Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Kehinde, Abisola Zainab
Master of Science - MSc
University of British Columbia
Breadfruit is an underutilized staple crop originating from Borneo and carried across the Pacific islands through generations of migration. My research investigated the potential of breadfruit as (1) a source of high-quality starch and, (2) a key ingredient for infant formula. To determine starch quality amongst cultivars A. altilis and hybrids, I analyzed starch yield, structure, swelling power, solubility, dietary fiber (DF), and starch-bound protein of 16 cultivars of A. altilis and hybrids. Hybrids differed significantly only in soluble DF content and starch solubility. Three cultivars, Yuley, Ulu fiti, and Ma’afala, showed distinct profiles in multivariate analysis of the cultivars by starch and fiber properties. To investigate breadfruit as a source of novel terpenes, I analyzed the glandular trichomes which is the site of synthesis of these important compounds across cultivars. My results showed that breadfruit contained both glandular and non-glandular trichomes and can be viewed successfully without sample preparation that involves chemical fixation and freeze-drying. Also, the younger leaves had a greater amount of glandular trichomes than the mature leaves and varied across cultivars. To identify breadfruit as a suitable food staple that can provide adequate nutrition for infant formula, I compared studies that determined the nutritional content of breadfruit to the standard for infant formula by the world health organization. My results showed that breadfruit contains nutrients that meet the recommended dietary allowance for children. The results of these experiments provide the basic scientific evidence necessary to support the development of novel bioproducts and breadfruit-based food products.
DEVELOPMENT OF NOVEL BIOPRODUCTS FROM BREADFRUIT by Abisola Zainab Kehinde B.Sc., University of Lagos, Nigeria, 2016 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) July 2021 © Abisola Zainab Kehinde, 2021 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: DEVELOPMENT OF NOVEL BIOPRODUCTS FROM BREADFRUIT submitted by Abisola Zainab Kehinde in partial fulfillment of the requirements of the degree of MASTER OF SCIENCE. Dr. Susan Murch, Chemistry, Irving K. Barber Faculty of Science Supervisor Dr. Thu-Thuy Dang, Biochemistry and Molecular Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Michael Deyholos, Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Soheil Mahmoud, Biochemistry and Molecular Biology, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Deborah Buszard, Biology, Irving K. Barber Faculty of Science University Examiner iii Abstract Breadfruit is an underutilized staple crop originating from Borneo and carried across the Pacific islands through generations of migration. My research investigated the potential of breadfruit as (1) a source of high-quality starch and, (2) a key ingredient for infant formula. To determine starch quality amongst cultivars A. altilis and hybrids, I analyzed starch yield, structure, swelling power, solubility, dietary fiber (DF), and starch-bound protein of 16 cultivars of A. altilis and hybrids. Hybrids differed significantly only in soluble DF content and starch solubility. Three cultivars, Yuley, Ulu fiti, and Ma’afala, showed distinct profiles in multivariate analysis of the cultivars by starch and fiber properties. To investigate breadfruit as a source of novel terpenes, I analyzed the glandular trichomes which is the site of synthesis of these important compounds across cultivars. My results showed that breadfruit contained both glandular and non-glandular trichomes and can be viewed successfully without sample preparation that involves chemical fixation and freeze-drying. Also, the younger leaves had a greater amount of glandular trichomes than the mature leaves and varied across cultivars. To identify breadfruit as a suitable food staple that can provide adequate nutrition for infant formula, I compared studies that determined the nutritional content of breadfruit to the standard for infant formula by the world health organization. My results showed that breadfruit contains nutrients that meet the recommended dietary allowance for children. The results of these experiments provide the basic scientific evidence necessary to support the development of novel bioproducts and breadfruit-based food products. iv Lay Summary My research investigated breadfruit (Artocarpus altilis) for food ingredients and bioproducts. Breadfruit is an abundant, staple but underutilized crop that has high nutritive value in terms of essential nutrients and can grow for more than 50 years once planted. There are hundreds of different cultivars of breadfruit. I hypothesized that different cultivars of breadfruit can be used to produce different types of products. I developed and optimized methods for the extraction and analysis of starch from the fruit and trichomes from the leaves. My research found that different cultivars of breadfruit produce different types of starch and different types of trichomes. Breadfruit is a sustainable, high-quality alternative to other starches used in infant formulas and can be a source of novel terpenes for novel bioproducts. v Preface A version of Chapter 6 is submitted for publication. I contributed to the design and conducted experiments, acquisition, analysis, and interpretation of the data, and manuscript preparation; Dr. Ying Liu contributed to the design, conducted experiments, acquisition, and interpretation of the data; Dr. AMP Jones contributed to the design, sample collection, method development, and acquisition of the data; Dr. Lauren A. E. Erland participated in the analysis of the data and manuscript preparation; Dr. Diane Ragone administers the germplasm collection and oversaw access to botanical resources, experimental design, sample collection, and field logistics. Dr. Susan J. Murch oversaw the design of the study, resources for the project, and manuscript preparation. All authors participated in the revision of the manuscript and provided approval of the final manuscript. A version of Chapter 7 is being prepared for publication. A version of Chapter 8 is being prepared for publication. Presentations: Data from Chapter 7 were presented at: Abisola Z. Kehinde, Lauren A.E. Erland, Diane Ragone, and Susan J. Murch (2021) Growing breadfruit flour for world food markets. NHPRS Annual Conference, June 2021 Data from Chapter 3, and 6 were presented at: Abisola Z. Kehinde and Susan J. Murch (2020) Variability and Diversity in Breadfruit (Artocarpus altilis and related hybrids). UBC life science poster session, November 2020 vi Abisola Z. Kehinde and Susan J. Murch (2020) Variability and Diversity in Breadfruit (Artocarpus altilis and related hybrids). CSPB Virtual Conference, November 2020. Abisola Z. Kehinde and Susan J. Murch (2020) Variability and Diversity in Breadfruit (Artocarpus altilis and related hybrids). CBGRC Conference, November 2020. Abisola Z. Kehinde and Susan J. Murch (2020) Breadfruit native starch exhibiting sphere aggregates. The Cluster of Research Excellence in Agricultural Technologies and Bioproducts (CREATe-BP) oral presentation, UBC Okanagan, Canada, July 2020. Data from Chapter 9 was presented at: Abisola Z. Kehinde, Ayelign Adal, Soheil S. Mahmoud, Susan J. Murch. Novel Bioproducts from Breadfruit (Artocarpus altilis). Inaugural Eminence Cluster Trainee Poster Session, UBC Okanagan, Canada, February 2020. Chapter 2 was presented at: Abisola Z. Kehinde and Susan J. Murch (2020). Developing an Infant Formula from Breadfruit. Three Minutes Thesis, University of British Columbia, Kelowna, March 10th, 2020. All breadfruit images used in this thesis was taken by Jan Vozenilek of Copper Sky Productions, Kelowna, BC, Canada, and Susan J. Murch and was reprinted with the permission of Susan J. Murch. vii Table of Contents Abstract .................................................................................................................................... iii Lay Summary ........................................................................................................................... iv Preface ....................................................................................................................................... v Table of Contents .................................................................................................................... vii List of Tables ........................................................................................................................... xv List of Figures ....................................................................................................................... xvii Acknowledgements .............................................................................................................. xxii Dedication ............................................................................................................................ xxiv Chapter 1: Introduction ............................................................................................................ 1 1.1 Breadfruit (Artocarpus altilis (Parkinson) Fosberg) ..................................................... 1 1.2 Botanical description .................................................................................................. 1 1.3 Ecological requirements ............................................................................................. 3 1.4 Breadfruit foods .......................................................................................................... 4 1.5 Nutritional properties .................................................................................................. 4 1.5.1 Fiber and glycemic index ................................................................................. 5 1.5.2 Carbohydrates ................................................................................................. 6 1.5.3 Minerals and vitamins ...................................................................................... 6 1.5.4 Protein ............................................................................................................. 7 1.6 Ma’afala ..................................................................................................................... 7 1.7 Breadfruit as a source of high-quality starch ............................................................... 7 1.8 Genomics of breadfruit (A. altilis) ...............................................................................10 1.9 Glandular trichomes in breadfruit ...............................................................................10 1.10 Breadfruit as a key ingredient for an infant formulation ............................................11 1.11 Overall objectives ....................................................................................................13 1.11.1 Objective 1: Breadfruit as a key ingredient for infant formula .......................13 viii 1.11.2 Objective 2: Breadfruit as a source of high-quality starch ...............................13 1.11.2.1 Optimization of breadfruit starch extraction .....................................13 1.11.2.2 Starch structural analysis ................................................................14 1.11.2.3 Pasting and textural properties ........................................................15 1.11.2.4 Cultivars of breadfruit (A. altilis (Parkinson) Fosberg and hybrids) have unique starch and fiber profiles. ................................................................................15 1.11.2.5 Characterization of nutritional properties of Ma’afala from different growing models .................................................................................................................16 1.11.2.6 Ma’afala (A. altilis) is an excellent source of fiber and starch in comparison to corn, rice, and wheat ..................................................................................16 1.11.3 Objective 3: Breadfruit as a source of high-quality terpenes and secondary metabolites ........................................................................................................................17 Chapter 2: Breadfruit as a key ingredient for an infant formulation .................................... 18 2.1 Synopsis ..................................................................................................................18 2.2 Materials and Methods ............................................................................................18 2.2.1 Search design and information sources .........................................................18 2.3 Results ....................................................................................................................19 2.3.1 Commercially available infant formulas ..........................................................19 2.3.2 WHO recommendations .................................................................................21 2.3.3 Comparison of breadfruit to WHO recommendations .....................................24 2.4 Discussion ...............................................................................................................37 2.5 Conclusion ...............................................................................................................37 Chapter 3: Method development and optimization .............................................................. 39 3.1 Synopsis ..................................................................................................................39 3.2 Materials and Methods ............................................................................................39 3.2.1 Breadfruit germplasm repository at Kahanu garden, Maui ..............................39 ix 3.2.2 Preparation of breadfruit flour ..................................................................44 3.2.3 Optimization of starch extraction for breadfruit flours...............................46 3.2.4 Protein assay .........................................................................................46 3.2.4.1 Chemicals ....................................................................................46 3.2.4.2 Isolation of proteins ......................................................................47 3.2.4.3 Bicinchoninic acid (BCA) protein assay ........................................47 3.2.5 Experimental design and statistical analysis ...........................................48 3.3 Results ...........................................................................................................48 3.3.1 Optimization of starch extraction protocols ..........................................48 3.3.2 Differences in the starch protein across 55 breadfruit cultivars…. .......51 3.4 Discussion .....................................................................................................54 3.5 Conclusion .....................................................................................................55 Chapter 4: Starch structural analysis ................................................................................... 56 4.1 Synopsis ........................................................................................................56 4.2 Materials and Methods ...................................................................................56 4.2.1 Scanning electron microscopy (SEM) of breadfruit starch ...................56 4.2.2 Image processing from SEM ...............................................................56 4.3 Results ...........................................................................................................57 4.3.1 Starch size and structure of different breadfruit cultivars .....................57 4.4 Discussion .....................................................................................................58 4.5 Conclusion .....................................................................................................59 Chapter 5: Pasting and textural properties .......................................................................... 