Preparations in Relation to Novel Protein Foods. https://doi.org/10.18174/121674 Herrero, P., Cortés-Francisco, N., Borrull, F., Caixach, J., Pocurull, E., & Marcé, R. M. (2014). Comparison of triple quadrupole mass spectrometry and Orbitrap high-resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks. J Mass Spectrom. 49(7), 585 - 96. https://doi.org/10.1002/jms.3377 Heuckeroth, S., Damiani, T., Smirnov, A., Mokshyna, O., Brungs, C., Korf, A., Smith, J. D., Stincone, P., Dreolin, N., Nothias, L. F., Hyötyläinen, T., OreÅ¡iÄ, M., Karst, U., Dorrestein, P. C., Petras, D., Du, X., van der Hooft, J. J. J., Schmid, R., & Pluskal, T. (2024). Reproducible mass spectrometry data processing and compound annotation in MZmine 3. Nature protocols, 19(9), 2597â2641. https://doi.org/10.1038/s41596-024-00996-y Ho, C.S., Lam, C. W., Chan, M. H., Cheung, R. C., Law, L. K., Lit, L. C., Ng, K. F., Suen, M. W., & Tai, H. L. (2003). Electrospray ionisation mass spectrometry: principles and clinical applications. The Clinical biochemist. Reviews, 24(1), 3â12. 151 Ho, S., Thoo, Y. Y., Young, D. J., & Siow, L. F. (2019). Stability and recovery of cyclodextrin encapsulated catechin in various food matrices. Food Chemistry, 275, 594â599. https://doi.org/10.1016/j.foodchem.2018.09.117 Holm, E. S., Adamsen, A. P. S., Feilberg, A., Schäfer, A., Løkke, M. M., & Petersen, M. A. (2013). Quality changes during storage of cooked and sliced meat products measured with PTR-MS and HS-GCâMS. Meat Science, 95(2), 302 â 310. https://doi.org/10.1016/j.meatsci.2013.04.046 Hossain, A., Dave, D., & Shahidi, F. (2020). Northern Sea cucumber (Cucumaria frondosa): A potential candidate for functional food, nutraceutical, and pharmaceutical sector. Mar. Drugs, 18, 274. https://doi.org/10.3390/md18050274 Huang, C. A., & Zayas, J. F. (1991). Phenolic acid contribution to taste characteristics of corn germ protein flour products. Journal of Food Science, 56(5), 1308â1315. https://doi.org/10.1111/j.1365-2621.1991.tb04759.x Huang, S., Martinez, M., & Bohrer, B. (2019). The compositional and functional attributes of commercial flours from tropical fruits (breadfruit and banana). Foods, 8, 586. https://doi.org/10.3390/foods8110586 Hufnagel, J. C., & Hofmann, T. (2008). Quantitative reconstruction of the nonvolatile sensometabolome of a red wine. Journal of Agricultural and Food Chemistry, 56(19), 9190â9199. https://doi.org/10.1021/jf801742w Ijarotimi, S. O., & Aroge, F. (2005). Evaluation of The Nutritional Composition, Sensory and Physical Properties of a Potential Weaning Food from Locally Available Food Materials-Breadfruit(Artocarpus Altilis) and Soybean (Glycine Max). In Pol. J. Food Nutr. Sci., 14, 4. Inui, T., Koharu, O., Hiroo, M., Takahiro, H., & Shigenori, K. (2017). Effect of harvest time on some in vitro functional properties of hop polyphenols, Food Chemistry, 225. https://doi.org/10.1016/j.foodchem.2017.01.002 Ishii, S., Kurokawa, A., Kishi, M., Yamagami, K., Okada, S., Ishimaru, Y., & Misaka, T. (2012). The response of PKD1L3/PKD2L1 to acid stimuli is inhibited by capsaicin and its pungent analogs. FEBS Journal, 279(10), 1857â1870. https://doi.org/10.1111/j.1742-4658.2012.08566.x Ishimaru, M., Haraoka, M., Hatate, H., & Tanaka, R. (2016). Simultaneous analysis of purine and pyrimidine compounds associated with the freshness and taste of marine foods. Food Analytical Methods, 9(1), 1â9. https://doi.org/10.1007/s12161-015-0341-1 AC, (2007). Iwaoka, W., Hagi, Y., Umano, K., & Shibamotoss, T. (1994). Volatile Chemicals Identified in Fresh and Cooked Breadfruit. In J. Agric. Food Chem., 42. https://doi.org/10.1021/jf00040a026 152 Jarrett, F. M. (1959). Studies in Artocarpus and Allied Genera, Iii. A Revision of Artocarpus Subgenus Artocarpus. In Source: Journal of the Arnold Arboretum, 40, 2. https://doi.org/10.5962/p.324661 Jeevan, K. P. (2012). Tandem mass spectrometry: applications and principles. Biochemistry, genetics, and molecular biology. Rijeka: InTech. https://doi.org/10.5772/1424 Ji, Y., Li, B., Qiao, M., Li, J., Xu, H., Zhang, L., & Zhang, X. (2020). Advances on the in vivo and in vitro glycosylations of flavonoids. Appl. Microbiol. Biotechnol.,104, 6587â6600. https://doi.org/10.1007/s00253-020-10667-z Jiang, W., Li, C., Cheng, J., Wei, S., & Wang, Y. (2023). Analysis of Quality Changes in Mulberry Fruit at Three Developmental Stages Using Targeted Metabolomics Based on LCâMS. Tropical Plant Biology, 16, 287-306. https://doi.org/10.1007/s12042-023-09345-x Jolad, S. D., Lantz, R. C., Solyom, A. M., Chen, G. J., Bates, R. B., & Timmermann, B. N. (2004). Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production. Phytochemistry, 65(13), 1937â1954. https://doi.org/10.1016/j.phytochem.2004.06.008 Jones, W. T., & Mangan, J. L., (1977). Complexes of the condensed tannins of sainfoin (Onobrychis viciifolia scop.) with fraction 1 leaf protein and with submaxillary mucoprotein, and their reversal by polyethylene glycol and pH. J. Sci. Food Agric. 28, 126â136 https://doi.org/10.1002/jsfa.2740280202 Jones, A.M.P., Murch, S.J., & Ragone, D. (2010). Diversity of breadfruit (Artocarpus altilis, Moraceae) seasonality: A resource for year-round nutrition, Economic Botany, 64, 340â351. https://doi.org/10.1663/0013-0001(2010)64[340:DOBAAM]2.0.CO;2 Jones, A. M. P., Ragone, D., Aiona, K., Lane, W. A., & Murch, S. J. (2011a). Nutritional and morphological diversity of breadfruit (Artocarpus, Moraceae): Identification of elite cultivars for food security. Journal of Food Composition and Analysis, 24(8), 1091â1102. https://doi.org/10.1016/j.jfca.2011.06.004 Jones, A. M. P., Ragone, D., Tavana, N. G., Bernotas, D. W., & Murch, S. J. (2011b). Beyond the Bounty: Breadfruit (Artocarpus altilis) for food security and novel foods in the 21st century. Ethnobotany Research and Applications, 9, 129â149. https://doi.org/10.17348/era.9.0.129-149 Jones, A. M., Klun, J. A., Cantrell, C. L., Ragone, D., Chauhan, K. R., Brown, P. N., & Murch, S. J. (2012). Isolation and identification of mosquito (Aedes aegypti ) biting deterrent fatty acids from male inflorescences of breadfruit (Artocarpus altilis (Parkinson) Fosberg). Journal of agricultural and food chemistry, 60(15), 3867â3873. https://doi.org/10.1021/jf301205w 153 Jones, A. M. P., Murch, S.J., Wiseman, J., & Ragone, D. (2013) Morphological diversity in breadfruit (Artocarpus, Moraceae): Insights into domestication, conservation, and cultivar identification. Genetic Resources and Crop Evolution, 60. https://doi.org/10.1007/s10722-013-9936-4 Liem, D. G., & Russell, C. G. (2019). The Influence of Taste Liking on the Consumption of Nutrient Rich and Nutrient Poor Foods. Front Nutr., 15, 6,174. https://doi.org/10.3389/fnut.2019.00174 Kader, A. (2001). Breadfruit Fruit Product Facts.. Available online at: https://postharvest.ucdavis.edu/Commodity_Resources/Fact_Sheets/Datastores/Fruit_English/?uid=11&ds=798 (assessed May 11, 2024). Kader, A. A. (2008). Flavour quality of fruits and vegetables. In Journal of the Science of Food and Agriculture, 88,11, 1863â1868. https://doi.org/10.1002/jsfa.3278 Kaufmann, A. & Bromirski, M. (2018). Selecting the best Q-Exactive Orbitrap mass spectrometer scan mode for your application. Thermoscientific. White Paper 65147. Kaufmann, A., & Teale, P. (2016). Capabilities and limitations of high-resolution mass spectrometry (HRMS): Time-of-flight and Orbitrapâ¢. In J. F. Kay, J. D. MacNeil, & J. Wang (Eds.), Chemical analysis of non-antimicrobial veterinary drug residues in food (Chapter 3). https://doi.org/10.1002/9781118696781.ch3 Kayoko, S., Hisae, O., Michiyo, F., & Others. (1998). Intestinal absorption of luteolin and luteolin 7-O-β-glucoside in rats and humans. FEBS Letters, 438(3), 220â224. https://doi.org/10.1016/S0014-5793(98)01351-6 Kebarle, P., & Verkerk, U. H. (2009). Electrospray: From ions in solution to ions in the gas phase, what we know now. Mass Spectrometry Reviews, 28(6), 898â917. https://doi.org/10.1002/mas.20247. Kehinde, A. Z., Erland, L. A. E., Liu, Y., Ragone, D., Jones, A. M. P., & Murch, S. J. (2022). South Pacific cultivars of breadfruit (Artocarpus altilis (Parkinson) Fosberg and A. mariannensis Trécul) and their hybrids (A. altilis Ã A. mariannensis) have unique dietary starch, protein, and fiber. Journal of Food Composition and Analysis, 105. https://doi.org/10.1016/j.jfca.2022.104214 Kerler, J., Winkel, C., Davidek, D., & Blank, I. (2010). Basic chemistry and process conditions for reaction flavours with particular focus on Maillard-type reactions. In Food Flavour Technology, 2nd ed.; Taylor, A.J., Linforth, R.S.T., Eds.; Wiley-Blackwell: Oxford, UK; pp. 51â88. Kinney, A. J. (2006). Metabolic engineering in plants for human health and nutrition. Curr. Opin. Plant Biol. 17:130â138 https://doi.org/10.1007/s12088-010-0047-0 Kinsella, L. E. (1989). Flavour perception and binding to food components. Pages 376â403 in Flavour Chemistry of Lipid Foods. D. B. Min and T. H. Smouse, ed. AOCS, Champaign, IL. 154 Knee, M. (2001). Fruit quality and its biological basis (Sheffield Biological sciences). Sheffield, UK: Blackwell. Kola, O., Kaya, C., Duran, H. & Altan, A. (2010). Removal of limonin bitterness by treatment of ion exchange and adsorbent resins. Food Sci. Biotechnol. 19(2), 411â416. Kuligowski J, Moreno-Torres M, & Quintás G. (2024). Improving insights from metabolomic functional analysis combining multivariate tools. Anal Chim Acta., 22, 1323-343062. https://doi.org/10.1016/j.aca.2024.343062 Kunst, A., Draeger, B. & Ziegenhorn, J. (1988). D-Glucose. âMethods of Enzymatic Analysisâ (Bergmeyer, H. U., ed.), 3rd ed., Vol. VI, pp. 163-172, VCH Publishers (UK) Ltd., Cambridge, UK. Labouisse, J. (2016). Ethnobotany of breadfruit in Vanuatu: review and prospects. Ethnobiology Letters 7(1), 14â23. Lako, J., V.C. Trenerry, M. Wahlqvist, N. Wattanapenpaiboon, S. Sotheeswaran, R. Premier. 2007. âPhytochemical flavonols, carotenoids and the antioxidant properties of a wide selection of Fijian fruit, vegetables and other readily available foods. Food Chem, 101, 1727-41 https://doi.org/10.1016/j.foodchem.2006.01.031 Land, G. (1996). In Understanding Natural Flavours, J.R.Piggott,A.Paterson(eds). Chapman and Hall: London, 298. Lawless, H. T., & Corrigan, C. J. (1994). Semantics of Astringency. In: Kurihara, K., Suzuki, N., Ogawa, H. (eds) Olfaction and Taste XI. Springer, Tokyo. Lawless, H. T., Horne, J., & Gias, P. (1996). Astringency of organic acids is related to pH. Chem Senses. 21(4), 397-403. https://doi.org/10.1093/chemse/21.4.397 Lea, P. J., & Miflin, B. J. (2011). Nitrogen assimilation and its relevance to crop improvement. Annual Plant Reviews, Volume 42: Nitrogen Metabolism in Plants in the Post-genomic Era, 1â40. https://doi.org/10.1002/9781444328608.ch1 Leakey, C. L. A., (1977). Breadfruit Reconnaissance Study in the Caribbean Region. CIAT/ Interamerican Development Bank, Cali, Colombia. Leakey, C., & Roberts-Nkrumah L. B. (2016). The introduction of the breadfruit (Artocarpus altilis (Parkinson) Fosberg)) to the West Indies-the role of Sir Joseph Banks. Trop Agric (Trin), 93(1), 32â40. Lee, R. A., Balick, M. J., & Ling, D. L. (2001). Cultural dynamism and changeâAn example from the Federated states of Micronesia. Econ Bot., 55, 9â13. https://doi.org/10.1007/BF02864542 155 Lee, E.-J., Kim, H., Lee, J. Y., Ramachandraiah, K., & Hong, G.-P. (2020). β-Cyclodextrin-Mediated Beany Flavor Masking and Textural Modification of an Isolated Soy Protein-Based Yuba Film. Foods, 9(6), Article 818. https://doi.org/10.3390/foods9060818 Levai, L. D., Tang, E. N., & Monono, Y. E. (2024). The physicochemical and nutritional properties of biscuits from low temperature dried breadfruit (Artocarpus altilis (Parkinson) Fosberg)) flour. Food Measure. https://doi.org/10.1007/s11694-024-02505-x Li, S., Park, Y., Duraisingham, S., Strobel, F. H., Khan, N., Soltow, Q. A., & Pulendran, B. (2013). Predicting network activity from high throughput metabolomics. PLoS computational biology, 9(7), e1003123. https://doi.org/10.1371/journal.pcbi.1003123 Liang, Q., Wang, C., Li, B., & Zhang, A. H. (2015). Metabolic fingerprinting to understand therapeutic effects and mechanisms of silybin on acute liver damage in rats. Pharmacogn. Mag. 11 (43), 586â593. https://doi.org/10.4103/0973-1296.161264 Lincoln, N. K., Cho, A., Dow, G., & Radovich, T., (2019). Early Growth of Breadfruit in a variety a 488 Ã Environment Trial. Agron J., 111, 3020â3027. https://doi.org/10.2134/agronj2019.02.0068 Linde, G. A., Junior, A. L., de Faria, E. V., Colauto, N. B., de Moraes, F. F., & Zanin, G. M. (2009).Taste modification of L-amino acids and protein hydrolysate by α-cyclodextrin. Food Res. Int; 42, 814â818. https://doi.org/10.1016/j.foodres.2009.03.001 Linde, G. A., Laverde A., & Colauto, N. B. (2011). Changes to Taste Perception in the Food Industry: Use of Cyclodextrins. In: Preedy V. R., Watson R. R., Martin C. R., editors. Handbook of Behavior, Food and Nutrition. Springer; Berlin/Heidelberg, Germany. pp. 99â118 https://doi.org/10.1007/978-0-387-92271-3 Liu, S. Q. (2002). Malolactic fermentation in wine â beyond deacidification. J. Appl. Microbiol. 92 589â601. https://doi.org/10.1046/j.1365-2672.2002.01589.x Liu, Z., Ezernieks, V., Reddy, P., Elkins, A., Krill, C., Murphy, K., Rochfort, S., & Spangenberg, G.A. (2020). Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes. Metabolites. Jul 17;10(7):294. https://doi.org/10.3390/metabo10070294 Liu, Y., Brown, P. N., Ragone, D., Gibson, D. L., & Murch, S. J. (2020). Breadfruit flour is a healthy option for modern foods and food security. PLoS ONE, 15(7). https://doi.org/10.1371/journal.pone.0236300 Liu, S., Grierson, D., & Xi, W. (2024). Biosynthesis, distribution, nutritional and organoleptic properties of bitter compounds in fruit and vegetables. Critical Reviews in Food Science and Nutrition, 64(7), 1934â1953. https://doi.org/10.1007/s00726-015-1914-4 156 Lonvaud-Funel, A. (1999). Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie Van Leeuwenhoek, 76, 317â331. https://doi.org/10.1023/A:1002088931106 Lucas, M., & Ragone, D. (2012). Will breadfruit solve the world hunger crisis? New developments in an innovative food crop. ArcNews, Summer, 6â7. Ma, B., Tromp, J., & Li, M. (2002). PatternHunter: Faster and more sensitive homology search. Bioinformatics, 18(3), 440â445. https://doi.org/10.1093/bioinformatics/18.3.440 Mackay, D. (1974). âBanks, Bligh and breadfruit.