60 5.1 Synopsis ........................................................................................................60 5.2 Materials and Methods ...................................................................................60 5.2.1 Gel textural analysis ............................................................................60 5.2.2 Pasting properties ...............................................................................60 x 5.2.3 Experimental design and statistical analysis ........................................61 5.3 Results ...........................................................................................................61 5.3.1 Textural properties vary across breadfruit cultivars. .............................61 5.3.2 Pasting properties vary across breadfruit cultivars. ..............................64 5.4 Discussion .....................................................................................................68 5.5 Conclusion .....................................................................................................69 Chapter 6: Cultivars of breadfruit (A. altilis (Parkinson) Fosberg and hybrids) have unique starch and fiber profiles ............................................................................................. 70 6.1 Synopsis ........................................................................................................70 6.2 Materials and Methods ...................................................................................70 6.2.1 Breadfruit germplasm repository at Kahanu garden, Maui ...................70 6.2.2 Preparation of breadfruit flour ..............................................................72 6.2.3 Isolation of breadfruit starch ................................................................73 6.2.4 Total Dietary Fibre (TDF)/Soluble Dietary Fibre (SDF)/ Insoluble Dietary Fibre (IDF) .................................................................................................................73 6.2.5 Total ash analysis................................................................................74 6.2.6 Protein determination of flour, starch, and fiber ...................................74 6.2.7 Scanning electron microscopy (SEM) of breadfruit starch ...................75 6.2.8 Image processing from SEM ...............................................................75 6.2.9 Swelling and solubility .........................................................................75 6.2.10 Experimental design and statistical analysis ......................................76 6.3 Results ...........................................................................................................76 6.3.1 Fruit morphology .................................................................................76 6.3.2 Dietary fiber .........................................................................................78 6.3.3 Hybridization did not impact starch yield. .............................................80 xi 6.3.4 Differences in the starch-bound protein across breadfruit cultivars of A. altilis and hybrids .......................................................................................................82 6.3.5 Starch size and structure of different breadfruit cultivars of A. altilis and hybrids ...............................................................................................................83 6.3.6 The swelling power and solubility of different breadfruit cultivars of A. altilis and hybrids .......................................................................................................87 6.3.7 Principal component analysis (PCA) identifies Yuley, Ulu fiti, and Ma’afala as distinct cultivars. .....................................................................................89 6.4 Discussion. ....................................................................................................94 6.5 Conclusion .....................................................................................................96 Chapter 7: Quality and consistency of Ma’afala as a source of flour ................................. 97 7.1 Synopsis ........................................................................................................97 7.2 Materials and Methods ...................................................................................97 7.2.1 Field collections ...................................................................................97 7.2.1.1 Ornamental horticulture ................................................................98 7.2.1.2 Breadfruit orchard ...................................................................... 100 7.2.1.3 Sustainable agroforestry farm samples ...................................... 100 7.2.1.4 Dryland windbreak samples ....................................................... 101 7.2.1.5 Botanic garden samples ............................................................ 103 7.2.1.6 Kahanu botanic garden samples ............................................... 103 7.2.2 Preparation of breadfruit samples ...................................................... 105 7.2.3 Isolation of breadfruit starch ............................................................. 105 7.2.4 Protein determination of flour, and starch ......................................... 106 7.2.5 Precipitation and temperature observations...................................... 106 7.2.6 Experimental design and statistical analysis ..................................... 106 7.3 Results ......................................................................................................... 107 xii 7.3.1 Fruit morphology of Ma’afala differs across various growing models. 107 7.3.2 Significant variability in the starch yield of Ma’afala from different growing models ....................................................................................................... 110 7.3.3 Differences in the starch protein of Ma’afala from different growing models. .................................................................................................................... 110 7.3.4 Protein content of Ma’afala flour differs significantly across growing models. .................................................................................................................... 110 7.3.5 Climate data of the different growing models of Ma’afala................... 111 7.4 Discussion ................................................................................................... 113 7.5 Conclusion ................................................................................................... 114 Chapter 8: Ma’afala (A. altilis) is an excellent source of fiber and starch in comparison to corn, rice, and wheat ............................................................................................................ 115 8.1 Synopsis ...................................................................................................... 115 8.2 Materials and Methods ................................................................................. 115 8.2.1 Flours ................................................................................................ 115 8.2.2 Isolation of starch .............................................................................. 116 8.2.3 Total Dietary Fiber (TDF)/Soluble Dietary Fiber (SDF)/ Insoluble Dietary Fiber (IDF) ............................................................................................................... 116 8.2.4 Total ash analysis.............................................................................. 117 8.2.5 Protein determination of flour, starch, and fiber ................................. 117 8.2.6 Swelling power and solubility ............................................................. 117 8.2.7 Scanning electron microscopy (SEM) of starch ................................. 117 8.2.8 Image processing from SEM ............................................................. 117 8.2.9 Experimental design and statistical analysis ...................................... 118 8.3 Results ......................................................................................................... 118 xiii 8.3.1 Breadfruit is a better source of dietary fiber as compared to traditional sources. ................................................................................................................... 118 8.3.2 Breadfruit has higher starch yields as compared to traditional flours. 119 8.3.3 Breadfruit starch is of higher purity than traditional sources with low levels of starch-bound protein. ................................................................................. 121 8.3.4 The swelling power and solubility of breadfruit in comparison to traditional sources ................................................................................................... 121 8.3.5 Starch granule structure and size ...................................................... 121 8.4 Discussion ................................................................................................... 124 8.5 Conclusion ................................................................................................... 126 Chapter 9: Characterization of glandular trichomes in breadfruit .................................... 127 9.1 Synopsis ...................................................................................................... 127 9.2 Materials and Methods ................................................................................. 127 9.2.1 Scanning electron microscopy (SEM) of breadfruit glandular trichomes ................................................................................................................. 127 9.2.2 Count of glandular trichomes ............................................................. 128 9.2.3 Isolation of breadfruit glandular trichomes ......................................... 128 9.2.4 Experimental design and statistical analysis ...................................... 129 9.3 Results ......................................................................................................... 129 9.3.1 Breadfruit contained glandular trichomes and can be viewed without chemical fixation. ..................................................................................................... 129 9.3.2 Number of trichomes did not differ across cultivars ........................... 131 9.3.3 The length of breadfruit leaf was not correlated to its number of trichomes ................................................................................................................. 132 9.3.4 Breadfruit glandular trichomes were successfully isolated ................. 133 9.4 Discussion ................................................................................................... 135 xiv 9.5 Conclusion ................................................................................................... 136 Chapter 10: Summary ........................................................................................................... 137 10.1 Objective 1 ............................................................................................................ 137 10.2 Objective 2 ............................................................................................................ 137 10.3 Objective 3 ............................................................................................................ 139 10.4 Conclusion ............................................................................................................ 139 Bibliography .......................................................................................................................... 141 Appendices ........................................................................................................................... 157 Appendix A: Supplemental table for chapter 2 ................................................................. 157 Appendix B: Supplemental figures for chapter 9 .............................................................. 180 xv List of Tables Table 1.1. Reported data of nutritional summary statistics for breadfruit conducted by Needham et al., (2020) ............................................................................................................................... 4 Table 2.1. Product information and ingredients list of twenty-seven (27) commercial infant formulas ....................................................................................................................................19 Table 2.2. Essential composition of infant formula according to CODEX adopted as a worldwide standard. ...................................................................................................................................22 Table 2.3. WHO’s essential nutrient requirement for infants in comparison to the reported nutritional content of breadfruit. .................................................................................................25 Table 3.1. Cultivars details of the 55 samples of the different breadfruit cultivars (Artocarpus altilis and hybrids of A. altilis x A. marianensis).. .......................................................................40 Table 4.1. SEM granule sizing of different breadfruit starch micrographs captured at 1000 magnification. ............................................................................................................................58 Table 5.1. Gel texture profile analysis of breadfruit flours ..........................................................62 Table 5.2. Correlation coefficients (r) between texture profile analysis of breadfruit flour ..........63 Table 5.3. Correlation coefficients (r) between starch yield and textural properties ...................63 Table 5.4. Pasting properties of different breadfruit flours using rapid-visco analyzer (maximum temperature of 95 °C)................................................................................................................66 Table 5.5. Correlation coefficients (r) between starch yield and pasting properties ...................67 Table 6.1. Breadfruit cultivars (Artocarpus altilis) and hybrids (A. altilis x A. marianensis) that were included in study ...............................................................................................................71 Table 8.1. Size of breadfruit starch in comparison to traditional starch sources at 5000x magnification ........................................................................................................................... 124 Table 9.1. Correlation coefficients (r) between leaf length and the number of glandular trichomes ............................................................................................................................................... 133 xvi Appendix Table A1. Ingredient lists for 27 infant formulas included in the survey……………157 xvii List of Figures Figure 1.1. Breadfruit (A. altilis cv Ma’afala) growing on a tree in Kahanu Garden in Hawaii. ..... 3 Figure 1.2. A short segment of amylose, a linear polymer of D-glucose residues in (α1-4) linkage. ...................................................................................................................................... 