â New Zealand Journal of History, 8(1), 61â77. MacMillan, H. G. (1946). Economic survey of Micronesia. Vol. 10: Agriculture and soils. Part 1. Agriculture general. Honolulu: U.S. Commercial Company. Maharaj, R, & Sankat C. (2004). Effect of shrink-wrapping and controlled atmosphere storage on the postharvest browning and quality of breadfruit. ASEAN Food J 13:29â40. Malo, D. (1951). Hawaiian Antiquities. N. Emerson (trans.). Honolulu, Bishop Museum Press Mall, V., Sellami, I., & Schieberle, P. (2018). New Degradation Pathways of the Key Aroma Compound 1-Penten-3-one during Storage of Not-from-Concentrate Orange Juice. Journal of Agricultural and Food Chemistry, 66(42), 11083â11091. . https://doi.org/10.1021/acs.jafc.8b04334 Matthews, .A, Grbin, P. R., Jiranek, V. (2007). Biochemical characterization of the esterase activities of wine lactic acid bacteria. Applied Microbiology and Biotechnology, 77, 329-337. https://doi.org/10.1007/s00253-007-1162-5 Maximize Market Research. (2024). Starch market: Global industry analysis and forecast (2024â2032). Retrieved April 30, 2025, from https://www.maximizemarketresearch.com/market-report/global-starch-market/112948/ Mayaki, O., Akingbala, J., Baccus-Taylor, G., & Thomas, S. (2003). Evaluation of breadfruit (Artocarpus communis) in traditional stiff porridge foods. Journal of Food Agriculture & Environment, 1, 54â59. McAlister, G. C, Phanstiel, D., Good, D. M, & Berggren, W. T., Coon, J.J. (2007). Implementation of electron-transfer dissociation on a hybrid linear ion trapâOrbitrap mass spectrometer. Anal. Chem. 79, 3525â34 https://doi.org/10.1021/ac070020k McMillin, K. W., Bidner, T. D., Felschle, S .E., Dugas, S. M. & Koh, K .C. (1991). Flavour and oxidative stability of ground beef patties as affected by source and storage. J. Food Sci. 56, 899-902. https://doi.org/10.1111/j.1365-2621.1991.tb14601.x 157 Medhe, S. (2018). Ionization techniques in mass spectrometry: A review. Mass Spectrometry & Purification Techniques, 4, Article 126. https://doi.org/10.4172/2469-9861.1000126 Mennella, J. A., Reed, D. R.., Mathew, P. S., Roberts, K. M., & Mansfield, C. J. (2015). A Spoonful of Sugar Helps the Medicine Go down: Bitter Masking by Sucrose among Children and Adults. Chem. Senses, 40(1), 17â25. https://doi.org/10.1093/chemse/bju053 Miller, P. E., & Denton, M. B. (1986). The quadrupole mass filter: basic operating concepts. J. Chem. Educ., 63, 617 â 622. https://doi.org/10.1021/ed063p617 Mishra, S., Sarkar, U., Taraphder, S., Datta, S., Swain, D., Saikhom, R., Panda, S., & Laishram, M. (2017). Principal component analysis. International Journal of Livestock Research, 1 https://doi.org/10.5455/ijlr.20170415115235 Mishra, P., Singh, U., Pandey, C. M., Mishra, P., & Pandey, G. (2019). Application of Studentâs t-test, analysis of variance, and covariance. Annals of Cardiac Anaesthesia, 22(4), 407â411. https://doi.org/10.4103/aca.ACA_94_19 Miyashita, T. & Etoh, H. (2013). Improvement of the bitterness and astringency of green tea by sub-critical water extraction. Food Sci. Technol. Res. 19, 471â478 https://doi.org/10.3136/fstr.19.471 Morris, H. R., Paxton, T., Dell, A., Langhorne, J., & Berg, M. (1996). High sensitivity collisionally activated decomposition tandem mass spectrometry on a novel quadrupole/orthogonal-acceleration time-of-flight mass spectrometer. Rapid Communications in Mass Spectrometry, 10(8), 889â896. https://doi.org/10.1002/(SICI)1097-0231(19960610)10:8 Morrissey, M. (2017). Breadfruit Stories: A tree's journey from Tahiti to the West Indies, 1760s to 1840. Trees That Feed Foundation. Morton, J. F. (1987). Breadfruit. In J. F. Morton (Ed.), Fruits of warm climates (pp. 50â58). Miami, USA: Morton. Moustache, A. M., & Moustache, M. (2007). History and use of breadfruit in the Seychelles. In Proceedings of the 1st International Symposium on Breadfruit Research and Development. Acta Horticulturae, 757, 135â140. https://doi.org/10.17660/ActaHortic.2007.757.18 Moco, S., Bino, R., De, V., Ric & Vervoort, J. J. M. (2007). Metabolomics technologies and metabolite identification. Trends in Analytical Chemistry, 26. 855-866. https://doi.org/10.1016/j.trac.2007.08.003 Monti, L. L., Bustamante, C. A., Osorio, S., Gabilondo, J., Borsani, J., Lauxmann, M. A., Maulión, E., Valentini, G., Budde, C. O., Fernie, A. R., Lara, M. V., & Drincovich, M. F. (2016). Metabolic profling of a range of peach fruit varieties reveals high metabolic diversity and 158 commonalities and differences during ripening. Food Chem., 190, 879â888. https://doi.org/10.1016/j.foodchem.2015.06.043 Mudge, E., Brown, P., & Murch, S. (2019). The Terroir of Cannabis: Terpene Metabolomics as a Tool to Understand Cannabis sativa Selections. Planta Medica, 85. https://doi.org/10.1055/a-0915-2550 Munin, A., & Edwards-Lévy, F. (2011). Encapsulation of natural polyphenolic compounds: A review. Pharmaceutics, 3(4), 793-829 https://doi.org/10.3390/pharmaceutics3040793 Murray, K. K., Boyd, R. K., Eberlin, M. N., Langley, G. J., Li, L., & Naito, Y. (2013). Definitions of terms relating to mass spectrometry (IUPAC Recommendations, 2013). Pure and Applied Chemistry, 85(7), 1515 - 1609. https://doi.org/10.1351/PAC-REC-06-04-06 Murray, N. J., Williamson, M. P., Lilley, T. H., & Haslam, E. (1994). Study of the interaction between salivary proline-rich proteins and a polyphenol by 1H NMR spectroscopy. Eur. J. Biochem., 219, 923-935 https://doi.org/10.1111/j.1432-1033.1994.tb18574.x Murch, S. J., Rupasinghe, H. P. V., Goodenowe, D., & Saxena, P. K. (2004). A metabolomic analysis of medicinal diversity in Huang-qin (Scutellaria baicalensis Georgi) genotypes: Discovery of novel compounds. Plant Cell Reports, 23(6), 419â425. https://doi.org/10.1007/s00299-004-0862-3 Murch, S. J., Ragone, D., Shi, W. L., Alan, A. R., & Saxena, P. K. (2007). In vitro conservation and micropropagation of breadfruit (Artocarpus altilis, Moraceae). In S. M. Jain & H. Häggman (Eds.), Protocols for micropropagation of woody trees and fruit (pp. 279â288). Springer. https://doi.org/10.1007/978-1-4020-6352-7_26 Murch, S. J., Ragone, D., Shi, W. L., Alan, A. R., & Saxena, P. K., (2008). In vitro conservation and sustained production of breadfruit (Artocarpus altilis, Moraceae): modern technologies for a traditional tropical crop. Sci. Nat. 95, 99â107. https://doi.org/10.1007/s00114-007-0295-2 Naczk, M., Oickle, D., Pink, D. & Shahidi, F. (1996). Protein Precipitating Capacity of Crude Canola Tannins:â Effect of pH, Tannin, and Protein Concentrations, Journal of Agricultural and Food Chemistry, 44(8), 2144-2148. https://doi.org/10.1021/jf960165k Narasimham, P. & John, P. J. (1995). Controlled low temperature vacuum dehydration as an alternative to freeze-drying of diced breadfruit. Journal of Food Science and Technology, India, 32(4), 305-309. Navarro, M., Malres, S., Labouisse, J. P., & Roupsard, O. (2007). Vanuatu breadfruit project: survey on botanical diversity and traditional uses of Artocarpus altilis. Acta Hortic., 757, 81â88. https://doi.org/10.17660/ActaHortic.2007.757.9 159 NBS, (2021). Nigeriaâs foreign trade data 2021. https://nairametrics.com/2022/03/21/nigerias-wheat-imports-surge-by-71-to-n1-29-trillion-in-2021/ (assessed 1 August 2022) Nelson-Quartey, F. C., Amagloh, F. K., Oduro, I. N. & Ellis, W. O. (2007). Formulation Of an Infant Food Based on Breadfruit (Artocarpus Altilis) And Breadnut (Artocarpus Camansi) . Acta Hortic., 757, 215-224 https://doi.org/10.17660/ActaHortic.2007.757.25 Niessen, W. M. A. & Falck, D. (2015). Introduction to Mass Spectrometry, a Tutorial. Wiley-VCH Verlag GmbH & Co. KGaA. Nguyen, T. M., Shafi, A., Nguyen, T., & Draghici, S. (2019). Identifying significantly impacted pathways: A comprehensive review and assessment. Genome Biology, 20(1), 203. https://doi.org/10.1186/s13059-019-1790-4 Nguyen, Q. D., La, Q. D., Nguyen, N. N., & Nguyen, T. N. L. (2023). Green removal of unpleasant volatiles from soapberry (Sapindus mukorossi) extracts by two-phase microbial fermentation fortified with pomelo peel waste. RSC Advances, 13, 13282â13291. https://doi.org/10.1039/D3RA01321G Nochera, C., & Caldwell, M. (1992). Nutritional Evaluation of Breadfruit-containing Composite Flour Products https://doi.org/10.1111/j.1365-2621.1992.tb06878.x Noestheden, M., Dennis, E. G., & Zandberg, W. F. (2017). Quantitating Volatile Phenols in Cabernet Franc Berries and Wine after On-Vine Exposure to Smoke from a Simulated Forest Fire. Journal of Agricultural and Food Chemistry, 66 (3), 695-703. https://doi.org/10.1021/acs.jafc.7b05426 Nomura, T., Y. Hano & M. Aida. (1998). Isoprenoid-substituted flavonoids from Artocarpus plants (Moraceae). Het-erocycles, 47(2),1179-1205. https://doi.org/10.3987/COM-97-S(N)112 Nochera, C. L., & Ragone, D. (2016). Preparation of a Breadfruit Flour Bar. Foods, 5,37. https://doi.org/10.3390/foods5020037 Nochera, C. L., & Ragone, D. (2019). Development of a breadfruit flour pasta product. Foods, 8(3), 110. https://doi.org/10.3390/foods8030110 Oke, E. K., Tijani O. A., Abiola, O. T., Adeoye, A. K, & Odumosu B. O. (2017). Effects of Partial Substitution of Wheat Flour with Breadfruit Flour on Quality Attributes of Fried Doughnut. The Journal of Agricultural Sciences, 13(1), 72 â 80. https://doi.org/10.4038/jas.v13i1.8274 Oladunjoye, I. O., Ologhobo, A. D. & Olaniyi, C. O. (2010). Nutrient composition, energy value and residual antinutritional factors in differently processed breadfruit (Artocarpus altilis) meal. African Journal of Biotechnology, 9, 4259-4263. https://doi.org/10.5897/AJB10.1158 Olapade, A. A & Umeonuorah, U. C. (2014 ). Niger. Food J. 32, 80 160 Olatunji, O., & Akinrele, I. A. (1978). Comparative Rheological Properties and Bread Qualities of Wheat Flour Diluted with Tropical Tuber and Breadfruit Flours. Cereal Chemistry, 55(1), 1â6. Olaoye, O., Onilude, A., & Oladoye, C. (2007). Breadfruit flour in biscuit making: Effects on product quality. African Journal of Food Science, 1, 20â23. https://doi.org/10.4314/ajfs.v1i1.43572 Olaoye, O. A. & Onilude, A. A. (2008). Microbiological, proximate analysis and sensory evaluation of baked products from blends of wheat-breadfruit flours. African Journal of Food, Agriculture, Nutrition and Development, 8(2), 192â203. https://doi.org/10.4314/ajfand.v8i2.19182 Omobuwajo, T. O. (2003). Compositional characteristics and sensory quality of biscuits, prawn crackers and fried chips produced from breadfruit. Innovative Food Science and Emerging Technologies, 4(2), 219â225. https://doi.org/10.1016/S1466-8564(03)00006-7 OâNeil, T. E. (1996). In Flavour-food interactions. In R. J. McGorrin & J. V. Leland (Eds.), Flavour-food interactions (p. 59). American Chemical Society. Outlaw, W. H., & Mitchell, C. T. (1988). Sucrose. In H. U. Bergmeyer (Ed.), Methods of enzymatic analysis (3rd ed., Vol. VI, pp. 96â103). VCH Publishers (UK) Ltd. Pan Q., Zhuo X., He C., Zhang Y., & Shi Q. (2020). Validation and evaluation of high-resolution orbitrap mass spectrometry on molecular characterization of dissolved organic matter. ACS Omega, 5(10), 5372â5379. https://doi.org/10.1021/acsomega.9b04411 Pang, Z., Lu, Y., Zhou, G., Hui, F., Xu, L., Viau, C., Spigelman, A. F., MacDonald, P. E., Wishart, D. S., Li, S., & Xia, J. (2024). MetaboAnalyst 6.0: Towards a unified platform for metabolomics data processing, analysis and interpretation. Nucleic Acids Research, 52(W1), W398âW406. https://doi.org/10.1093/nar/gkae253. Pangborn, R. M. (1963). Relative taste intensities of selected sugars and organic acids. J. Food Sci., 28, 726â733. https://doi.org/10.1111/j.1365-2621.1963.tb03321.x Parkinson, S. (1773). A Journal of a Voyage to the South Seas in His Majestyâs Ship the Endeavour. London: W. Bowyer. Parthasarathy, V. A., Chempakam, B. & Zachariah, T. J. (2008). Chemistry of spices. CABI ISBN 978-1-84593-405-7 UK Peleg, H., Gacon, K., Schlich, P., & Noble, A. C. (1999). Bitterness and astringency of flavan-3-ol monomers, dimers, and trimers. Journal of the Science of Food and Agriculture, 79(8), 1123â1128. https://doi.org/10.1002/(SICI)1097-0010(199906)79:8. 161 Peralbo-Molina, Ã., Solà -Santos, P., Perera-Lluna, A., & Chicano-Gálvez, E. (2023). Data processing and analysis in mass spectrometry-based metabolomics. In Methods in Molecular Biology (Vol. 2571, pp. 207â239). Springer. https://doi.org/10.1007/978-1-0716. Picart-Armada, S., Ferna´ndez-Albert, F., & Vinaixa, M. (2017). Null diffusion-based enrichment for metabolomics data. PLoS One 12: e0189012. https://doi.org/10.1371/journal.pone.0189012 Pierpoint, W. S. (1969). O-quinones are formed in plant extracts. Their reaction to bovine serum albumin. Biochem. J. 112-619. https://doi.org/10.1042/bj1120619 Pluskal, T., Castillo, S., Villar-Briones, A., & Oresic, M. (2010). MZmine 2: A modular framework for processing, visualizing, and analyzing mass spectrometry-based molecular profile data. BMC Bioinformatics, 11, 395. https://doi.org/10.1186/1471-2105-11-395 Preininger, M. (2006). Interactions of flavour components in foods. In A. G. Gaonkar and A. McPherson (Eds), Ingredient Interactions: Effects on Food Quality (pp. 478-528). Boca Raton: CRC Press. Przeor, M., Flaczyk, E., Kmiecik, D., Buchowski, M. S., Staniek, H., TomczakGraczyk, A., Kobus-Cisowska J., Gramza-MichaÅowska A., & Foksowicz-Flaczyk, J. (2020). Functional properties and antioxidant activity of morus alba L. leaves var. Zolwinska Wielkolistna (WML-P)âThe effect of controlled conditioning process. Antioxidants (Basel) 9, 668. https://doi.org/10.3390/antiox9080668 Powell, D. (1977). The Voyage of the Plant Nursery, H.M.S. Providence, 1791-17931. Eco Bot, 31, 387â431. https://doi.org/10.1007/BF02912555 Prakash, V. (1990). Leafy spices. Florida: CRC Press, pp. 35â42. Privalov, P. L. (1979). Adv. Protein Chem. 33, 167 - 241. Progenesis QI for proteomics-Whatâs new in the latest release? (2017) https://doi.org/10.1016/S0065-3233(08)60460-X PubChem, (2024). https://pubchem.ncbi.nlm.nih.gov/ (Assessed 8 February 2024). Purewal, S. S., & Sandhu, K. S. (2021). Debittering of citrus juice by different processing methods: A novel approach for food industry and agro-industrial sector. Sci. Hort, 276, 109750. https://doi.org/10.1016/j.scienta.2020.109750 Rafiei, A., & Sleno, L. (2014). Comparison of peak picking workflows for untargeted liquid chromatography/high-resolution mass spectrometry metabolomics data analysis. Rapid Commun Mass Spectrom, 29, 119â127. https://doi.org/10.1002/rcm.6801 162 Raiola, A., Rigano, M. M., Calafiore, R., Frusciante, L., & Barone, A. (2014). Enhancing the health-promoting effects of tomato fruit for biofortified food. Mediators of Inflammation, 2014, 139873. Ragone, D. (1991). Ethnobotany of breadfruit in Polynesia. In Cox P. A. and S. A. Banack [eds.], Islands, Plants, and Polynesians: An Introduction to Polynesian Ethnobotany, 203-220. Dioscorides Press, Portland, USA. Ragone, D. (1995). Description of Pacific Island breadfruit cultivars. Acta Horticulturae 413: 93-98. Ragone, D. (1997). Complementary conservation and use of plant genetic resources View project Building human and institutional capacity to conserve and utilize agricultural diversity more efficiently View project Diane Ragone National Tropical Botanical Garden. Ragone, D. (2001). Chromosome Numbers and Pollen Stainability of Three Species