9 Figure 1.3. A short segment of Amylopectin showing both its linear polymer of D-glucose residues in (α1-4) linkage and its (α1-6) branch point. ................................................................ 9 Figure 3.1. Preparation of breadfruit flour ..................................................................................45 Figure 3.2. Mean with standard error bars of the starch yield of different breadfruit flour mass. The different superscript differs (p ≤ 0.05) using Welch’s t-test .................................................49 Figure 3.3. The starch yield of 55 breadfruit cultivars ................................................................50 Figure 3.4. Mean with standard error bars of the starch yield of 55 breadfruit cultivars of A. altilis and hybrids ...............................................................................................................................51 Figure 3.5. Mean with standard error bars of starch-bound protein of 55 breadfruit cultivars. ....53 Figure 3.6. Mean with standard error bars of starch-bound protein of 55 breadfruit cultivars of A. altilis and hybrids ......................................................................................................................53 Figure 4.1. The scanning electron micrographs of six different breadfruit starches are shown in the order of Ulu, Tuutou taatoe, Aveloloa, Unidentified 17, Yap, and Samoan at 1000x magnification. ............................................................................................................................57 Figure 5.1. The appearance of breadfruit flours from different cultivars when slurred with water. .................................................................................................................................................65 Figure 5.2. The pasting curve of the different breadfruit flours ...................................................67 Figure 6.1. Fruit characteristics of breadfruit starches by cultivar and hybridization ..................77 Figure 6.2. Fiber characteristics of breadfruit flour. (A, D) Insoluble dietary fiber, (B, E) Soluble dietary fiber, and (C, F) Total dietary fiber. ................................................................................80 Figure 6.3. Characteristics of breadfruit starch by cultivar .........................................................82 xviii Figure 6.4. Scanning electron micrographs of cultivars of breadfruit starches at 5000x magnification .............................................................................................................................85 Figure 6.5. Characteristics of starch granules by cultivar and hybridization ...............................87 Figure 6.6. Processing potential characteristics of breadfruit starches by cultivar and hybridization ..............................................................................................................................88 Figure 6.7. Principal component analysis (PCA) and correlation analysis of IDF, SDF, TDF, starch protein, starch bound protein in total protein, total protein, swelling power, solubility, fiber content, fruit whole weight, and edible fruit weight of the 16 cultivars. .......................................92 Figure 6.8. Comparison of breadfruit fiber content to other common flours using the USDA database ..................................................................................................................................93 Figure 7.1. Map of Ma’afala collection sites ...............................................................................98 Figure 7.2. Harvest of breadfruit samples from the trees at the Mini-Golf Course ......................99 Figure 7.3. Orchard style Ma’afala plantation .......................................................................... 100 Figure 7.4. Ma’afala trees grown in a sustainable agroforestry model ..................................... 101 Figure 7.5. Ma’afala trees grown as a windbreak on an exposed cliff ..................................... 102 Figure 7.6. Ma’afala tree grown in a botanic garden for display ............................................... 103 Figure 7.7. Ma’afala clones grown in the common garden test model before it started bearing fruit .......................................................................................................................................... 105 Figure 7.8. Fruit and nutritional characteristics of Ma’afala from the different growth models.. 108 Figure 7.9. Nutritional properties of Ma’afala from two extreme models .................................. 109 Figure 7.10. Record of (A) Precipitation and (B) Temperature observations from the National Centers for Environmental Information, U.S. Department of Commerce, NOAA, NESDIS of Ma’afala Locations. ................................................................................................................. 112 Figure 8.1. Mean with standard error bars of (a) Insoluble dietary fiber, (b) Soluble dietary fiber, and (c) Total dietary fiber of breadfruit flour compared with traditional flours. .......................... 119 xix Figure 8.2. Mean with standard error bars of (a) Yield, (b) Protein, (c) Swelling power, and (d) Solubility of breadfruit starch compared with traditional starch ................................................ 120 Figure 8.3. Scanning electron micrographs (SEM) of breadfruit starch compared with traditional starch at 5000x magnification .................................................................................................. 122 Figure 8.4. Characteristics of starch granules by flour type ..................................................... 123 Figure 9.1. Types of Trichomes in Breadfruit ........................................................................... 130 Figure 9.2. SEM images of Breadfruit glandular trichomes using different sample preparation130 Figure 9.3. Mean with standard error bars of the number glandular trichomes across breadfruit cultivars................................................................................................................................... 131 Figure 9.4. The number of glandular trichomes and the leaf length across breadfruit cultivars. ............................................................................................................................................... 132 Figure 9.5. Optical Microscopy of glandular trichomes from lavender and breadfruit using the same extraction method. ......................................................................................................... 134 Appendix Figure B1. Image of breadfruit A. Young leaf; B. SEM image of glandular trichomes.………………………………………………. ……………………………………………180 Appendix Figure B2: Image of breadfruit A. Fully expanded leaf; B. SEM image of glandular trichomes………………………………………………………………………………………………180 Appendix Figure B3: SEM images of Ma’afala glandular trichomes. A. Young leaf; B. Mature leaf………………………………………………………………………………………………….. 181 xx List of Abbreviations Aa Artocarpus altilis Aa✕Am Aa ✕ A. mariannensis BCA Bicinchoninic acid BF Breadfruit BSA Bovine serum albumin ° C degree Celsius DW Dry weight FDA Food and Drug Administration GUL Guidance upper levels H0 Null hypothesis H1 Alternative hypothesis HEPES 4-(2-Hydroxyethyl)-piperazine ethane sulfonic acid HCl Hydrochloric acid IDF Insoluble dietary fiber Max Maximum MES 2-(N-morpholino) ethanesulfonic acid xxi Min Minimum mls Milliliters n Sample size NaCl Sodium chloride RDA Recommended dietary allowance. SDF Soluble dietary fiber SEM Scanning electron microscope St.Dev Standard deviation TDF Total dietary fiber TRIS Tris(hydroxymethyl)-aminomethane USDA United States Department of Agriculture WHO World Health Organization xxii Acknowledgements I would like to thank everyone that supported me during my graduate studies at UBC Okanagan and respectfully acknowledge the traditional and unceded territory of the Syilx Okanagan people where my program was conducted. First, I would start by thanking my Supervisor, Mentor, and Advisor, Dr. Susan Murch for her constant support, guidance, and mentoring. Dr. Murch gave me the privileged opportunity to work in her lab without any prior Canadian experience on a topic that excited me. She made me a better scientist and gave me wings to fly in the science world, I would forever be grateful. Newly from Nigeria, she constantly ensured that I was comfortable in Canada and particularly ensured I never got a “Frostbite”. I also want to express my gratitude to my committee members: Dr. Thuy Dang, Dr. Michael Deyholos, and Dr. Soheil Mahmoud for their valued support and guidance throughout my program. I would also like to thank Dr. Diane Ragone for sharing the breadfruit germplasm collection at the National Tropical Botanical Garden. I would also like to thank Dr. Lauren Erland who provided me with invaluable support for data analysis, writing, and guidance during my program. My sincere gratitude to all the past and present members of the PlantSMART Lab: Dr. Lauren Erland, Dr. Ali Akbari Sehat, Dr. Stephanie Bishop, Jill Forsyth, Brent Lashuk, Adrian Monthony, Shilong Pan, Ryland Giebelhaus, Kaede Hirabayashi, and Simona Mastroianni for their invaluable support, countless gifts, fun activities, group lunch, providing answers to my numerous questions, driving me to see Kelowna’s scenery, and most importantly providing a conducive environment for my graduate program. I also want to thank Dr. Max Jones, who collected the breadfruit samples used in this project. I would like to give photo credit to Jan Vozenilek of Copper Sky Productions, Kelowna, BC, Canada, and Susan J. Murch for all the beautiful breadfruit images used in this thesis. I would like to specifically thank Dr. Ayelign Adal from Mahmoud Research Group for his profound help and training with the Isolation, RNA extraction, and purification of glandular trichomes and for also providing useful mentorship. I would like to thank Dr. Soheil Mahmoud, xxiii Cluster of Research Excellence in Agricultural Technologies and Bioproducts (CREATe-Bioproducts), and the UBC Eminence Program for providing the resources and initiatives for the trichome project. I acknowledge the immense support of collaborators on my project: Dr. Ying Liu from British Columbia Institute of Technology, Dr. Lauren Erland, Dr. Ayelign Adal, Dr. Soheil Mahmoud, Dr. Isaac Li, and Adam Yasunaga from UBC Okanagan, Dr. Shiqi Huang, Dr. Benjamin Bohrer and Dr. Max Jones from the University of Guelph. I would like to thank Dr. Fred Menard, Dr. Christine Schreyer, Dr. Julien Picault, College of Graduate Studies, and Centre for Scholarly Communication center for their help with my Three Minute Thesis Competition which improved my communication skills. I would also like to thank the FiLTER laboratory, UBC Okanagan, and especially Dr. Sudip Shrestha for the help with training and use of Scanning Electron Microscopy. Additionally, I would like to thank all the members of the Chemistry, Biology, and Biochemistry Department who have assisted me with my graduate program, including Karen Krysko, Susan Black, and Natalie Kirkwood. I would also like to greatly acknowledge all the sponsors of the International Peace Scholarship for their financial support towards my education. My research program was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) and UBC Okanagan. Finally, I would like to thank all my family and friends for their outpour of love in ensuring my emotional and physical well-being. Thanks to my Mom, Yinka, Taiwo, and Kehinde for the love, support, constant encouragement, and our long talks to ensure I am not missing home. Thanks to my roomies- Somi and Reshma for always noticing my mood changes, giving me lots of food, advice and always making me stay in a happy mood. Special thanks go to my late father, who was very excited and proud to see me start my research program and showered me with affection and financial support. I am forever grateful to everyone mentioned and those that I did not include their names, for making my graduate school journey a memorable one. xxiv Dedication I dedicate this work to Allah, who is the most compassionate to me. 1 Chapter 1: Introduction 1.1 Breadfruit (Artocarpus altilis (Parkinson) Fosberg) Breadfruit is an underutilized staple crop originating from Borneo and recently introduced into worldwide distribution for food security and economic benefits. While the tree is mostly grown for the fruit, all parts of the tree have economic value. Wood from the breadfruit tree resistant to termites and is used for the construction of carvings, canoe, and cosmetics (Donsing et al., 2009, 2008; Ragone, 1997), and cloth can be made from the bast fibers (Jones et al., 2011b). The other parts of the plant, including crushed leaves, latex, and roots, have been used medicinally to treat skin conditions, liver disease, fever, purgative, fungal infections, and high blood pressure (Ragone, 1997). Male inflorescences are burnt traditionally to repel insects (Ragone, 1997) and a study by Jones et al., (2012) confirmed its efficacy as mosquito repellants primarily due to its fatty acid constituents. Breadfruit leaves have been found to contain a geranylated chalcone that showed a promising antiplatelet activity by inhibiting platelet aggregation and inducing platelet disaggregation in the initial and late phases of aggregation, respectively (Fakhrudin et al., 2020). Breadfruit peel and the core was found to be highly efficient in the biosorption of cationic dyes (Priyantha et al., 2018). The purpose of this work is to evaluate breadfruit as a novel source of food and non-food bioproducts. 1.2 Botanical description The origin of breadfruit is most likely Borneo which was then distributed throughout the Pacific Islands (Lincoln et al., 2018). Breadfruit belongs to the genus Artocarpus in the Moraceae, which is comprised of 37 genera and 1050 species (Berg et al., 2006; Lincoln et al., 2018; Zerega et al., 2005). The common name breadfruit consists of Artocarpus altilis and Artocarpus altilis X Artocarpus mariannensis (hybrids). Genetic data suggest that Artocarpus altilis diploid cultivars may have been introduced to Micronesia from the south over millennia which was followed by hybridization and human selection leading to breadfruit diversity in 2 Micronesia (Lincoln et al., 2018; Zerega et al., 2004). The diploid (2n=2x=~56) produces few or many seeds and the triploids (2n=3x=~84) are seedless (Ragone, 2001). The survival and importance of the hybrids were because of their enhanced vigor and tolerance to salinity (Ragone, 1997) and many hybrid cultivars may show superior growth rate, yield, and reproductive success (Lincoln et al., 2018). Artocarpus camansi Blanco and Artocarpus mariannensis Trécul have been identified as breadfruit wild relatives (Lincoln et al., 2018). A single mature tree produces > 400kg of fruit per year continuously for more than 70 years (Jones et al., 2011b; Lincoln et al., 2018; Liu et al., 2014). It has been estimated that there are more than 300 different cultivars of breadfruit bred by the peoples of the Pacific Islands over centuries (Jones et al., 2011b; Ragone, 1997). Hundreds of traditional cultivars have been selected and named based on fruit/leaf morphology, fruiting season, flavor, horticultural needs, cooking and storage qualities over the millennia (Lincoln et al., 2018). Breadfruit cultivars can be diploids or triploids hybrids, vegetatively or seed-propagated fertile diploids, and vegetatively propagated seedless triploids (Zerega et al., 2015). Germplasm collections are assembled over the decades to conserve breadfruit as many cultivars are seedless and the seed of others cannot be dried, frozen, or stored (Lincoln et al., 2018; Ragone, 1997). 3 Figure 1.1. Breadfruit (A. altilis cv Ma’afala) growing on a tree in Kahanu Garden in Hawaii. GPS 20°47’57.07” N, 156°02’18.42” W. Bar = 2.5”. Photo credit: Jan Vozenilek, Copper Sky Productions, Kelowna, BC, Canada. 