of Pacific Island Breadfruit (Artocarpus, Moraceae). American Journal of Botany, 88, 693â696. https://doi.org/10.2307/2657070 Ragone, D. (2002). Breadfruit storage and preparation in the Pacific Islands. In S. Yoshida & P. J. Matthews (Eds.), Vegeculture in Eastern Asia and Oceania (JCAS Symposium Series 16, pp. The Japan Center for Area Studies. Ragone D., G. Tavana, J. Stevens, P. Stewart, R. Stone, Cox, P. & Cox, P. (2004). Nomenclature of breadfruit cultivars in Samoa: Saliency, ambiguity, and monomiality. Journal of Ethnobiology 24, 33-50. https://doi.org/10.2993/0278-0771(2004)24%5B33:NOBCIS%5D2.0.CO;2 Ragone, D. (2006). Artocarpus altilis (breadfruit). Pp. 85-100 in Traditional Trees of Pacific Islands: Their culture environment and use. Edited by C.R. Elevitch. Permanent Agriculture Resources, Holualoa, Hawaii. Ragone, C. G., & Cavaletto, C. G. (2006). Sensory Evaluation of Fruit Quality and Nutritional Composition of 20 Breadfruit (Artocarpus, Moraceae) Cultivars. https://doi.org/10.1663/0013-0001(2006)60[335:SEOFQA]2.0.CO;2 Ragone, D. (2007). Breadfruit: Diversity, conservation, and potential. Acta Horticulturae, 757, 19-30. https://doi.org/10.17660/ActaHortic.2007.757.1 Ragone, D. (2011). Farm and forestry production and marketing profile for breadfruit (Artocarpus altilis). In: Elevitch CR editor. Specialty Crops for Pacific Island Agroforestry. Holualoa: Permanent Agriculture Resources (PAR). 163 Ragone, Diane & Wiseman, Jim. (2007). Developing and applying descriptors for breadfruit germplasm. Acta horticulturae, 757, 161-167. https://doi.org/10.17660/ActaHortic.2007.757.22 Ragone, D., & B. Raynor. (2009). Breadfruit and its traditional cultivation and use on Pohnpei. In Ethnobotany of Pohnpei: Plants People and Island Culture (M. J. Balick and collaborators, eds.):63â88. Honolulu: University of Hawaiâi Press. Rahmah, W., & Waluyo, B. (2019). Genetic distance and diversity of breadfruit (Artocarpus altilis) based on morphological characters in Bawean Island, Indonesia. Biodiversitas Journal of Biological Diversity. https://doi.org/10.13057/biodiv/d201122 Ramdath, D. D., Isaacs, C. L. R., Teelucksingh, S., & Wolever, T. M. S. (2004). Glycemic index of selected staples commonly eaten in the Caribbean and the effects of boiling v. crushing. British Journal of Nutrition, 91(6), 971â977. https://doi.org/10.1079/BJN20041102 Raynor, W.C., Fownes, J.H. (1991). Indigenous agroforestry of Pohnpei. Agroforest Syst 16, 139â157. https://doi.org/10.1007/BF00129745 Reineccius, G. (2006). Flavour Chemistry & Technology. Boca Raton: CRC Press Ricci, A., Parpinello, G., Schwertner, A. P., Teslic, N., Brilli, C., Pizzi, A., & Versari, A. (2017). Quality assessment of food grade plant extracts using MALDI-TOF-MS, ICP-MS and spectrophotometric methods. Journal of Food Composition and Analysis, 59, 95-104. https://doi.org/10.1016/j.jfca.2017.01.014 Rizzi, G. P. (2006). Formation of strecker aldehydes from polyphenol-derived quinones and α-L-amino acids in a nonenzymic model system. J. Agric. Food Chem., 54, 1893â1897 https://doi.org/10.1021/jf052781z Rizzo, P. V., Del Toro-Gipson, R. S., Cadwallader, D. C. & Drake, M. A. (2022). Identification of aroma-active compounds in Cheddar cheese imparted by wood smoke, Journal of Dairy Science, 105, 7. https://doi.org/10.3168/jds.2021-21697 Roberts, K., Badrie, N. & Roberts-Nkrumah, L. B. (2007). Colour and sensory characteristics of fried chips from three breadfruit (Artocarpus altilis) cultivars. Acta Hort. 757, 225-231. https://doi.org/10.17660/ActaHortic.2007.757.30 Roberts, L. D., Souza, A. L., Gerszten, R. E., & Clish, C. B. (2012). Targeted Metabolomics. Curr. Protoc. Mol. Biol., 98 (30), 1â24. https://doi.org/10.1002/0471142727.mb3002s98 Roberts-Nkrumah, L. B. (1997). Towards a description of the breadfruit germplasm in St Vincent. Fruits, 52(1), 27â35. https://doi.org/10.17660/ActaHortic.2007.757.30 Roberts-Nkrumah, L. B. & Badrie, N. (2005). Breadfruit consumption, cooking methods and cultivar preference among consumers in Trinidad, West Indies. Food Quality and Preference, 16(3), 267â274. 164 Roberts-Nkrumah, Laura B. (2008). Breadfruit Germplasm Collection and Evaluation at the University of the West Indies." Overcoming Challenges to Develop Sustainable Agri-Food Systems in the Tropics Port of Spain, Trinidad and Tobago, November 30 - December 5, 2008. https://doi.org/10.37234/HJPVNOBX Roberts-Nkrumah, (2015). Breadfruit and breadnut Orchard Establishment and Management â A Manual for Commercial Production. Rome: Food and Agriculture Organization of the United Nations. Robinson, T. L. (2007). Recent advances and future directions in orchard planting systems. Acta Horticulturea, 732, 367-381. https://doi.org/10.17660/ActaHortic.2007.732.57 Robinson, P. K. (2015). Enzymes: Principles and Biotechnological Applications. Essays in Biochemistry, 59, 1â41. https://doi.org/10.1042/bse0590001 Rodríguez, L. G. R., Gasga, V. M. Z., Pescuma, M., Van Nieuwenhove, C., Mozzi, F., & Burgos, J. A. S. (2021). Fruits and fruit by-products as sources of bioactive compounds: Benefits and trends of lactic acid fermentation in the development of novel fruit-based functional beverages. Food Research International, 140, 109854. https://doi.org/10.1016/j.foodres.2020.109854 Rubino, F. M. (2020). Center-of-Mass iso-energetic collision-induced decomposition in tandem triple quadrupole mass spectrometry. Mol. (Basel, Switz. 25 (9), 2250. https://doi.org/10.3390/molecules25092250 Ruiz-Perez, D.; Guan, H.; Madhivanan, P.; Mathee, K.; Narasimhan, G. (2020). So, You Think You Can PLS-DA?, preprint, https://doi.org/10.1101/207225. Sahu, S. K., Liu, M., Yssel, A., Kariba, R., Muthemba, S., Jiang, S., Song, B., Hendre, P.S, Muchugi, A, Jamnadass, R, Kao S,M, Featherston J, Zerega, N.J.C., Xu, X., Yang, H., Deynze, A.V., Peer, Y.V., Liu, X., & Liu, H., (2019). Draft Genomes of Two Artocarpus Plants, Jackfruit (A. heterophyllus) and Breadfruit (A. altilis). Genes (Basel). 11(1), 27. https://doi.org/10.3390/genes11010027 Sahu, P. K., Sao, R., Mondal, S., Vishwakarma, G., Gupta, S. K., Kumar, V., ... & Das, B. K. (2020). Next generation sequencing based forward genetic approaches for identification and mapping of causal mutations in crop plants: A comprehensive review. Plants, 9(10), 1355. https://doi.org/10.3390/plants9101355 Å amec, D., Karalija, E., Å ola I., Bok V. V., & Salopek-Sondi B. (2021). The role of polyphenols in abiotic stress response: The influence of molecular structure. Plants;10:118 https://doi.org/10.3390/plants10010118 Santello, V. (2021). Volcano Plot Explanation. https://doi.org/10.13140/RG.2.2.16945.25446 Sanz, C., Olias, & Perez, A. (1997). Aroma biochemistry of fruits and vegetables. In: Phyto-chemistry of Fruit and Vegetables. New York, NY, USA: Oxford University Press Inc.; 1997. pp. 125-155. ISBN: 0198577907 165 Sari, D., & Lestari, R. (2017). The production of breadfruit flour: effect of heater temperature to the drying rate and time of the breadfruit. Journal Bahan Alam Terraranan, 6, 20â4. https://doi.org/10.15294/jbat.v6i1.7168 Sarni-Manchado, P., Cheynier, V., & Moutoune, M. (1999). Interactions of grape seed tannins with salivary proteins. J Agric Food Chem. Jan;47(1):42-7. https://doi.org/10.1021/jf9805146 Sauerborn, J. (2002). Site Productivity, the Key to Crop Productivity. Journal of Agronomy and Crop Science, 188, 363â367. https://doi.org/10.1046/j.1439-037X.2002.00556.x Schindler, S., Zelena, K., Krings, U., Bez, J., Eisner, P., & Berger, R. G. (2012). Improvement of the aroma of pea (Pisum sativum) protein extracts by lactic acid fermentation. Food Biotechnology, 26(1), 58â74. https://doi.org/10.1080/08905436.2011.645939 Schiffman, S. S., & Dackis, C. (1975). Taste of nutrients: L-amino acids, vitamins, and fatty acids. Percept. Psychophys. 