1.3 Ecological requirements Breadfruit thrives best in wet tropical regions with more than 1500mm rainfall and temperature above 15°C (Jones et al., 2010). A simple form of propagation and nursery that does not depend on electricity or running water but involves the combination of planting pot and bottomless PET-bottle proved to create a good environment for the promotion of root development of breadfruit (Van Leeuwea and Batista Gomes, 2021). A study that explored breadfruit potential in future climate scenarios found it to increase in area and quality (Mausio et al., 2020). 4 1.4 Breadfruit foods The fruit can be eaten at all stages of maturity, baked, steamed, roasted, boiled, and dried (Jones et al., 2010; Ragone, 1997). Breadfruit flour was used to make an alternative snack for individuals with diabetes mellitus and when compared to various levels of inclusion of wheat flour, 100 % of breadfruit cookies were found to have the highest resistant starch and amylose (Fitriani et al., 2021). Breadfruit flour has also been used to develop products like pasta (Nochera and Ragone, 2019) and probiotic beverages (Gao et al., 2019). 1.5 Nutritional properties A recent review by (Needham et al., 2020) derived summary statistics for 41 studies that determined the proximate analysis and including the location of raw (dried) mature breadfruit as shown in Table 1. The nutritional composition was found to be per past review by Turi et al., (2015) and study by Jones et al., (2011a). Table 1.1. Reported data of nutritional summary statistics for breadfruit conducted by Needham et al., (2020) Nutritional content n Mean St. Dev Max Min (kCal/100 g) Energy 18 209 111 393 103 (g/100 g) Water 15 68.9 6.4 83.6 59 Crude Protein 27 3.6 2.3 11.4 0.6 Actual Protein 12 3.3 2.3 7.4 0.1 Total Protein 39 3.5 2.2 11.4 0.1 Fat 26 1.4 1.5 4.9 0.2 5 Nutritional content n Mean St. Dev Max Min Total Fat 32 1.4 1.4 4.9 0.2 Starch 12 59.9 22.8 80.9 9.3 Total Carbohydrates 23 57.2 24.4 88.2 17.5 Crude Fiber 23 4.2 2.5 9 0.9 Dietary Fiber 11 3.7 2.6 8.13 0.9 Total Fiber 34 4 2.5 9 0.9 (mg/100 g) Ash 25 2.1 1.2 3.9 0.3 Calcium 24 37.2 28.4 123 1 Phosphorus 19 73.5 56.5 188 1.7 Magnesium 12 64.4 48 165 10 Sodium 11 58.8 72.5 240 2.8 Potassium 12 849 648 2060 67 Iron 20 4.4 7.9 29 0.3 Zinc 8 0.7 0.8 2.2 0.1 Thiamine 9 0.1 0.1 0.2 0.1 Riboflavin 10 0.1 0.1 0.2 0.1 Niacin 10 1.4 0.5 2.4 0.6 1.5.1 Fiber and glycemic index Breadfruit contains fiber that is more than most staples and could help regulate blood sugar in humans (Turi et al., 2015). Although there has been no study on the glycemic index (GI) of processed breadfruit products, Cooked breadfruit has low to moderate GI which could be beneficial to control diabetes (Bahado-Singh et al., 2006; Dan Ramdath et al., 2004; Turi et al., 2015). 6 1.5.2 Carbohydrates Cooked breadfruit contains 18.1 -37 g of total carbohydrates, which is comparable to other cooked starch crops such as potato (21.55 g/100 g), white rice (28.59 g/100 g), sweet potato (20.71 g/100 g), taro (34.60/100 g), and plantain (31.15 g/100 g; Turi et al., 2015). The reported total carbohydrates of 100g breadfruit flour (50 – 88.2 g) were however higher than both 100g cooked fruit (18.1 -37 g) and 100g fresh fruit (14.3 – 70.1 g; Turi et al., 2015). 1.5.3 Minerals and vitamins Breadfruit is also a good source of pro-vitamin A carotenoids having the potential to delay the onset of diabetes in adults and mitigate vitamin A deficiencies within populations that are unable to meet daily requirements through consumption of meat or dairy (Jones et al., 2013a; Turi et al., 2015). Breadfruit has significantly more calcium, cobalt, iron, potassium, and magnesium than refined wheat, refined rice, and cornflour (Jones et al., 2011a). Breadfruit’s calcium (283–1491µg/g) was more than 16 folds higher than corn (17 µg/g), 4 folds of refined rice (61 µg/g), and refined wheat (198 µg/g). The cobalt (3.6-31 ng/g) was higher than corn (below detection limit) and rice (10 ng/g). Breadfruit had greater iron (6.2 – 21.2µg/g) than corn (5.9 µg/g), refined rice flour (5.8 µg/g), refined wheat (7.9 µg/g). Its potassium (7.5-16.2 mg/g) content was significantly greater than corn (1.1 mg/g), refined rice flour (1.0 mg/g), and refined wheat (0.9 mg/g). Also, it had significantly higher magnesium (630 – 2281 µg/g) than corn (327 µg/g), refined rice flour (490) µg/g), and refined wheat (342 µg/g). 100 g of boiled breadfruit can provide vitamins and minerals up to 9.3% (B1), 3.3% (B2), 7.0% (B3), 20.2% (C), 3.0% (Ca),22.5% (Cu), 6.1% (Fe), 7.5% (Mg), 15.0% (Mn), 4.1% (P), 14.9% (K), 2.9%(Na), and 0.9% (Zn) of one's daily mineral requirements when compared to FDA’s requirements (Turi et al., 2015). 7 1.5.4 Protein Liu et al. (2015) found that breadfruit (Ma’afala cultivar) protein is complete, containing all the essential amino acid (>568 mg/g protein) required for human health higher than many other staples including wheat (336 mg/100g protein), corn (413 mg/100g protein), rice (374 mg/100g protein), soybean (443 mg/100g protein), potato (326 mg/100g protein), and yellow pea (366 mg/100g protein). Among the essential amino acids found in all the measured 49 cultivars, phenylalanine, leucine, isoleucine, and valine were in abundance (Liu et al., 2015). Using an in vitro multi-stage enzyme digestion model, breadfruit protein was found to be more digestible than wheat protein (Liu et al., 2020). Breadfruit protein extract has been shown to have metal chelating activity and reversed anxiety behavior in adult zebrafish (Gonçalves et al., 2020). 1.6 Ma’afala Ma’afala is the first commercial cultivar selected because it contains protein containing all the essential amino acids at levels higher than many other staples including wheat, corn, rice, soybean, and yellow pea (Liu et al., 2015). With the highly efficient in-vitro plant propagation system developed by Murch et al. (2008), over 150,00 Ma’afala trees have been mass propagated and distributed to more than 50 countries. Flour produced from the cultivar Ma’afala is more digestible than wheat flour, has a high-energy value, is gluten-free, and has no toxicity in the chemical and histological analysis (Liu et al., 2020). These data show the potential of Ma’afala in combating global food insecurity. 1.7 Breadfruit as a source of high-quality starches Starch is simply glucose polymers joined by glycosidic bonds which are ordered in a three-dimensional semi-crystalline structure known as a starch granule (Martin and Smith, 1995) 8 and is used in diverse products including in pharmaceuticals, confectionaries, adhesives, foods, beverages, textiles, fuels, thin films, thermoplastic materials, cosmetics, and animal feeds (Kaur et al., 2012). The internal architecture of starch granules consisting of growth rings, blocklets, and crystalline and amorphous lamellae is similar among plants despite that the diversity of starch granule size and morphology in plants (Bertoft, 2017). Driven by a compounded growth of 5.3 %, the starch market worldwide is projected to grow by 47. 3 million metric tons (Analysts, 2020), with the most common sources of starch being corn, potato, wheat, rice, and cassava (Waterschoot et al., 2015). Breadfruit has the potential to meet the high industrial demands of starch as a mature fruit is made up of 84 % carbohydrates, of which 60 % are starch (Oladunjoye et al., 2010). Breadfruit was found to contain increased starch synthesis genes when compared to closely related species (Sahu et al., 2020). There is the enormous potential of breadfruit starch for products such as coatings for post-harvest storage (Bezerra et al., 2019), stabilizer for high-oil-load emulsions useful in food industries for low-fat food products such as margarine and butter (Anwar et al., 2020), bioethanol production (Betiku and Taiwo, 2015), synthetic polymers in drug delivery (Okunlola and Adewusi, 2019), drug tablet formulation (Adebayo et al., 2008) and nanoparticles of high commercial value (Andrade et al., 2020). Breadfruit starch has been modified using a microwave-assisted chemical method to give a low glycemic index and better functional properties (Otemuyiwa and Aina, 2021). Breadfruit starch has previously been found to have higher swelling power, water binding capability, water absorption index, bulk density, and dispersibility capability when compared to both broken rice and cassava starches (Koh and Long, 2012). Limitations of native starch such as water insolubility, the high tendency of retrogradation, and syneresis have led to the need for starch modification to make it suitable for industrial applications (Ashogbon and Akintayo, 2014). To fully exploit the potential of breadfruit starch, chemical and physical modifications have been used to enhance its compositional and functional attributes with the latter being preferred (Adebowale et al., 2005; Andrade et al., 9 2020; Awokoya et al., 2018; Tan et al., 2017); however, there are limitations of some of these modified starches, for example, acetylation, oxidation, and heat–moisture-treatment improved the swelling power of the native breadfruit starch, but reduced its solubility and gelation properties (Adebowale et al., 2005). Figure 1.2. A short segment of amylose, a linear polymer of D-glucose residues in (α1-4) linkage. Figure 1.3. A short segment of Amylopectin showing both its linear polymer of D-glucose residues in (α1-4) linkage and its (α1-6) branch point. 10 1.8 Genomics of breadfruit (A. altilis) A simple definition of the genome is an organism’s complete set of DNA, including all of the genes that make up the genome and each genome contains all the information needed to build and maintain that organism (Genetics Home Reference website, 2020). However, this definition is deemed incomplete and a more expansive definition of the “ genome as an informational entity, often but not always manifest as DNA, encoding a broad set of functional possibilities that, together with other sources of information, produce and maintain the organism” was suggested (Goldman and Landweber, 2016). The genetic diversity of breadfruit has been characterized using isozymes, microsatellite markers, and amplified fragment length polymorphism (AFLPs) (Ragone, 1991; Zerega et al., 2015, 2006, 2005, 2004). Another genetic study on breadfruit was performed by generating an assembled and annotated reference transcriptome which revealed transcriptomic signals of positive selection that may have resulted from local adaptation or natural selection (Laricchia et al., 2018). Most recently, high-quality genomic information of breadfruit has been published consisting of 2034 expanded gene families with enriched pathways in starch and sucrose metabolism, photosynthesis, and others (Sahu et al., 2020). The sequencing was done using a whole-genome shotgun sequencing strategy (Sahu et al., 2020). 1.9 Glandular trichomes in breadfruit Trichomes are epidermal outgrowth on aerial surfaces of plants and provide important functions in plants such as insects’ repellant, allelopathy, pollinator attraction, plant defense, protection from harsh climatic conditions, promotion of seed dispersal, and water absorption (Mahmoud and Croteau, 2001; Wagner et al., 2004). In many plants, glandular trichomes are the site of essential oil production, and storage and contain several secondary metabolites such as terpenes and sesquiterpenoid hydrocarbons (Lange et al., 2000; Wagner et al., 2004). Terpenes are hydrocarbons formed from the polymerization of isoprene (five-carbon unit) and 11 perform important functions in plants as hormones, photosynthetic pigments, defense, membrane components, and electron carriers (Harborne and Tomas-Barberan, 1990; Sarker et al., 2013). Monoterpenes are a class of terpene with two five-carbon units and have been reported to give the characteristic flavor and aroma of mint oils (Lange et al., 2000). Sesquiterpenes contain three five-carbon units and have industrial value due to their insecticidal, anticancer, antimicrobial, and anti-inflammatory properties (Li-Weber et al., 2002; Majdi et al., 2011; Trusheva et al., 2010). In traditional uses, breadfruit leaves are used for the treatment of fever, high blood pressure, urinary infection, skin ailment, fungal infection, teas for treatment of headaches, and extracted juices are used to treat ear infections (Navarro et al., 2007; Ragone, 1997). The breadfruit genome has been sequenced and stated to have expanded pathways of plant-host interaction (Sahu et al., 2020). These could be a result of enriched terpene genes as GC-MS analysis of breadfruit male inflorescence extracts revealed many compounds including several terpenes such as linalool, terpineol, and fatty acids previously associated with insecticidal or insect repellent activity (Glover, 2015; Jones et al., 2012). Monoterpenes such as limonene, phellandrene, and sabinene have also been identified in the fruit samples of Artocarpus altilis (Soifoini et al., 2018). Methods for extraction of glandular trichomes from leaves include using modified bead abrasion (Lane et al., 2010; Lange et al., 2000; Mahmoud et al., 2004; Sarker et al., 2013). 1.10 Breadfruit as a key ingredient for an infant formulation One emerging potential application of breadfruit starch is the development of novel breadfruit-based infant formulas. Although many infants can get all the required nutrients from human milk, there can be a need for infant formula when there is no breastmilk, or it is unsuitable for infants with special nutritional and metabolic needs such as allergy (Carver, 12 2003). Infant formulas are usually made from animal milk, corn, wheat, or soy but these are either having allergens, deficient in some nutrients or contain anti-nutrients (Dupont, 2003; Ijarotimi and Aroge, 2005; Ivarsson et al., 2013; Lifschitz and Szajewska, 2015). Breadfruit has a potential advantage as it is gluten-free, soy-free, GMO-free with the potential to combat global malnutrition (Liu et al., 2020, 2015). From the late 1920s to date, corn syrup is the most commonly used modified starch added to milk formulas as it was widely available, inexpensive, and readily accepted (Greer, 2018). Breadfruit syrup might offer an alternative as maltodextrin has been obtained from breadfruit with the potential to initiate a saccharification reaction in the production of glucose syrup (GI et al., 2018). Another common starch used in infant formula is rice starch due to its palatability, hypoallergenic, digestibility, and non-GMO (Christian et al., 1999; Gelski, 2018; Wani et al., 2012), but its limitations include arsenic content (Biswas et al., 2020), breadfruit starch may represent a safer alternative. Several studies have been carried out on the formulation and processing of weaning foods from breadfruit (Esparagoza and Tangonan, 1993; Ijarotimi and Aroge, 2005; Nelson-Quartey et al., 2007). In Esparagoza and Tangonan, (1993), a blend of breadfruit and banana was evaluated on only the taste, flavor, and general appearance of the formulation but not its nutritional composition. Ijarotimi and Aroge, (2005) analyzed breadfruit as a complementary food but was found not to contain adequate protein supply, thereby incorporating soybean to improve its protein quality. Only sensory analysis and proximate analysis were done on complementary infant food formulated with breadfruit and other locally available food materials such as malted, roasted maize, and roasted groundnuts in Nelson-Quartey et al., (2007). However, it was found to be comparable to commercial infant food with respect to protein and fat. The breadfruit cultivars used in all the above studies were unable to meet up to recommended dietary allowance and thereby it was necessary to incorporate other food materials. The nutritional quality varies across breadfruit cultivars (Jones et al., 2011a) with the Ma’afala cultivar recently discovered to have complete protein with higher quality than wheat, corn, rice, soybean, and yellow pea. 13 1.11 Overall Objectives Breadfruit is an abundant crop with high commercial value, but more data is needed to realize the crop potential. My thesis investigated 3 overall objectives. 1. Breadfruit as a key ingredient for infant formula 2. Breadfruit as a source of high-quality starches 3. Breadfruit as a source of high-value terpenes and secondary metabolites 1.11.1 Objective 1: Breadfruit as a key ingredient for infant formula Literature has reported the potential of breadfruit for infant foods but an infant formula comparable to commercial formulations based on animal milk, corn, wheat, or soy has not been developed. The following were my hypothesis statements: H1: An infant formulation based on breadfruit will meet the WHO’s standard for infant formula which will be sustainable, gluten-free, and soy-free to combat malnutrition globally. H0: The breadfruit-based infant formula will not meet the WHO’s standard for infant formula. To investigate this hypothesis, my specific objectives were to review the literature and government regulatory requirements for ingredients in infant formula and to compare with known values for breadfruit flours. 1.11.2 Objective 2: Breadfruit as a source of high-quality starches 1.11.2.1 Optimization of breadfruit starch extraction There are estimated to be >300 unique cultivars of breadfruit including both diploids and triploids but data on the characteristics of breadfruit starch across cultivars of A. altilis and hybrids is limited. The following were my hypothesis statements: 14 H1: Different cultivars of breadfruit have different starch and the mass of flour influences the starch yield. H0: There is no difference in starch between cultivars of breadfruit and the mass of flour does not affect the starch yield. My specific objectives were: (1) To develop and optimize methods for starch extraction from breadfruit (2) To compare starch yield, and starch-bound protein, for 55 breadfruit cultivars. (3) To determine important characteristics of breadfruit starches by comparison to ploidy and other botanical characteristics 1.11.2.2 Starch structural analysis There is limited data on the molecular structure of breadfruit starch across cultivars of A. altilis and hybrids. My specific hypotheses were: H1: Size and shape of starch granules influence the difference in the starch yield across breadfruit cultivars. H0: The structure of the starch granules did not affect the starch yield differences across cultivars. To investigate this, my specific objective was to determine the starch shape and size of the small cluster of the breadfruit cultivars. 15 1.11.2.3 Pasting and textural properties Based on the available literature, I predicted that the pasting and textural properties of breadfruit starches vary across a range between cultivars. My specific hypotheses were: H1: Pasting and textural properties vary between cultivars and influence starch yield. H0: There is no influence of pasting and textural properties on the differences in the starch yield. To investigate this hypothesis, my specific objectives were: 1. to compare pasting and textural properties of breadfruit flours from different cultivars. 2. to determine the relationships between pasting and textural properties and starch yield. 1.11.2.4 Cultivars of breadfruit (A. altilis (Parkinson) Fosberg and hybrids) have unique starch and fiber profiles. The preliminary results from 1.11.2.1 led to the extraction of a large quantity of starch for further characterization of the following hypothesis statements: H1: Breadfruit cultivars of A. altilis and hybrids have variable starch and fiber profiles. H0: There is no difference in starch and fiber profiles across breadfruit cultivars of A. altilis and hybrids. To investigate this, my specific objectives were: (1) to compare the dietary fiber content and profile between different breadfruit cultivars of A. altilis and hybrids and (2) to compare the starch yield, starch protein, structure, swelling, and solubility of different breadfruit cultivars of A. altilis and hybrids. 16 1.11.2.5 Effect of growing conditions on nutritional properties of Ma’afala Based on the available literature, I predicted that different growing models would influence the nutritional properties of trees grown from a single clone of Ma’afala. The following were my hypothesis statements: H1: All Ma’afala trees have similar nutritional content irrespective of their growth models. H0: Ma’afala trees from different growing models differ in nutritional content. Clones from a single Ma’afala (Maternal) grown in different models were used and my specific objectives were to (1) determine the fruit weight and % edible weight of clones and maternal tree; (2) determine the starch yield, the protein content of the starch and flour from the clones and maternal tree, and (3) compare the precipitation and temperature of the locations 1.11.2.6 Ma’afala (A. altilis) is an excellent source of fiber and starch in comparison to corn, rice, and wheat There is limited information on the comparison of the excellent commercial breadfruit cultivar- Ma’afala’s starch to the widely used traditional starches. The following were my hypothesis statements: H1: Ma’afala cultivar is comparable to traditional sources of starch. H0: Traditional sources of starch are better in quality than Ma’afala. To investigate this, my specific objectives were: (1) to compare Ma’afala to corn, rice, whole wheat, and all-purpose wheat flour on insoluble dietary fiber, soluble dietary fiber, and total dietary fiber and (2) to compare Ma’afala to corn, rice, whole wheat, and all-purpose wheat flour on starch yield, starch protein, structure, swelling, and solubility. 17 1.11.3 Objective 3: Breadfruit as a source of high-value terpenes and secondary metabolites Breadfruit glandular trichomes have not been studied in detail and the following was my hypothesis statements: H1: Breadfruit contains glandular trichomes and they vary across cultivars. H0: Breadfruit does not have glandular trichomes. To investigate this, my specific objectives were: (1) to develop a microscope method to view and quantify trichomes on breadfruit leaves; (2) to determine the correlation of length of breadfruit leaf to the number of trichomes; (3) to determine if the number of glandular trichomes varies cultivars, and (4) to developed methods for isolation of trichomes from breadfruit leaves. 18 Chapter 2: Breadfruit as a key ingredient for an infant formulation 2.1 Synopsis To date, there is no report on the nutritional quality of breadfruit in comparison to WHO’s standard for infant formula. I hypothesized that breadfruit is a suitable food staple that can provide adequate nutrition for infant formula. I compared studies that determined the nutritional content of breadfruit to the standard for infant formula by the world health organization (Codex Alimentarius CODEX STAN 72 – 1981, 2011), and my results showed that breadfruit can meet the recommended dietary allowance (RDA) for children in protein, carbohydrates, fat, vitamins, and minerals. The percentage of the required nutrients the breadfruit formula meets was calculated using the minimum and maximum reported value of breadfruit nutritional information. 2.2 Materials and Methods 2.2.1 Search design and information sources A search of the scientific, commercial and regulatory literature was performed using Web of Science™, Food and Drug Administration (FDA) database, Health Canada database, and European Union food databases to determine the baseline nutrient requirements of infant formulas. A systematic search of the scientific literature was conducted to determine the minimum and maximum reported values of nutrients in breadfruit. The maximum reported value of breadfruit nutrients was used to calculate the percentage of RDA (Codex Alimentarius CODEX STAN 72 – 1981, 2011) that a breadfruit formula has the potential to meet. The minimum reported value of breadfruit nutrients was used as well to account for the lowest case scenario. 19 2.3 Results 2.3.1 Commercially available infant formulas Out of the 23 analyzed powdered infant formulas as shown in Table 2.1, a common trend in the main ingredients was majorly from corn sources as corn syrup solids (4), corn syrup (2), and corn maltodextrin (2). The next common main ingredients were skim milk (5) and lactose (5). The other type of main ingredients that were used were referred to as milk ingredients (4) and partially hydrolyzed reduced minerals whey protein concentrate (1). Table 2.1. Product information of twenty-seven (27) commercial infant formulas included in the survey Product Name Recommended age Use Recommendation Main Ingredient Similac Total Comfort 0-12 months Fussiness Corn Maltodextrin Similac Step 2 6-24 months Routine formula Skim Milk Similac Step 1 0-12 months Routine formula Skim Milk Similac simply nurture 0-12 months Routine formula Skim Milk Similac Sensitive Lactose Sensitivity 0-12 months Fussiness Corn Syrup Similac Neosure 0-12 months Premature babies Skim Milk Similac Lower iron 0-12 months Routine formula Skim Milk Similac Isomil with DHA 0-12 months Soy formula Corn Syrup 20 Product Name Recommended age Use Recommendation Main Ingredient Similac Alimentum 0+ Food allergy or colic symptoms Corn Maltodextrin Similac Advance Step 2 6-24 months Routine formula Lactose Similac Advance Step 1 0-12 months Routine formula Lactose Puramino A+ 0-24 months Tummy Troubles, Birth+, hypoallergenic, Amino acid-based Corn Syrup Solids Pregestimil A+ 0-12 months Tummy Troubles, fat malabsorption, Allergy, Birth+ Corn Syrup Solids Nutramigen® A+® with LGG® 0+ months Tummy Troubles, Birth+, hypoallergenic Corn Syrup Solids Nestlé® Good Start® 1 Baby Formula 0 months and up Newborn Partially Hydrolyzed Reduced Minerals Whey Protein Concentrate Gerber® Good Start® A2 Powder Infant Formula 0-12 months For Advanced Digestive Support Lactose 21 Product Name Recommended age Use Recommendation Main Ingredient Enfamil Lower Iron Infant Formula 0-12 months When lower iron is preferred Lactose Enfamil Infant Formula 0-12 months Newborn, Birth+ Modified Milk Ingredients Enfamil A+® Infant Formula 0-12 months Newborn, Birth + Lactose Enfamil A+® Infant Formula 0-12 months Newborn, Birth + Water Enfamil A+ Soy 0-24 months Tummy Troubles, Birth + Water Enfamil A+ Lactose Free 0-12 months Tummy Troubles, Birth+ Water Enfamil A+ Gentlease 0-12 months Tummy Troubles, Birth+ Corn Syrup Solids Enfamil A+ for frequent Spit Up 0-12 months Tummy Troubles, Birth+ Milk Ingredients Enfamil A+ EnfaCare 0-12 months Designed for low birth weight or premature babies, Newborn Water Enfamil A+ 2 Infant Formula 6-18 months Next stage for a growing baby Milk Ingredients Enfamil 2 Infant Formula 6-18 months Balanced nutrition for your growing baby Milk Ingredients Note: Detailed ingredient list is in Appendix 1. 22 2.3.2 WHO recommendations Table 2.2. Essential composition of infant formula according to CODEX adopted as a worldwide standard. NUTRIENTS UNITS /100kcal WHO Minimum WHO Maximum Guidance Upper Levels (GUL) Protein g 1.8 3 _ Total Fats g 4.4 6 _ Total carbohydrates g 9 14 _ Linoleic Acid mg 300 _ 1400 α-Linolenic acid mg 50 N.S _ Vitamin A μg RE 60 180 _ Vitamin D3 μg 1 2.5 _ Vitamin E mg α-TE 0.5 _ 5 Vitamin K μg 4 _ 27 Thiamin μg 60 _ 300 Riboflavin μg 80 _ 500 Niacin μg 300 _ 1500 Vitamin B6 μg 35 _ 175 Vitamin B12 μg 0.1 _ 1.5 23 NUTRIENTS UNITS /100kcal WHO Minimum WHO Maximum Guidance Upper Levels (GUL) Panthothenic acid μg 400 _ 2000 Folic acid μg 10 _ 50 Vitamin C mg 10 _ 70 Biotin μg 1.5 _ 10 Iron mg 0.45 _ _ Calcium mg 50 _ 140 Phosphorus mg 25 _ 100 Magnesium mg 5 _ 15 Sodium mg 20 60 _ Chloride mg 50 160 _ Potassium mg 60 180 _ Manganese μg 1 _ 100 Iodine μg 10 _ 60 Selenium μg 1 _ 9 Copper μg 35 _ 120 Zinc mg 0.5 _ 1.5 24 NUTRIENTS UNITS /100kcal WHO Minimum WHO Maximum Guidance Upper Levels (GUL) Choline mg 7 _ 50 Myo-inositol mg 4 _ 40 L-Carnitine mg 1.2 N.S. _ 2.3.3 Comparison of breadfruit to WHO recommendations An infant formula composed of 75 % breadfruit flour contains Niacin, Manganese, Sodium, Magnesium, Calcium, Iron that met WHO requirement without exceeding guidance upper levels (GUL) or Max (Table 2.3). Breadfruit had Copper, Phosphorus, Manganese, Potassium that met the requirement but either exceeds the Max or GUL (Table 2.3). The number of ingredients included in the 27 commercially available formulas ranged from 33 to 53 with the average being 45 (Appendix 1). Although 75 % breadfruit flour for infant formula was slightly not enough to meet the protein, carbohydrate, or fat, there is a 25% allowance for the addition of limiting ingredients (Table 2.3). It was also unable to meet 7 other nutrients (Vitamin A, Thiamin, Riboflavin, Folic acid, Vitamin C, Chloride, and Zinc), however, these 7 nutrients were each added as ingredients in all the 27 commercial formulations except Riboflavin which was in 26 formulations (Table 2.3). 14 of the nutrients recommended by WHO have not been previously analyzed in breadfruit (Table 2.3). 25 Table 2.3. WHO’s essential nutrient requirement for infants in comparison to the reported nutritional content of breadfruit. Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Protein g 1.8 3 _ 2 18 0.14 1.4 No No Skim Milk - 7, Whey Protein Hydrolysate- 9, Whey Protein Concentrate- 8, Modified Milk Ingredients - 5, Milk Ingredients- 3, Hydrolyzed Casein* (From Milk)- 2, Other Amino Acids- 1, Soy Protein Isolate- 2, Casein Hydrolysate (Derived from Milk)- 1, Milk Protein 26 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Concentrate- 1, Milk Protein Isolate- 1, Partially Hydrolyzed Modified Milk Ingredients (Soy)- 1, Nonfat Dry Milk- 1, and Partially Hydrolyzed Reduced Minerals Whey Protein Concentrate -1 Total carbohydrates g 9 14 _ 50 88 3.75 6.6 No No Lactose- 18, Corn Syrup Solids- 15, Galactooligossacharides- 7, Polydextrose- 4, Sucrose- 4, Corn Maltodextrin- 4, Modified Corn Starch - 3, Corn Syrup- 2, Modified 27 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Tapioca Starch- 1, Glucose- 1, Rice Starch- 1 (Milk can add to the Total Carbohydrates but I included them in Protein) Total Fats g 4.4 6 _ 0.5 12 0.04 0.9 No No Soy Oil - 27, Coconut Oil- 25, High Oleic Sunflower Oil- 24, C. Cohnii Oil- 20, M. Alphina Oil - 20, Palm Olein Oil- 13, High Oleic Safflower Oil- 10, Medium Chain Triglycerides-5, and Mono and Diglycerides- 5 28 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Linoleic Acid mg 300 _ 1400 0.15 N.S 0.01 N.S No ND Same as in Total Fats α-Linolenic acid mg 50 N.S _ 2.13 N.S 0.16 N.S No ND Same as in Total Fats Vitamin A μg RE 60 180 _ ND 12.5 ND 0.94 ND No Vitamin A Palmitate- 25, and Vitamin A Acetate- 2 Vitamin D3 μg 1 2.5 _ ND N.S ND N.S ND ND 27 29 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Vitamin E mg α-TE 0.5 _ 5 0.1 N.S 0.01 N.S No ND 27 (D-Α-Tocopheryl Acetate), 1 (Tocopherols) and 23 (Mixed Tocopherols) Vitamin K μg 4 _ 27 0.5 N.S 0.04 N.S No ND 27 Thiamin μg 60 _ 300 290 560 21.75 42.00 No No Thiamine Hydrochloride- 25, and Thiamine Mononitrate- 2 Riboflavin μg 80 _ 500 160 390 12.00 29.25 No No 26 30 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Niacin μg 300 _ 1500 2300 4380 172.50 328.50 No Yes Vitamin B6 μg 35 _ 175 100 N.S 7.50 N.S No ND 27 Vitamin B12 μg 0.1 _ 1.5 ND N.S ND N.S ND ND 27 Panthothenic acid μg 400 _ 2000 457 N.S 34.28 N.S No ND 27 Folic acid μg 10 _ 50 ND 3.1 ND 0.23 ND No 27 31 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Vitamin C mg 10 _ 70 ND 22.7 ND 1.70 ND No 25 (Ascorbyl Palmitate) and 25 (Ascorbic Acid) Biotin μg 1.5 _ 10 N.S N.S N.S N.S ND ND 27 Iron mg 0.45 _ _ 0.5 12 0.04 0.90 No Yes Calcium mg 50 _ 140 5 800 0.38 60.00 No Yes 32 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Phosphorus mg 25 _ 100 74 1920 5.55 144.00 No Yes Magnesium mg 