17, 140â146. https://doi.org/10.3758/BF03203878 Schmid, R., Heuckeroth, S., Korf, A., Smirnov, A., Myers, O., Dyrlund, T. S., Bushuiev, R., Murray, K. J., Hoffmann, N., Lu, M., Sarvepalli, A., Zhang, Z., Fleischauer, M., Dührkop, K., Wesner, M., Hoogstra, S. J., Rudt, E., Mokshyna, O., Brungs, C., Ponomarov, K., & Pluskal, T. (2023). Integrative analysis of multimodal mass spectrometry data in MZmine 3. Nature biotechnology, 41(4), 447â449. https://doi.org/10.1080/08905436.2011.645939 Schlebecker, J. T. (1987). âFarming at the time of the constitutionâ. In Our American Land: 1987 Yearbook of Agriculture, ed. by William Whyte, pp. 26â30. Washington, DC: United States Department of Agriculture. Schwaiger-Haber, M., Stancliffe, E., Arends, V., Thayagaranjan, B., Sindelar, M. & Patti, G. J. (2021). Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer, ACS Meas. Sci. Au 2021, 1, 1, 35 â 45. https://doi.org/10.1021/acsmeasuresciau.1c00007 Sentandreu, E., Peris-Díaz, M., Sweeney, S., Chiou, J., Muñoz, N., & Tiziani, S. (2018). A survey of Orbitrap all ion fragmentation analysis assessed by an R metabolist package to study small-molecule metabolites. bioRxiv. https://doi.org/10.1101/257147 Shahidi, F., & Abad, A. (2019). Lipid-derived flavours and off-flavours in food. Food Science & Nutrition, 182-192. https://doi.org/10.1002/fsn3.1294 Shannon, P., Markiel, A., Ozier, O., Baliga, N. S, Wang, J. T, Ramage, D, Amin, N., Schwikowski, B., & Ideker, T. (2019). Cystoscape: a software environment for integrated models of biomolecular interaction networks. Shahidi, Fereidoon, and Abrehem Abad. "Lipid-derived flavours and off-flavours in food: 182-192. Genome Research, 13(11), 2498-504. https://doi.org/10.1101/gr.1239303 Shi, W.L., P.K. Saxena, S.J. Murch & D. Ragone. 2007. Mass-propagation and bioreactor-based technologies for germplasm conservation, evaluation and international distribution of 166 breadfruit. In Proceedings of the 1st International Symposium on Breadfruit Research and Development. Acta Horticulturae, 757, 169-176. https://doi.org/10.17660/ActaHortic.2007.757.23 Shi, Y., Singh, A., Kitts, D. D., & Pratap-Singh, A. (2021). Lactic acid fermentation: A novel approach to eliminate unpleasant aroma in pea protein isolates. LWT, 150, 111927. https://doi.org/10.1016/j.lwt.2021.111927 Siebert, K. J. (1999). Protein-polyphenol hazes in beverages. Food Technology, 53, 54-57 Singh, D. P., Zerihun, A., Kelly, D., Cain, N. M., Nankervis, P., & Downey, M. O. (2012). GC-MS-based analytical method for detection of smoke taint-associated phenols in smoke-affected wines. Current Bioactive Compounds, 8(3), 190â199. https://doi.org/10.2174/157340712802762483 Smith, N. J. H., J.T. Williams, D. L. Plucknett, & Talbot, T. P. (1992). Tropical forests and their crops. Comstock Publishing Associates, Ithaca, New York Smith, C. A., Want, E. J., O'Maille, G., Abagyan, R., & Siuzdak, G. (2006). XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Analytical chemistry, 78(3), 779â787. https://doi.org/10.1021/ac051437y Souleyre, E. J. F., Chagné, D., Chen, X., Tomes, S., Turner, R. M., Wang, M. Y., Maddumage, R., Hunt, M. B., Winz, R. A., Wiedow, C., Hamiaux, C., Gardiner, S. E., Rowan, D. D., & Atkinson, R. G. (2014). The AAT1 locus is critical for the biosynthesis of esters contributing to âripe appleâ flavour in âRoyal Galaâ and âGranny Smithâ apples. Plant Journal, 78(6), 903â915. https://doi.org/10.1111/tpj.12518 Souza. D. S., Souza. J. D. R. P., Coutinho. J. P., Ferrão. S. P. B., Souza. T. S., & Silva, A. A. L. (2012). Development of instant flour from breadfruit pulp (Artocarpus altilis). Ciência Rural. 42(6). https://doi.org/10.1590/S0103-847820120006000XX Spencer, C. M., Cai, Y., Martin, R., Gaffney, S. H., Goulding, P.N., Magnolato, D., Lilley, T. H.; Haslam, E. (1988). Polyphenol complexations some thoughts and observations. Phytochemistry, 27, 2397-2409 https://doi.org/10.1016/0031-9422(88)87002-9 Statista, (2022). Wheat consumption worldwide in 2021/2022, by country. (assessed 1 August 2022) https://www.statista.com/statistics/1094056/total-global-rice-consumption/ Sumner, L. W., Amberg, A., & Barrett, D. (2007). Proposed minimum reporting standards for chemical analysis. Metabolomics, 3, 211â221. https://doi.org/10.1007/s11306-007-0082-2 Suppavorasatit, I., & Cadwallader, K. R. (2012). J. Agric. Food Chem. 2012, 60, 7817. https://doi.org/10.1021/jf3020006 167 Sweeney, M. T., Thomson, M. J., Cho, Y. G., Park, Y. J., Williamson, S. H., Bustamante, C. D., McCouch, S. R. (2007). Global dissemination of a single mutation conferring white pericarp in rice. PLoS Genet 3:e133 https://doi.org/10.1371/journal.pgen.0030133 Swiegers, J. H., Bartowsky E. J., Henschke P. A., & Pretorius I. S. (2005). Yeast and bacterial modulation of wine aroma and flavour. Aust. J. Grape Wine Res. 11 139â173. https://doi.org/10.1111/j.1755-0238.2005.tb00285.x Szejtli, J. & Szente, L. (2005). Elimination of bitter, disgusting tastes of drugs and foods by cyclodextrins. European Journal of Pharmaceutics and Biopharmaceutics, 61(3), 115-125. https://doi.org/10.1016/j.ejpb.2005.05.006 SzymaÅska, E., Saccenti, E., Smilde, A. K., & Westerhuis, J. A. (2012). Double-check: Validation of diagnostic statistics for PLS-DA models in metabolomics studies. Metabolomics, 8(Suppl 1), 3â16. https://doi.org/10.1007/s11306-011-0330-3 Takahashi, N., Phinney, B. O., & MacMillan, J. (1991). Gibberellins. Springer-Verlag. https://link.springer.com/book/10.1007/978-3-642-75811-5 Tang, J., Li, X., Zhang, Y., Yang, Y., Sun, R., Li, Y., Gao, J., & Han, Y. (2022). Differential flavonoids and carotenoids profiles in grains of six Poaceae crops. Foods (Basel, Switzerland), 11(14), 2068. https://doi.org/10.3390/foods11142068 Thompson, A. K., Been, B. O., & Perkins, C. (1974). Storage of fresh breadfruit. Tropical Agriculture (Trinidad), 51(3), 407-415 Trumbo, P., Appleton, K., de Graaf, K., Hayes, J., Baer, D., Beauchamp, G., Dwyer, J., Fernstrom, J., Klurfeld, D., Mattes, R., & Wise, P. (2020). Perspective: Measuring Sweetness in Foods, Beverages, and Diets: Toward Understanding the Role of Sweetness in Health. Advances in Nutrition. 