5 _ 15 10 200 0.75 15.00 No Yes Sodium mg 20 60 _ 2 598 0.15 44.85 No Yes Chloride mg 50 160 _ ND 4.9 ND 0.37 ND No Potassium Chloride -18, Sodium Chloride - 15, Magnesium Chloride -15, Calcium Chloride -5 33 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Potassium mg 60 180 _ 67 2830 5.03 212.25 No Yes Manganese μg 1 _ 100 80 2630 6.00 197.25 Yes Yes Iodine μg 10 _ 60 N.S N.S N.S N.S ND ND Potassium iodide -16, Sodium Iodide -3 Selenium μg 1 _ 9 0.6 N.S 0.05 N.S No ND Sodium Selenite -18, Sodium Selenate -9 34 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient Copper μg 35 _ 120 100 4950 7.50 371.25 No Yes Zinc mg 0.5 _ 1.5 0.13 2.97 0.01 0.22 No No 27 Choline mg 7 _ 50 9.8 N.S 0.74 N.S No ND Choline Bitartrate- 11, and Choline Chloride- 20 Myo-inositol mg 4 _ 40 N.S N.S N.S N.S ND ND 27 35 Nutrients Units /100kcal Minimum (WHO) Maximum (WHO) GUL Amount per 100g Breadfruit flour (Min) Amount per 100g Breadfruit flour (Max) Amount in Formula for 75% BF flour mix (per 75 ml serving)-Min Amount in Formula for 75% BF flour mix (per 75 ml serving)- Max Meets or Exceeds Requirement (Min BF) Meets or Exceeds Requirement (Max BF) Number of commercial formulations out of 27 that included the nutrient L-Carnitine mg 1.2 N.S _ N.S N.S N.S N.S ND ND 25 *Protein, Total fats, Total carbohydrates, Thiamine, Riboflavin, Niacin, Folic acid, Vitamin C, Iron, Calcium, Phosphorus, Magnesium, Sodium, Chloride, Potassium, Manganese, Copper, and Zinc content of breadfruit flour was extrapolated from Turi et al., (2015). *Value of 0 -12.5 retinol equivalents per 100 g of fresh fruit was reported by Jones et al., (2013a). *Linoleic and Linolenic acid values per 100g of ripe breadfruit were extrapolated from Golden and Williams, (2001). *Vitamins D3, E, K, B6, B12, Pantothenic acid, Selenium, and Choline were extrapolated from U.S. Department of Agriculture, Agricultural Research Service, FoodData Central, 2019 (raw breadfruit per 100g), https://fdc.nal.usda.gov/ (Selenium was calculated 36 from different food; From average values for food category; No adjustment; Retention factors not used. Choline was based on another form of the food or similar food; Concentration adjustment; No adjustment; Retention factors not used. Vitamin B-12 was assumed zero (An insignificant amount or not naturally occurring in a food, such as fiber in meat). Vitamin E (alpha-tocopherol) was based on another form of the food or similar food; Concentration adjustment; No adjustment; Retention factors not used. Vitamin D (D2 + D3) was assumed zero (An insignificant amount or not naturally occurring in a food, such as fiber in meat). Vitamin K (phylloquinone) was based on another form of the food or similar food; Concentration adjustment; No adjustment; Retention factors not used). *RDA is according to CODEX STAN 72-1981. * On average, every child should eat about 75 mls of formula per day per pound of body weight. * About 10 grams of powdered formula in 75 mls (by most recipes). NS = not specified and ND = not detected or below the limits of quantification. ND = Not Determined 37 2.4 Discussion Breadfruit has been initially researched for the formulation of weaning food, a food made for infants above 6 months of age but it was not compared to all the essential nutrients required by WHO’s standard for infant formula (Esparagoza and Tangonan, 1993; Ijarotimi and Aroge, 2005; Nelson-Quartey et al., 2007). The objective of this study was therefore to compare the literature reported nutritional value of breadfruit to WHO’s standard and commercial infant formulation. Corn was the most common source of main ingredients in all the commercial infant formulas which were similar to the report of Greer, (2018). This study shows that breadfruit can be used as the main ingredient in infant formula as a formulation with 75 % breadfruit met 9 of the WHO’s nutritional requirements. Although breadfruit did not meet the remaining nutrients, there is an allowance for the additional individual nutrients as observed in all the infant formulas that used corn. Rice starch was used as a second main ingredient in one of the commercial infant formulas that were labeled as a recommendation for spit-up in infants, but it also had additional ingredients. This study suggests that breadfruit might be an alternative to rice starch which is known to contain arsenic content that is not safe for infants (Biswas et al., 2020). Future studies should determine the nutrients in breadfruit that were not previously analyzed. This will be useful in knowing the exact number of limiting nutrients that a WHO standard infant formula made with 75 % breadfruit will need to include in its 25 % allowance. 2.5 Conclusion Breadfruit can be used as an ingredient for infant formula as 75% met the WHO’s infant formula requirements of Niacin, Manganese, Sodium, Magnesium, Calcium, Iron, Copper, 38 Phosphorus, Manganese, and Potassium. It was slightly not enough in meeting the requirements of protein, carbohydrate, or fat, however, there is 25% available for the inclusion of these ingredients. The addition of individual nutrients is common in the commercial formula as all the 27 infant formulas analyzed in this study had an average of 45 added ingredients. 39 Chapter 3: Method development and optimization 3.1 Synopsis There are estimated to be >300 unique cultivars of breadfruit including both diploids and triploids (Jones et al., 2011b; Ragone, 1997) and there is limited study on the starch variability across the cultivars. The first step in this study was to optimize the methods for the extraction of starch. I then hypothesized that starch characteristics vary across cultivars. I determined starch yield, starch-bound protein, and ploidy of 55 cultivars. The results of this experiment showed that 100g mass of flour yielded significantly more starch than 20g of flour. There were also differences in the starch yield and starch protein across the 55 cultivars. 3.2 Materials and Methods 3.2.1 Breadfruit germplasm repository at Kahanu garden, Maui Samples were harvested, from Kahanu Garden in Maui (20847057.0700N, 156802018.4200W) and transported to the University of British Columbia, Kelowna, Canada as described in Jones et al., (2011). The Breadfruit Institute germplasm collection at Kahanu is the largest and most diverse collection of breadfruit in the world (Murch et al., 2008b; Ragone, 1991, 1997) with > 330 accessions of breadfruit (A. altilis and A. altilis X A. mariannensis hybrids), breadnut (A. camansi Blanco), and dugdug (A. mariannensis Trecul.). The collection includes trees from at least 34 Pacific islands, the Philippines, Indonesia, and Seychelles. The trees used in this study were planted between 1978 and 2004 at 15 m elevation and samples harvested between 2008 - 2012. 40 Table 3.1. Cultivars details of the 55 samples of the different breadfruit cultivars (Artocarpus altilis and hybrids of A. altilis x A. marianensis). Hybrids are highlighted in red. Kahanu Garden Grid number Cultivar Name Ploidy Species Country / Island Group Island Date planted N6 Samoan 1 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 C5 Karawa 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 H8 Apuapua 3 Artocarpus altilis Society Islands Raiatea 03-26-1986 E4 Unidentified 01 3 Artocarpus altilis Unknown Unknown 03-26-1986 B7 Tuutou taatoe 3 Artocarpus altilis Society Islands Raiatea 03-26-1986 V4 Meriaur 3 Artocarpus altilis Palau Koror 07-26-1985 P5 Unidentified 07 2 Artocarpus altilis Fiji Unknown 07-26-1985 T3 Enua 3 Artocarpus altilis Cook Islands/WS Upolu Vailima 03-26-1986 E5 Meiarephe 3 Artocarpus altilis x A. mariannensis Pohnpei, FSM Pohnpei 03-26-1986 I8 Anahonaho 3 Artocarpus altilis Society Islands Raiatea 03-26-1986 KM1 Aveloloa 2 Artocarpus altilis Samoa Upolu Nafanua Agriculture Station 12-14-2000 41 Kahanu Garden Grid number Cultivar Name Ploidy Species Country / Island Group Island Date planted M6 Manua 3 Artocarpus altilis Samoa Savaii 03-26-1986 R8 Unidentified 25 (Lipet 2) 3 Artocarpus altilis Pohnpei, FSM Pohnpei 07-09-1987 U7 Karawa (Unidentified 12) 2 Artocarpus altilis Fiji Vitu Levu 07-26-1985 F6 Mei aueka 3 Artocarpus altilis Marquesas Islands Nuku Hiva 03-26-1986 N6-2 Samoan 1 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 01 Mei 1 (Piipia) 3 Artocarpus altilis x A. mariannensis Pohnpei, FSM Pohnpei Agriculture Station 09-11-1975 X4 Lemae/Lemai 3 Artocarpus altilis Mariana Islands Saipan 02-08-1985 D8 Lipet 3 Artocarpus altilis x A. mariannensis Pohnpei, FSM Pohnpei 07-09-1987 Heiau1 Ulu 3 Artocarpus altilis Hawaii Maui, Hana 12-31-1965 A8 Uto ni viti 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 I6 Tuutou ooa 3 Artocarpus altilis Society Islands Raiatea 03-26-1986 A7 Atu 3 Artocarpus altilis Cook Islands Rarotonga 03-26-1986 42 Kahanu Garden Grid number Cultivar Name Ploidy Species Country / Island Group Island Date planted B8 Mei kopumoko 3 Artocarpus altilis Marquesas Islands Nuku Hiva 03-26-1986 U2 Mei puau 3 Artocarpus altilis Marquesas Islands Nuku Hiva 07-26-1985 O7 Meisei 3 Artocarpus altilis Pohnpei, FSM Pohnpei 07-26-1985 G8 Huero 3 Artocarpus altilis Society Islands Raiatea 03-26-1986 P8 Kea 3 Artocarpus altilis Tonga Nukualofa 03-26-1986 S7 Puurea 3 Artocarpus altilis Society Islands Raiatea 07-26-1985 T5 Mei maoi 3 Artocarpus altilis Marquesas Islands Nuku Hiva 03-26-1986 V8 Afara 3 Artocarpus altilis Society Islands Tahaa 07-09-1987 B5 Meinpwahr 3 Artocarpus altilis x A. mariannensis Pohnpei, FSM Pohnpei 03-26-1986 R4 Puou (Uto Samoa) 2 Artocarpus altilis Fiji Viti Levu 07-26-1985 Heiau1 Ulu 3 Artocarpus altilis Hawaii Maui, Hana 12-31-1965 F7 Samoan 2 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 KM3 Unidentified 17 2 Artocarpus altilis Samoa Upolu Vailima 12-14-2000 53 Puou 2 Artocarpus altilis Samoa Upolu 12-30-1974 Y1 Hamoa 3 Artocarpus altilis Society Islands Tahaa 02-08-1985 43 Kahanu Garden Grid number Cultivar Name Ploidy Species Country / Island Group Island Date planted V7-2 Teahimatoa 3 Artocarpus altilis Society Islands Tahaa 07-26-1985 A5-2 Yap 3 Artocarpus altilis x A. mariannensis Palau Koror 03-26-1986 Heiau2 Ulu 3 Artocarpus altilis Hawaii Maui, Hana 12-31-1965 S5 Unidentified 10 3 Artocarpus altilis Unknown Unknown 07-26-1985 S9 Sagosago 2 Artocarpus altilis Samoa Savaii 07-09-1987 V7 Teahimatoa 3 Artocarpus altilis Society Islands Tahaa 07-26-1985 Heiau1 Ulu 3 Artocarpus altilis Hawaii Maui, Hana 12-31-1965 W3 Uto dina 2 Artocarpus altilis Fiji/WS Upolu Vailima 07-26-1985 B6 Mei puou 3 Artocarpus altilis Marquesas Islands Nuku Hiva 03-26-1986 V3 Patara 3 Artocarpus altilis Society Islands Tahaa 07-26-1985 A5-3 Yap 3 Artocarpus altilis x A. mariannensis Palau Koror 03-26-1986 35 Ulu fiti 2 Artocarpus altilis Rotuma/WS Upolu Vailima 02-08-1985 K9 Siviri 3 2 Artocarpus altilis Vanuatu Efate 07-09-1987 V6 Furau 2 Artocarpus altilis Rotuma/WS Upolu Vailima 07-26-1985 44 Kahanu Garden Grid number Cultivar Name Ploidy Species Country / Island Group Island Date planted W3-2 Uto dina 2 Artocarpus altilis Fiji/WS Upolu Vailima 07-26-1985 E7 Unidentified 02 2 Artocarpus altilis Samoa Savaii 03-26-1986 A8 Uto ni viti 2 Artocarpus altilis Fiji Viti Levu 03-26-1986 3.2.2 Preparation of breadfruit flour The fruit was picked, the peduncles were removed, and the fruit inverted for about 1 h to allow the latex to drain/coagulate (Figure 3.1A; Jones et al., 2013b, 2011a; Liu et al., 2014). The fruit was weighed, peeled with a household vegetable peeler (Figure 3.1B), quartered longitudinally, and the seeds (when present) were removed (Figure 3.1C). One-half of each harvested fruit was cut into 5–10 cm pieces, frozen, and shipped to UBC where they were stored at -86 ° C. One half of each fruit was sliced into 2 mm sections using a mandolin vegetable slicer (Figure 3.1D; Benriner, Philadelphia, PA) and dried on screen trays in a dark drying room at 22–47 ° C and 19–47 % relative humidity at Kahanu Garden before shipping to UBC. The dehydrated slices of fruit were ground into flour using the finest setting of a commercial coffee grinder (Bunn1 model G3, Aurora, ON). The flour was stored at room temperature in air-tight Ziplock bags until analysis. As part of a study of the shelf life of the flours, samples were assessed for degradation. Discoloration, odors, or evidence of decay were 45 not noted indicating that the shelf life of breadfruit flour can exceed 10 years in a cool, dry, dark, air-tight storage facility. Figure 3.1. Preparation of breadfruit flour 46 3.2.3 Optimization of starch extraction for breadfruit flours The previous method of starch isolation (Adebowale et al., 2005) was employed. Briefly, breadfruit flour was dispersed in 50ml ePure water and sieved (75 μm nut milk bag, Wielding Trading Co, Ltd, China). Impurities were removed from the starch by 3 x repeated washing with ePure water. The supernatant was decanted, and the pellet was air-dried at 23±°C for 48 hours to obtain the final starch extract. The starch yield was calculated by Equation 3.1. 𝒀𝒊𝒆𝒍𝒅 (%) = 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒔𝒕𝒂𝒓𝒄𝒉 (𝒈)𝒎𝒂𝒔𝒔 𝒐𝒇 𝒇𝒍𝒐𝒖𝒓 (𝒈) ∗ 𝟏𝟎𝟎 Equation 3.1 This was then optimized by using two mass categories on three cultivars (Ulu, Ulu fiti, and Yap). The first mass category was 20g and the second mass category was 100g with a modification to the water ratio (1:10). This optimum method was then employed in any further starch extraction studies. However, 20g flour was employed for starch extraction of the fifty-five different samples due to insufficient samples across some of the cultivars. 3.2.4 Protein assay 3.2.4.1 Chemicals HEPES (Fisher Scientific, Mississauga, ON), Tween 20 (MP Biomedicals, California, USA), BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific, Montreal, Canada), bovine serum albumin protein standard solution (Thermo Scientific, Montreal, Canada), NaCl (Fisher Scientific, Mississauga, ON). 47 3.2.4.2 Isolation of proteins Protein was extracted from the isolated breadfruit and control starches according to the method of Jones et al., (2011). In brief, 10 mg of each starch extract was weighed into a 1.5 mL microcentrifuge tube (Fisher Scientific, Ottawa, ON) with 0.5 mL protein extraction buffer (20 mM HEPES, 150 mM NaCl, 0.3% Tween 20). The mixture was vortexed and sonicated for 15 min at room temperature (FS20, Fisher Scientific). The tubes were centrifuged for 10 min at 13,000 RPM (Galaxy 16DH; VWR, Westchester, PA). The soluble proteins (supernatants) were decanted and assayed for the protein content. 3.2.4.3 Bicinchoninic acid (BCA) protein assay The protein content of the supernatant was determined with a Pierce™ BCA Protein Assay Kit (Fisher) and with a serial dilution of bovine serum albumin (BSA; 0, 0.01, 0.05, 0.1, 0.15, 0.25, 0.375, and 0.5mg/mL) protein standard for quantification (Fisher) as described previously (Jones, 2011; Liu, 2014). A 25 µL aliquot of each standard solution or protein extract was dispensed into a 96 well microplate (Bio-Rad Laboratories, Ontario, Canada) in a randomized manner. BCA working reagent (200 µL; 50:1, v/v, Reagent A/Reagent B) was added to each well and the plates were gently shaken for 30 seconds, covered with film, incubated at 37°C-50°C for 1hr, and then cooled to room temperature. The absorbance was measured using a Synergy HT Multi-Mode Microplate Reader (BioTek Instruments, Vermont, USA) using Gen5: Microplate Data Collection and Analysis Software (BioTek) at 562 nm. Protein content was determined in triplicate for each standard and sample. The protein content was calculated by comparing it to a standard curve of varying concentrations of BSA standard solutions (125 - 2000 µg/mL). The protein working solution was used as a blank to determine any contribution from the reagents. 48 3.2.5 Experimental design and statistical analysis The experiments on the starch yield and starch protein were conducted on 55 cultivars. The starch-protein content was conducted in triplicates for each cultivar and significant differences were done using ANOVA with Prism (v9.0, GraphPad Prism LLC). All results were plotted in Prism (v9.0, GraphPad Prism LLC). Significant differences for averages of A. altilis and all hybrids of A. altilis x A. marianensis were determined by Welch’s t-test at α = 0.05 using Prism (v9.0, GraphPad Prism LLC). Significant differences across the starch yield from the two different flour mass were determined by Welch’s t-test at α = 0.05 using Prism (v9.0, GraphPad Prism LLC). 3.3 Results 3.3.1 Optimization of starch extraction protocols Fifty-five cultivars of breadfruit were evaluated for optimization of starch extraction and starch characterization protocols (Table 3.1). The amount of flour used in the initial starch extraction significantly affected the results. The starch yield from the 100g flour (15 %) was more than seven times greater than the starch yield obtained from the 20g flour (2.1 %). The starch yield from the 100g flour was statistically different from the 20g flour (Figure 3.2). Studies of 20g samples of 55 cultivars revealed significant differences in the starch yield ranging from 1 % to 6.5 % with an average yield of 3.2% (Figure 3.3). The cultivated variety Samoan yielded the highest amount of starch and the cultivar Uto ni viti had the lowest starch yield. When the cultivars were grouped by A. altilis and hybrids, the mean starch yield of A. altilis (3.2 %) was not significantly different from A. altilis X A. mariannensis (3.2 %; Figure 3.4). 49 Figure 3.2. Mean with standard error bars of the starch yield of different breadfruit flour mass. The different superscript differs (p ≤ 0.05) using Welch’s t-test. Treatments were starch extracted from breadfruit using two masses- 20g and 100g of flour. 50 Figure 3.3. The starch yield of 55 breadfruit cultivars. Black bars represent A.altilis while grey bars represent the hybrids. 51 Figure 3.4. Mean with standard error bars of the starch yield of 55 breadfruit cultivars by grouped A. altilis and hybrids. The significant differences (p ≤ 0.05) were shown using Welch’s t-test. Black bars represent A. altilis while grey bars represent the hybrids. 3.3.2 Differences in the starch-bound protein across breadfruit 55 cultivars The breadfruit starch yield of the 55 cultivars ranged from 1 % to 6.5 % Samoan with an average yield of 3.2% (Figure 3.5). Samoan had the highest yield while Uto ni viti had the 52 lowest yield. When grouped by A. altilis and hybrids, the mean starch yield of A. altilis (3.2 %) was not significantly different from A. altilis X A. mariannensis (3.2 %; Figure 3.6). 53 Figure 3.5. Mean with standard error bars of starch-bound protein of 55 breadfruit cultivars. Black bars represent A. altilis while grey bars represent the hybrids. Figure 3.6. Mean with standard error bars of starch-bound protein of 55 breadfruit cultivars grouped by A. altilis and hybrids. The significant differences (p ≤ 0.05) were shown using Welch’s t-test. Black bars represent A. altilis while grey bars represent the hybrids. 54 3.4 Discussion There is limited information available on starch and flour characteristics across breadfruit cultivars. I analyzed the starch extractions of 55 cultivars from A. altilis and hybrids. The starch yield of the cultivars in this study varied despite being grown under the same conditions and the average of the 55 cultivars was within the range of the 3.19% yield reported by Awokoya et al., (2018) and less than the 14.3% from Akanbi et al., (2009). Hybridization did not affect the starch yield and starch-bound protein in this study. An important quality consideration in the starch isolation process is the starch yield and this process influences the functionality of the starch (Vithu et al., 2020). A study by Vithu et al., (2020) showed that starch yield from 100g sweet potato slices increased from 9.063 % to 16.371 % when the number of filtration washing was increased from was once to twice with an optimal yield of 13. 967 %. The study of Lumdubwong and Seib, (2000) showed that alkaline protease digestion increased starch recovery from 20 g wet-milled rice flour from 85. 4 % (only alkaline) to 95.1 %. In another study of isolation of starch from 100g, long-grain rice flour through alkaline steeping gave 71.6 %. (Wang and Wang, 2004). However, the alkaline method of starch isolation results in a large number of alkaline wastes, and the wastewater treatment is expensive (Wang and Wang, 2004). My study was done to optimize the starch isolation process from breadfruit flour to get a better quantity of starch without the addition of chemicals. I hypothesized that the starting mass of flour affects starch yield. Breadfruit starch has been previously extracted from fruits in several studies (Bezerra et al., 2019, Nwokocha and Williams, 2011, Bezerra et al., 2019, Adebowale et al., 2005, Gbadamosi and Oladeji, 2013, Marta et al., 2019, Rahayu et al., 2019). The yield was reported only in few studies as 14.26 % (Akanbi et al., 2009) and 18.5 % (Rincón, 2005) without the mention of the starting mass of fruit. However, to the best of my knowledge, this is the first time there is a report on differences in starch yield arising from method factors. My results showed that starting mass of flour influenced the starch yield as the yield from 100g flour differs significantly 55 from that of 20g. My results also show the importance of considering starting mass of flour when comparing the starch yield of plant sources in the literature. The results from this study help to maximize the efficiency of starch extraction from flour and further similar work described in this thesis was done using this optimized method. 3.5 Conclusion The starch yield, the protein concentration of starch varied among different cultivars but not across the group of A. altilis and hybrids. The starch yield was optimized using a higher amount of flour. 56 Chapter 4: Starch structural analysis 4.1 Synopsis The size and shape of starch granules can also influence the yield, textural, physical, and pasting properties of starches (Singh et al., 2007; Zhu, 2015). I hypothesized that the size and shape of starch granules differ across breadfruit cultivars. To investigate this, I determined the starch structure, size, and starch characteristics on a subset of breadfruit cultivars representing the diversity across A. altilis and hybrids. 4.2 Materials and Methods 4.2.1 Scanning electron microscopy (SEM) of breadfruit starch Suspensions of dry starch from 6 different cultivars were dusted onto aluminum stubs using double-sided adhesive tape (Loos et al., 1981). They were coated with gold using a sputter coater (Cressington sputter coater HR, 208) to 20 nm thickness. Samples were then viewed using a scanning electron microscope (Tescan Mira3 XMU field emission gun) at an acceleration potential of 10 - 20 kv. The micrographs were captured at a magnification of 1k, 2k, and 5k using automatic image capture software. All variables such as brightness, contrast, beam intensity, were kept constant for each magnification during the microscopy. 4.2.2 Image processing from SEM The SEM images were processed with custom scripts written in Matlab® to determine the size of the starch granules. Filtering parameters such as circularity, eccentricity, normalized area, and major axis length were employed in processing the images. The area and major axis length of the images were determined in pixels and converted to µm. 57 4.3 Results 4.3.1 Starch size and structure of different breadfruit cultivars Breadfruit starch granules appeared polygonal and circular in shape in most cultivars (Figure 4.1). The exception was the Aveloloa cultivar which had granules that appeared to be made up of only circular shapes (Figure 4.1). Sizes of breadfruit starches ranged from 7.8 µm to 10.3 µm in length as shown in Table 4.1. Figure 4.1. The scanning electron micrographs of six different breadfruit starches are shown in the order of Ulu, Tuutou taatoe, Aveloloa, Unidentified 17, Yap, and Samoan at 1000x magnification. A B C D E F 58 Table 4.1. SEM granule sizing of different breadfruit starch micrographs captured at 1000 magnification. Cultivar Name Mean Area (µm2) Median Area (µm2) Standard Error Area (µm2) Mean Major Axis Length (µm) Median Major Axis Length (µm) Standard error Major Axis Length (µm) Ulu 39.6 32.6 1.4 7.8 7.4 0.1 Yap 42.6 34.6 1.8 8 7.8 0.2 Aveloloa 43 30.9 2.5 7.9 7.4 0.2 Samoan 51 40.5 2.7 8.8 8.5 0.3 Unidentified 17 57.5 53.2 1.8 9.4 9.5 0.2 Tuutou taatoe 69.1 71.8 2 10.3 10.8 0.2 4.4 Discussion Based on the yield data from the 55 cultivars in chapter 2, 6 significant representative subsets were chosen for further analysis on starch characteristics. The starch structure of the 6 cultivars was similar in polyhedral shapes and comparable to literature (Loos et al., 1981; Nwokocha and Williams, 2011; Rincón, 2005; Tan et al., 2017) except for cultivar Aveleloa which had granules that appeared more globular. The globular shape observed might be due to the influence of a different shape of amylopectin molecules unlike the other starches (Jane, 2007). This can be exploited because the shape of 59 starch granules affects starch reactivity by making the granule susceptible to acid or enzymes (Oates, 1997). Further starch characteristics were unable to be performed on these 6 cultivars due to insufficient samples. Future studies should determine if the digestibility or amylopectin shape of Aveloloa is different from the other breadfruit starches. 4.5 Conclusion The starch granules of the different breadfruit cultivars were mostly polygonal and circular shaped except Aveloloa which was made up of globular granules. The average granule sizes varied between breadfruit starches and thus can be exploited for hydration, swelling capacity, digestibility, and amylose content. 60 Chapter 5: Pasting and textural properties 5.1 Synopsis The flour from the different flours varied in appearance upon slurry. I, therefore, hypothesized that pasting and textural properties varied among the cultivars and can influence their starch yield. To investigate this hypothesis, I determined the correlation of pasting and textural properties of breadfruit flour to its starch yield 5.2 Materials and Methods 5.2.1 Gel textural analysis Gel hardness, adhesiveness, springiness, cohesiveness, gumminess, and resilience will be determined on the flour gel formed in the RVA canister using a Texture Analyzer (Model TA-XT Icon, Stable Micro Systems, Texture Technologies Corp., South Hamilton, Massachusetts, USA). After RVA testing, the paddle will be removed, and the flour paste in the canister will be covered by parafilm and stored at 4 °C for 24 h. Each flour gel in the canister will be compressed to 10 mm (trigger force = 0.03 N) with a cylinder probe (8 mm in diameter) at a speed of 0.5 mm/s. Gel textural properties will be recorded. 5.2.2 Pasting properties The pasting properties of the breadfruit flour suspension were analyzed according to the methodology of (Huang et al., 2019) with modifications. Pasting properties were conducted with a Rapid-Visco Analyser (#RVA-4, Newport Scientific Inc.; Hägersten, Sweden), where the maximum temperature was 95°C. The moisture content of breadfruit starch was first measured by a moisture meter (MB45, OHAUS Corp.; Parsippany, New Jersey, USA) to obtain the correct starch sample weight and amount of water required for the test. The starch suspension (28 g 61 total weight for the first method) was poured into an aluminum sample cup and the cup, with a plastic stirring paddle, was inserted into the RVA machine. The sample temperature was held at 50°C for 1 min, increased to 95°C, held at 95°C for 2.5 min, lowered to 50°C, and held at 50°C for 2 min. 5.2.3 Experimental design and statistical analysis Experiments on pasting and textural properties were conducted on only a subset of 15 cultivars. The results of both pasting and textural properties were analyzed in Excel™. Pearson correlation coefficient analysis was performed to determine the co-linearity among texture properties, pasting properties, and starch yield using Excel™. 5.3 Results 5.3.1 Textural properties vary across breadfruit cultivars. There was a strong negative correlation between hardness and adhesiveness (-0.92; Table 5.2). There was a strong positive correlation between the hardness and both gumminess and chewiness (0.97; Table 5.2). Gumminess was positively correlated with chewiness with a coefficient of 1 (Table 5.2). The correlations, therefore, indicate that the higher the hardness, the more the adhesiveness, gumminess, and chewiness. This was observed as across the measured textural properties of 15 cultivars, Kea had the maximum hardness, adhesiveness, gumminess, and chewiness while Uto ni viti had the lowest hardness, springiness, gumminess, and chewiness (Table 5.1). Springiness showed a positive correlation with a coefficient of 0.55 and 0.53 to cohesiveness and resilience respectively (Table 5.2). Yap had the maximum springiness, cohesiveness, and resilience and lowest adhesiveness. Although Ulu (Heiau2) also had equal resilience as Yap (Table 5.1). Unidentified 02 (E7) had the lowest cohesiveness with Puurea having the lowest resilience (Table 5.1). This reflects the correlation coefficient as it is 62 not a strong positive correlation. There was no correlation between each of the textural properties and the starch yield except resilience (-0.5; Table 5.3). Table 5.1. Gel texture profile analysis of breadfruit flours Cultivar Name Hardness (N) Adhesiveness (g.sec) Springiness (%) Cohesiveness (%) Gumminess Chewiness Resilience (%) Kea 0.129 -0.627 91.6 56.3 0.073 0.067 4.7 Puou 0.077 -0.364 92.3 55.9 0.043 0.04 4.2 Karawa 0.067 -0.439 90.6 56.9 0.038 0.035 3.7 Teahimatoa 0.102 -0.579 92.4 56.2 0.058 0.053 4.1 Ulu 0.103 -0.524 91.6 55.1 0.057 0.052 4.8 Apuapua 0.092 -0.564 91.5 58.7 0.054 0.049 4.3 Mei aueka 0.083 -0.45 91 55.9 0.046 0.042 4.1 Puurea 0.077 -0.446 92.1 53.2 0.041 0.038 3.6 Unidentified 01 0.095 -0.576 91.5 57.8 0.055 0.05 3.8 Yap 0.112 -0.601 91.5 52.4 0.058 0.053 4.7 Patara 0.081 -0.456 91.6 56 0.045 0.041 4.4 Uto ni viti 0.054 -0.342 89.5 55 0.029 0.026 4.3 Yap 0.059 -0.314 93 62.4 0.037 0.034 4.8 Uto ni viti 0.051 -0.342 90.4 55.5 0.028 0.025 4.5 Unidentified 02 0.074 -0.389 92 43.1 0.032 0.029 3.9 63 Table 5.2. Correlation coefficients (r) between texture profile analysis of breadfruit flours Hardness Adhesiveness Springiness Cohesiveness Gumminess Chewiness Resilience Hardness -0.92 0.54 0.25 0.97 0.97 0.45 Adhesiveness -0.52 -0.42 -0.93 -0.93 -0.4 Springiness 0.55 0.61 0.62 0.53 Cohesiveness 0.46 0.47 0.59 Gumminess 1 0.53 Chewiness 0.52 Table 5.3. Correlation coefficients (r) between Starch Yield and Textural Properties Hardness (N) Adhesiveness (g.sec) Springiness (%) Cohesiveness (%) Gumminess Chewiness Resilience (%) Correlation with starch yield (%) 0.2 -0.4 -0.3 0.4 0.3 0.3 -0.5 64 5.3.2 Pasting properties vary across breadfruit cultivars. The viscosity of the flours from the different cultivars appeared differently when slurred with the same amount of water (Figure 5.1). Across the measured pasting properties of 15 cultivars, Kea had the highest peak, trough, final, and setback viscosity, and Unidentified 02 (E7) had the lowest of these viscosities (Table 5.4). The setback viscosity ranged from 1160 cP to 2251cp with an average of 1491P (Table 5.4). Unidentified 02 (E7) had the least setback viscosity. Pasting temperature which is the minimum temperature required to cook the flour ranged from 75.1°C (Puurea) to 78.45° C (Uto ni viti) with an average of 76.6°C. The breakdown viscosity was from 39cP to 152cP with an average of 93.9cP (Table 5.4). Puou had the lowest breakdown viscosity while Yap had the highest breakdown viscosity. Final viscosity measures the ability of the flour to be viscous at the end of the cooking and cooling cycle. Final viscosity ranged from 1877 cP to 4768cp with an average of 3146 cP (Table 5.4). Peak time is the time taken for a peak viscosity to occur and the flours had similar peak times ranging from 6.3 minutes (Ulu) to many cultivars with 7 minutes with an average of 6.8 minutes (Figure 5.2). There was a correlation, although not strong, between each of the pasting properties and the starch yield except setback viscosity, peak, and pasting temperature (Table 5.5). 