12. https://doi.org/10.1093/advances/nmaa151 Trygg, J., & Wold, S. (2002). Orthogonal projections to latent structures (O-PLS). Journal of Chemometrics, 16(3), 119â128. https://doi.org/10.1002/cem.695 Tsugawa, H., Cajka, T., Kind, T., Ma, Y., Higgins, B., Ikeda, K., Kanazawa, M., VanderGheynst, J., Fiehn, O., & Arita, M. (2015). MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nature methods, 12(6), 523â526. https://doi.org/10.1038/nmeth.3393 TTFF, (2022). Annual Report 2021-2022. Trees That Feed Foundation (assessed 27 July 2022) Turi, C. E., & Murch, S. J. (2013). Targeted and untargeted phytochemistry of Ligusticum canbyi: Indoleamines, phthalides, antioxidant potential, and use of metabolomics as a hypothesis-generating technique for compound discovery. Planta Medica, 79(14), 1370â1379. https://doi.org/10.1055/s-0033-1350643 Turi, C. E., Axwik, K. E., Smith, A., Jones, A. M. P., Saxena, P. K., & Murch, S. J. (2014). Galanthamine, an anti-cholinesterase drug, effects plant growth and development in 168 Artemisia tridentata Nutt. Via modulation of auxin and neurotransmitter signaling. Plant Signaling and Behavior, 9(4). https://doi.org/10.4161/psb.28645 Turi, C. E., Liu, Y., Ragone, D., & Murch, S. J. (2015). Breadfruit (Artocarpus altilis and hybrids): A traditional crop with the potential to prevent hunger and mitigate diabetes in Oceania. In Trends in Food Science and Technology (Vol. 45, Issue 2, pp. 264â272). Elsevier Ltd. https://doi.org/10.1016/j.tifs.2015.07.014 Turi, C. E., Finley, J., Shipley, P. R., Murch, S. J., & Brown, P. N. (2015). Metabolomics for phytochemical discovery: Development of statistical approaches using a cranberry model system. Journal of Natural Products, 78, 953 â 966. https://doi.org/10.1021/np500667z Tusher, V. G., Tibshirani, R., and Chu, G. (2001). Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116â5121. https://doi.org/10.1073/pnas.091062498 Tusialofa, S. (1992). Bwiro biscuits on Makin Island. IRETAâs South Pacific Agric. News 10(6):5 US EPA, (2020). Sources of Greenhouse Gas Emissions. https://www.epa.gov/ghgemissions/sources-greenhouse-gas-emissions (accessed 1 August 2022) Voltaire. (1769). Précis du siècle de Louis XV. Tome second. Geneva: s.n https://gallica.bnf.fr/ark:/12148/bpt6k75051r van Ruth, S. M., & Roozen, J. R. (2002). Delivery of flavours from food matrices. In A. J. Taylor (Ed), Food Flavour Technology (pp. 168-184). Sheffield: Sheffield Academic Press Ltd. Voltaire, (1770). âArbre a painâ. In Questions sur lâencyclopédie par des amateurs. Seconde Partie, pp. 104â07. Geneva: Cramer. Accessed via Gallica, the digital library of the National Library of France, at https://gallica.bnf.fr/ark:/12148/bpt6k1231486. Wang, C. Y., Liu, Y. W., Jia, J. Q., Sivakumar, T. R., Fan, T., & Gui, Z. Z. (2013) Optimization of fermentation process for preparation of mulberry fruit wine by response surface methodology. African Journal of Microbiology Research, 7:227â236. https://doi.org/10.5897/AJMR12.2090 Wang, K., & Arntfield. S. D. (2015). Food Res. Int.2015,77,1. https://doi.org/10.1016/j.foodres.2015.03.030 Wang, K., & Arntfield, S. D. (2017). Effect of proteinâflavour binding on flavour delivery and protein functional properties: A special emphasis on plant-based proteins. Flavour and Fragrance Journal, 32(2), 92â101. https://doi.org/10.1002/ffj.3365 169 Wang, Y., Li, C., Lin, J., Sun, Y., Wei, S., & Wu, L. (2022). The impact of different withering approaches on the metabolism of flavour compounds in oolong tea leaves. Foods, 11(23), 3601. https://doi.org/10.3390/foods11233601 Wang, L., Zhang, S., Li, J., Zhang, Y., Zhou, D., Li, C., He, L., Li, H., Wang, F., & Gao, J. (2022). Identification of key genes controlling soluble sugar and glucosinolate biosynthesis in Chinese cabbage by integrating metabolome and genome-wide transcriptome analysis. Front Plant Sci.;13:1043489. https://doi.org/10.3389/fpls.2022.1043489 Wang, T., An, J., Chai, M., Zhu, Z., Jiang, Y., Huang, X., & Han, B. (2023). Volatile metabolomics reveals the characteristics of the unique flavour substances in oats. Food Chemistry, 20, 101000. https://doi.org/10.1016/j.fochx.2023.101000 Watanabe, K., Shuto, T., Sato, M., Onuki, K., Mizunoe, S., Suzuki, S., Sat, T., Koga, T., Suico, M. A., Kai, H., & Ikeda, T. (2011). Lucidenic acids-rich extract from antlered form of Ganoderma lucidum enhances TNFα induction in THP-1 monocytic cells possibly via its modulation of MAP kinases p38 and JNK. Biochem Biophys Res Commun., 408(1), 18-24. https://doi.org/10.1016/j.bbrc.2011.03.107 Wester, P. J. (1922). The breadfruit: A plea for the preservation of varieties. Journal of Heredity, 13(3), 129â135. https://doi.org/10.1093/oxfordjournals.jhered.a102181 Widanagamage, R. D., Ekanayake, S., & Welihinda, J. (2009). Carbohydrate-rich foods: Glycemic indices and the effect of constituent macronutrients. International Journal of Food Sciences and Nutrition, 60(4), 215â223. https://doi.org/10.1080/09637480701755600 Widowati, S., Widaningrum, W., & Sulistyaningrum, A. (2023). Improving Organoleptic and Physicochemical Properties of Breadfruit Flour through Tannin Reduction. E3S Web of Conferences. 444. Wiechelman, K. J, Braun, R. D., & Fitzpatrick, J. D. (1988). Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem. Nov 15;175(1):231-7. https://doi.org/10.1016/0003-2697(88)90383-1 Whistler, W. A. (1988). Ethnobotany of Tokelau: The plants, their Tokelau names, and their uses. Economic Botany, 42(2), 155â176. https://doi.org/10.1007/BF02858917 Widaningrum, B. M., Flanagan, B. A., Williams, F., Sonni, D., Mikkelsen, & Gidley, M. J. (2020). Bioact. Carbohydrates Diet. Fibre, 23, 100223. https://doi.org/10.1016/j.bcdf.2020.100223 Williams, E. W., Gardner, E. M., Harris, R., Chaveerach, A., Pereira, J. T., & Zerega, N. J. C. (2017). Out of Borneo: Biogeography, phylogeny, and divergence date estimates of Artocarpus (Moraceae). Annals of Botany, 119(4), 611â627. https://doi.org/10.1093/aob/mcw249 Williamson, M. P. (1994). The structure and function of proline-rich regions in proteins. Biochem. J., 297, 249-260. https://doi.org/10.1042/bj2970249 170 World Bank (2007). World Development Report: Agriculture for Development. World Bank, Washington DC. Wood, E. J., Archer. M. C., & Palmer, J. K. (1989). An experiment in enzyme characterization: banana polyphenol oxidase Practical Biochemistry for Colleges Pergamon Press Cambridge UK Wootton, M., & Tumaalii, F. (1984). Composition of flours from Samoan breadfruit. Journal of Food Science, 49(5), 1396e1397. https://doi.org/10.1111/j.1365-2621.1984.tb15000.x Worrell, D., Carrington, S., & Huber, D. (2002). The use of low temperature and coatings to maintain storage quality of breadfruit, Artocarpus altilis (Parks.) Fosb. Postharvest Biology and Technology, Postharvest Biol Technol, 25, 33â40. https://doi.org/10.1016/S0925-5214(01)00160-4 WPR, (2022). Rice production by Country 2022. https://worldpopulationreview.com/country-rankings/rice-production-by-country (assessed 1 August 2022) Wu, T., Tang, Q., Gao, Z., Yu, Z., Song, H., Zheng, X., & Chen, W. (2013) Blueberry and mulberry juice prevent obesity development in C57BL/6 mice. PLoS One 8:e77585. https://doi.org/10.1371/journal.pone.0077585 Xia, J., Psychogios, N., Young, N. & Wishart, D. S. (2009). MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucleic Acids Res, 37, 652â660. https://doi.org/10.1093/nar/gkp356 Xia, J., Mandal, R., Sinelnikov, I. V. , Broadhurst, D. & Wishart, D. S. (2012). MetaboAnalyst 2.0âA comprehensive server for metabolomic data analysis. Nucleic Acids Res. , 40, 127â133. https://doi.org/10.1093/nar/gks374 Xia, J., Sinelnikov, I.V. , Han, B. & Wishart, D. S. (2015). MetaboAnalyst 3.0âMaking metabolomics more meaningful. Nucleic Acids Res., 43, 251â257. https://doi.org/10.1093/nar/gkv380 Xia, J., & Wishart, D. S. (2010). MetPA: A web-based metabolomics tool for pathway analysis and visualization. Bioinformatics, 26(18), 2342â2344. https://doi.org/10.1093/bioinformatics/btq418 Xie, L., Deng, Z., Zhang, J., Dong, H., Wang, W., Xing, B., & Liu, X. (2022). Comparison of flavonoid O-glycoside, C-glycoside and their aglycones on antioxidant capacity and metabolism during in vitro digestion and in vivo. Foods, 11(6), 882. https://doi.org/10.3390/foods11060882 Xu, J., Zhou, S., Gong, X., Song, Y., van Nocker, S., Ma, F., & Guan, Q. (2018) Single-base methylome analysis reveals dynamic epigenomic differences associated with water deficit in apple. Plant Biotechnol J 16:672â687. https://doi.org/10.1111/pbi.12820 171 Yamamoto, T. & Inui-Yamamoto, C. (2023). The flavour-enhancing action of glutamate and its mechanism involving the notion of kokumi. npj Sci Food 7,3. https://doi.org/10.1038/s41538-023-00182-6 Yamaguchi, S. (2012). Arachidonic Acid Enhance Taste and Palatability of Foods. Oleoscience, 12. 283-288. Yauk, Y. K., Souleyre, E. J. F., Matich, A. J., Chen, X., Wang, M. Y., Plunkett, B., Dare, A. P., Espley, R. v., Tomes, S., Chagné, D., & Atkinson, R. G. (2017). Alcohol acyl transferase 1 links two distinct volatile pathways that produce esters and phenylpropenes in apple fruit. Plant Journal, 91(2), 292â305. https://doi.org/10.1111/tpj.13564 Ye, J., Erland, L. A. E., Gill, S. K., Bishop, S. L., Verdugo-Meza, A., Murch, S. J., & Gibson, D. L. (2021). Metabolomics-guided hypothesis generation for mechanisms of intestinal protection by live biotherapeutic products. Biomolecules, 11(5). https://doi.org/10.3390/biom11050738 Ye, J., Ren, G., Li, W., Zhong, G., Zhang, M., Yuan, J., & Lu, T. (2019). Characterization, and identification of prenylated flavonoids from Artocarpus heterophyllus Lam. roots by quadrupole time-of-flight and linear trap quadrupole orbitrap mass spectrometry. Molecules, 24(24), 4591. https://doi.org/10.3390/molecules24244591 Yeang, H. Y. (2007). The sunshine-mediated trigger of synchronous flowering in the tropics: The rubber tree as a study model. The New Phytologist, 176(4), 730â735. https://doi.org/10.1111/j.1469-8137.2007.02226.x Yelluh Meat (2024). https://www.yelluhmeat.com/our-story (assessed 17 May 2024) Zhang, Y., Hoon, M. A., Chandrashekar, J., Mueller, K.L., Cook, B., Wu, D., Zuker, C. S., & Ryba, N.J. (2003). Coding of sweet, bitter, and umami tastes: different receptor cells sharing similar signaling pathways. Cell, 112(3), 293â301. https://doi.org/10.1016/S0092-8674(03)00071-0 Zhang, Y. (2013). Ascorbic Acid in Plants: Biosynthesis, Regulation and Enhancement, Springer: New York, 2013; pp 1-6. https://doi.org/10.1007/978-1-4614-4127-4_1 Zhang, Y., Li, X., & Chen, H. (2023). Caffeine synthesis and its mechanism and application by microbial fermentation. Foods, 12(12), 2334. https://doi.org/10.3390/foods12122334 Zhang, A. H., Sun H., Yan G. L., Yuan Y., Han Y., & Wang X. J. (2014). Metabolomics study of type 2 diabetes using ultra-performance LC-ESI/quadrupole-TOF high-definition MS coupled with pattern recognition methods. J. Physiol. Biochem., 70(1), 117â128. https://doi.org/10.1007/s13105-013-0286-z Zhao, C. J., Schieber, A., & Gänzle, M. G. (2016). Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations â A review. Food Research International, 89, 39â47. https://doi.org/10.1016/j.foodres.2016.08.042 172 Zerega, N. J. C., Ragone, D., & Motley, T. J. (2004). Complex Origins of Breadfruit (Artocarpus altilis, Moraceae): Implications for Human Migrations in Oceania. In Source: American Journal of Botany, 91, 5. https://doi.org/10.3732/ajb.91.5.760 Zerega, N., Ragone, D., & Motley, T. (2006). Breadfruit origins, diversity, and human-facilitated distribution (pp. 213â238). Zerega, N., Wiesner-Hanks, T., Ragone, D., Irish, B., Scheffler, B., Simpson, S., & Zee, F. (2015). Diversity in the breadfruit complex (Artocarpus, Moraceae): Genetic characterization of critical germplasm. Tree Genetics & Genomes, 11, 1-26. Zhou, M., Chen, Q., Bi, J., Wang, Y., & Wu, X. (2017). Degradation kinetics of cyanidin 3- O-glucoside and cyanidin 3-O-rutinoside during hot air and vacuum drying in mulberry (Morus alba L.) fruit: A comparative study based on solid food system. Food Chem, 229:574â579. https://doi.org/10.1016/j.foodchem.2017.02.131 Zhu, G., Wang, S., Huang, Z., Zhang, S., Liao, Q., Zhang, C., Lin, T., Qin, M., Peng, M., Yang, C., Cao, X., Han, X., Wang, X., van der Knaap, E., Zhang, Z., Cui, X., Klee, H., Fernie, A. R., Luo, J., & Huang, S. (2018). Rewiring of the Fruit Metabolome in Tomato Breeding. Cell, 172(1-2), 249â261. https://doi.org/10.1016/j.cell.2017.12.022 Zubarev, R. A. & Makarov, A. (2013). Orbitrap Mass Spectrometry. Anal. Chem. 2013, 85, 5288 â 5296 https://doi.org/10.1021/ac402481d 173 Appendix Figure S1. Normalization of the Features/Sample Data. 174 Figure S2. Normalization of the Features/Sample Data. 175 Figure S3. Principal component analysis (PCA) for the Mild and Intense Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). This analysis was performed on MetaboAnalyst Ver. 6.0. The PCA plots did not find an individual distinguishing feature that accounted for significant variance. Two dimensional plots of (PCs) are as follows: 1 & 2 (a), 1 & 3 (b), 1 & 4 (c), 2 & 3 (d), 2 & 4 (e) and 3 & 4 (f). Figure S4. Relative intensities of the significant metabolitesâ hits in the alpha-linolenic pathway between Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The metabolites were hits in the pathway mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Yellow denotes mild and blue denotes intense. Bars represents Mean±Standard error calculated from the nine replicates in each variety. 176 Figure S5. Relative intensities of the significant metabolitesâ hits in the biosynthesis of various plant secondary metabolites between Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The metabolites were hits in the pathway mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Yellow denotes mild and blue denotes intense. Bars represents Mean±Standard error calculated from the nine replicates in each variety. Figure S6: Relative intensities of the significant metabolites' hits in the diterpenoid biosynthesis between the Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The metabolites were hits in the pathway mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Yellow denotes mild and blue denotes intense. Bars represents Mean±Standard error calculated from the nine replicates in each variety. 177 Figure S7. Relative intensities of the significant metabolitesâ hits in the phenylpropanoid biosynthesis between the Mild and Intense breadfruit (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis). The metabolites were hits in the pathway mapped to KEGG (Kyoto Encyclopedia of Genes and Genomes). Yellow denotes mild and blue denotes intense. Bars represents Mean±Standard error calculated from the nine replicates in each variety. Figure S8. Gibberellins Acids Putatively Identified in the Mild And Intense Breadfruit. 178 Figure S9. Chromatogram for the Refence Standards.. The column has an efficiency considered normal for nonanal and suitable for the adsorption of saturated aldehydes. Figure S10. Chromatogram for Ethanol and Hexane Blanks for the Reference Standards. (a) Ethanol (b) hexane. 179 Figure S11. Sugars in Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locations. (a) D-fructose (b) sucrose. Bars represents Mean±Standard error. Figure S12. Total Flavonoids in Ten Commercial Breadfruit Flour (Artocarpus altilis, Artocarpus altilis Ã Artocarpus mariannensis) from different geographical locations. Rutin as the equivalent. Bars represents Mean±Standard error.
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Murch, S. J. (Susan J.)
Flavour chemistry in breadfruit (Artocarpus altilis Parkinson (Fosberg)) and its hybrids (Artocarpus altilis × Artocarpus mariannensis)
Text