65 Figure 5.1. The appearance of breadfruit flours from different cultivars when slurred with water using the method as described in Chapter 3, section 3.2.3. 66 Table 5.4. Pasting properties of different breadfruit flours using Rapid-Visco Analyser (maximum temperature of 95 °C) Cultivar Name Final Viscosity, cP Peak viscosity, cP Trough viscosity, cP Breakdown viscosity, cP Setback, cP Peak Time, min Pasting Temperature, °C Kea 4768 2601 2517 84 2251 6.87 76.75 Puou 3729 2342 2303 39 1426 6.67 76.8 Karawa 3626 1959 1879 80 1747 6.67 76.75 Teahimatoa 3521 2007 1916 91 1605 6.4 76.7 Ulu 3445 1909 1760 149 1685 6.33 76 Apuapua 3429 1965 1873 92 1556 6.67 75.85 Mei aueka 3354 1956 1886 70 1468 6.73 76.05 Puurea 3354 1935 1866 69 1488 6.67 75.1 Unidentified 01 3327 1961 1911 50 1416 6.8 75.15 Yap 3279 1952 1875 77 1404 6.73 75.95 Patara 3189 1669 1550 119 1639 7 76.8 Uto ni viti 3110 1811 1679 132 1431 7 78.45 Yap 2678 1584 1432 152 1246 7 76.7 Uto ni viti 2339 1126 1036 90 1303 7 76.8 Unidentified 02 1877 844 717 127 1160 7 76.75 67 Table 5.5. Correlation coefficients (r) between Starch Yield and Pasting Properties Final Viscosity, cP Peak viscosity, cP Trough viscosity, cP Breakdown viscosity, cP Setback, cP Peak Time, min Pasting Temperature, °C Correlation with starch yield (%) 0.5 0.5 0.6 -0.5 0.3 -0.3 -0.3 Figure 5.2. The pasting curve of the different breadfruit flours 68 5.4 Discussion During the starch extraction from the flours of the 55 cultivars, some of them varied in an appearance on slurry with water. This led to the hypothesis that the pasting and textural properties varied among the cultivars. My data shows that the pasting and textural properties were different among the flours. These results are consistent with previous studies that had shown that textural properties varied among different cultivars of corn (Sandhu and Singh, 2007). These varying differences might be due to different carbohydrate contents (Huang et al., 2019) which were observed in this study as the starch yield across the cultivars was different. The texture is important in the food industry as consumers usually have preferences for certain textural attributes (Brennan et al., 2004). Also, the level of textural attributes required depends on the type of desired product, for example, high adhesiveness made it more difficult to manage the sliced product flow of low-fat sausage, due to high stickiness (Álvarez and Barbut, 2013), and the lower adhesiveness made it more difficult for corn masa to stick to the shaping rollers for tortilla production (Topete-Betancourt et al., 2020). The absence of a well-defined peak in the pasting curve of all the breadfruit flours shows that starch granules have not completely ruptured at 95°C and hence, the continuous increase in viscosity on cooling. According to Huang and Bohrer, (2020), increasing the temperature to 130°C gave a higher peak as compared to the peak at 95°C but the viscosity decreased significantly on cooling. This suggests that the temperature applied affects the time it takes for a well-defined peak to be attained and if the temperature is not fully increased to its maximum capacity, the breadfruit flour can continue to increase in viscosity even on cooling. This can be advantageous in food applications that requires a stable viscosity such as infant formula. The pasting and textural analysis was not done in triplicate, so a conclusion as to if they differ significantly was unable to be made. Further replicate analysis should be done to select distinct cultivars in terms of pasting and textural properties for food and industrial applications. 69 5.5 Conclusion The textural and pasting properties of breadfruit flour varied among different cultivars. This can be exploited in the food and manufacturing industry that requires high viscosity, a low rate of retrogradation, and different degrees of textural properties. 70 Chapter 6: Cultivars of breadfruit (A. altilis (Parkinson) Fosberg and hybrids) have unique starch and fiber profiles 6.1 Synopsis Nutritional and physical properties are reported to vary significantly across breadfruit cultivars (Jones et al., 2013a, 2011a; Liu et al., 2015) but the starch and flour characteristics across cultivars of A. altilis and hybrids have not been studied in detail. I hypothesized that breadfruit cultivars of A. altilis and hybrids have variable starch and fiber profiles and content. To investigate this, I did the following: (1) compared the dietary fiber content and profile between different breadfruit cultivars of A. altilis and hybrids and (2) compared the starch yield, starch protein, structure, swelling, and solubility of different breadfruit cultivars of A. altilis and hybrids. 6.2 Materials and Methods 6.2.1 Breadfruit germplasm repository at Kahanu garden, Maui The samples were harvested, from Kahanu Garden in Maui (20847057.0700N, 156802018.4200W) and the details are fully described in Chapter 3, section 3.2.1. 71 Table 6.1. Breadfruit cultivars (Artocarpus altilis) and hybrids (A. altilis x A. marianensis) that were included in the study. All samples were harvested from representative trees in the collections at Kahanu Garden, Maui. Cultivar Name Species Ploidy Country / Island Group Island Ulu fiti Artocarpus altilis 2 Samoa Upolu Roihaa Artocarpus altilis 3 Society Islands Huahine Lipet Artocarpus altilis x. A. mariannensis 3 Federated States of Micronesia Pohnpei Lemae Artocarpus altilis x. A. mariannensis 3 Mariana Islands Rota Otea Artocarpus altilis 3 Society Islands Moorea White Artocarpus altilis 3 Seychelles Mahe Yap Artocarpus altilis x A. mariannensis 3 Palau Koror Puaa Artocarpus altilis 3 Society Islands Moorea Ma’afala Artocarpus altilis 2 Samoa Upolu Apu Artocarpus altilis 3 Society Islands Raiatea 72 Cultivar Name Species Ploidy Country / Island Group Island Ulu elise 1 Artocarpus altilis x. A. mariannensis 2 Tokelau Fakaofo Meinpadahk Artocarpus altilis x. A. mariannensis 3 Federated States of Micronesia Pohnpei Midolab Artocarpus altilis x. A. mariannensis 3 Palau Babeldaob Yuley Artocarpus altilis x. A. mariannensis 3 Federated States of Micronesia Yap Meinpohnsakar Artocarpus altilis x. A. mariannensis 3 Federated States of Micronesia Pohnpei 6.2.2 Preparation of breadfruit flour The details of the preparation of breadfruit flour from the fruit are fully described in Chapter 3, section 3.2.2. 73 6.2.3 Isolation of breadfruit starch Starch was isolated from breadfruit flour using the optimized method as described in Chapter 3, section 3.2.3. 6.2.4 Total Dietary Fibre (TDF)/Soluble Dietary Fibre (SDF)/ Insoluble Dietary Fibre (IDF) The total dietary fiber assay kit (Sigma-Aldrich, St. Louis, USA) was used for fiber analysis according to manufacturer instructions and following the American Association of Cereal Chemists (AACC) method NO. 32-07 (AACC, 2000). The experiment was conducted in triplicate. The flour samples (1 g) and an empty glass bottle without flour (blank) were homogenized in 40 mL MES – TRIS buffer (0.05 M MES; PhytoTechnology Laboratories, Kansas, USA with 0.05 M TRIS; Fisher Scientific, Mississauga, ON) and incubated with 200 µL α-amylase solution at 95 - 100 ° C for 35 min in a water bath. After cooling to 60 ° C, 100 µL protease solution (Fisherbrand, Mississauga, ON) was added to the sample, followed by a 30 min incubation at 60 ° C. The samples were adjusted to pH 4.1 - 4.8 with HCl (0.325 M) before 300 µL amyloglucosidase solution was added. The samples were incubated with amyloglucosidase for 30 min at 60 ° C and filtered through nylon woven filters (mesh opening: 53 µm, open area: 36 %, thickness: 60 µm; Spectra/Mesh®, Spectrum Laboratories, Inc.). After filtration, both the residue and filtrate were used to determine the total dietary fiber (TDF) content. The residue was washed and rinsed with 95 % ethanol and acetone and oven-dried at 100 ± 5 ° C overnight for insoluble dietary fiber (IDF) calculation, followed by protein and ash correction. The filtrate was adjusted to 80 g with water and mixed with 320 mL preheated (60 ° C) 95 % ethanol for 4 h. Soluble dietary fiber (SDF) was precipitated, filtered, and oven-dried at 100 ± 5 ° C overnight. Protein and ash correction was performed for the calculation of SDF content. The IDF, SDF, and TDF were calculated using Equations 6.1, 6.2, and 6.3 respectively. 74 𝑰𝑫𝑭 (%) = (𝒓𝒆𝒔𝒊𝒅𝒖𝒆 𝒘𝒆𝒊𝒈𝒉𝒕−𝒑𝒓𝒐𝒕𝒆𝒊𝒏 𝒘𝒆𝒊𝒈𝒉𝒕−𝒂𝒔𝒉 𝒘𝒆𝒊𝒈𝒉𝒕−𝒃𝒍𝒂𝒏𝒌 𝒘𝒆𝒊𝒈𝒉𝒕)𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕× 𝟏𝟎𝟎 Equation 6.1 𝑺𝑫𝑭 (%) = (𝒓𝒆𝒔𝒊𝒅𝒖𝒆 𝒘𝒆𝒊𝒈𝒉𝒕−𝒑𝒓𝒐𝒕𝒆𝒊𝒏 𝒘𝒆𝒊𝒈𝒉𝒕−𝒂𝒔𝒉 𝒘𝒆𝒊𝒈𝒉𝒕−𝒃𝒍𝒂𝒏𝒌 𝒘𝒆𝒊𝒈𝒉𝒕)𝒔𝒂𝒎𝒑𝒍𝒆 𝒘𝒆𝒊𝒈𝒉𝒕× 𝟏𝟎𝟎 Equation 6.2 𝑻𝑫𝑭 (%) = 𝑰𝑫𝑭 (%) + 𝑺𝑫𝑭 (%) Equation 6.3 6.2.5 Total ash analysis Total ash content was determined as described in AACC method 08-01 (AACC, 2000). Residues of SDF and IDF samples were weighed in pre-weighed and pre-dried porcelain crucibles (Coorstek, Golden, CO) before (W1) and after incineration (𝑊2) in an isotemp programmable muffle furnace (Fisher Scientific, Ottawa, ON) at 550 ° C for 5 hours to determine the total ash weight (Equation 6.4). 𝑨𝒔𝒉 𝒘𝒆𝒊𝒈𝒉𝒕 (𝒈) = 𝐖𝟏 (𝒈) − 𝑾𝟐(𝒈) Equation 6.4 6.2.6 Protein determination of flour, starch, and fiber Determination of total protein content of flours, starch-bound protein, and the oven-dried fiber residue (SDF and IDF) was performed using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit, (Thermo Scientific, Montreal, Canada). The procedure of the isolation of protein and BCA assay are fully described in Chapter 3, section 3.2.4.2, and section 3.2.4.3 respectively. 75 6.2.7 Scanning electron microscopy (SEM) of breadfruit starch The breadfruit starches were imaged using SEM with details fully described in Chapter 4, section 4.2.1 6.2.8 Image processing from SEM The SEM images were processed using the measure function in ImageJ (Schindelin et al., 2012) to determine the size of the starch granules. Filtering parameters such as scale and length were employed in processing the images. A line was manually drawn across the longest dimension where the shape is irregular. Compound granules were measured as a single particle with the use of a line tool. 6.2.9 Swelling and solubility To calculate swelling power 1 % aqueous suspension of each starch sample was heated to 70 ° C for 1 h with constant stirring in a water bath (Huang et al., 2019). Samples were then centrifuged at 3,000 × g for 10 min and the weight of sediment was measured for the determination of the swelling power (Equation 6.6). The supernatants were then evaporated at 100 ± 10 ° C for 24 h using a drying oven (Innova®44, Eppendorf, Connecticut, USA) and the weight of the remaining dry solids was determined for the calculation of solubility (Equation 6.5). 𝑺𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚 (%) = 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐝𝐫𝐲 𝐬𝐨𝐥𝐢𝐝 (𝒈) 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐭𝐡𝐞 𝐭𝐨𝐭𝐚𝐥 𝐬𝐭𝐚𝐫𝐜𝐡 𝐬𝐚𝐦𝐩𝐥𝐞 (𝒈) ∗ 𝟏𝟎𝟎 Equation 6.5 𝑺𝒘𝒆𝒍𝒍𝒊𝒏𝒈 𝒑𝒐𝒘𝒆𝒓 (𝒈/𝒈) = 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐭𝐡𝐞 𝐰𝐞𝐭 𝐬𝐞𝐝𝐢𝐦𝐞𝐧𝐭𝐬(𝒈) 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐭𝐡𝐞 𝐭𝐨𝐭𝐚𝐥 𝐬𝐭𝐚𝐫𝐜𝐡 𝐬𝐚𝐦𝐩𝐥𝐞 (𝒈) ∗ (𝟏−𝒔𝒐𝒍𝒖𝒃𝒊𝒍𝒊𝒕𝒚𝟏𝟎𝟎) Equation 6.6 76 6.2.10 Experimental design and statistical analysis Treatments were from 7 cultivars of Aa and 9 cultivars of Aa ✕ Am (hybrid); each cultivar was from a single tree. The experiments on IDF, SDF, TDF, starch protein, starch-bound protein in total protein, total protein, swelling power, solubility, fiber content, fruit whole weight, and edible fruit weight were conducted in triplicates for each cultivar. All results were plotted in Prism (v9.0, GraphPad Prism LLC). Significant differences for averages of Aa and all hybrids of Aa✕Am were determined by Welch’s t-test at α = 0.05 using Prism (v9.0, GraphPad Prism LLC). Significant differences across cultivars were done using ANOVA and Tukey’s multiple comparisons test with Prism (v9.0, GraphPad Prism LLC). To show the variability among cultivars, multivariate analysis was done on all the replicate analyses, processed in Excel™, and submitted to MetaboAnalyst (v5.0; Chong et al., 2019b, 2019a). Data were scaled by auto-scaling (mean-centered and divided by the standard deviation of each variable) and subjected to principal component and correlation analysis. 6.3 Results 6.3.1 Fruit morphology The fruit whole weight was variable among the 16 cultivars and ranged from 781.7 g (Ulu elise) to 3540.7 g (Lipet; Figure 6.1A), there was no overall difference with hybridization status (Figure 6.1C). Percent edible fruit weight was also found to be variable but to a lesser extent than whole fruit weight ranging from 70.2 % (Ulu fiti) to 89.6 % (Apu; Figure 6.1B), which almost equal distribution with hybridization status (Figure 6.1D). Although Lipet (Aa✕Am) had a significantly higher fruit whole weight than all cultivars, its % edible fruit weight was only significantly higher than Ulu Fiti, Lemae, Ma’afala, and Ulu elise (Figure 6.1D). The most marked difference among cultivars for percent edible fruit weight was Ulu fiti (Aa) and Ulu elise 77 (Aa✕Am) which had 10% lower edible fruit weight than the other cultivars investigated (Figure 6.1C). Figure 6.1. Fruit characteristics of breadfruit starches by cultivar and hybridization. (A, C) Fruit Whole Weight; (B, D) % Edible Fruit Weight. Black bars represent A. altilis while grey bars represent the hybrids. Data are plotted as mean of n= 3, error bars represent standard error. A significant difference ( = 0.05) is indicated using Welch’s t-test within the A. altilis and hybrids group. 78 6.3.2 Dietary fiber The hybrid cultivar Yuley had the highest dietary fiber content across all fiber types investigated: soluble, insoluble, and total, while two Aa cultivars, Ma’afala and Puaa, consistently had the lowest (Figure 6.2 A-C). IDF varied from 5.9 % (Ma’afala) to 11.4 % (Yuley; Figure 6.2A), SDF from 0.2 % (Puaa) to 7.0 % (Yuley; Figure 5.2B), and TDF from 6.2 % (Ma’afala) to 17% (Yuley; Figure 6.2C). Midolab also performed well across dietary fiber types (Figure 6.2 A-C) while several other cultivars showed variability across fiber types. For example, White, Apu (both Aa), and Yap (Aa✕Am) show high IDF (Figure 6.2A), but midrange SDF (Figure 6.2B) and TDF (Figure 6.2C). Generally, the hybrids had the same or higher SDF than Aa cultivars (e.g., high in Sewan, Midolab, and Yuley in particular) pushing the hybrid average for SDF significantly higher than Aa (Figure 6.2E). The consistently high levels of SDF in these hybrids are also reflected in the TDF (Figure 6.2C, F), though the increased TDF of the hybrids was not significant (Figure 6.2F). 79 80 Figure 6.2. Fiber characteristics of breadfruit flour. (A, D) Insoluble dietary fiber, (B, E) Soluble dietary fiber, and (C, F) Total dietary fiber. Black bars represent A. altilis while grey bars represent the hybrids. Error bars represent the standard error of the means. A significant difference ( = 0.05) is indicated using Welch’s t-test within the A. altilis and hybrids group. 6.3.3 Hybridization did not impact starch yield. The starch yield ranged from 3.3% (Meinpohnsakar (Aa✕Am) to 17.1% (Ulu fiti; Figure 6.3A). The hybrids generally had slightly lower yields, particularly the cultivars with high fiber content such as Yuley, Midolab, and Sewan (Figure 6.2B). When averages were calculated by A. altilis and hybrids, the mean starch yield of Aa (12.5 %) was not significantly different from Aa✕Am (9.1 %; Figure 6.3E). 81 82 Figure 6.3. Characteristics of breadfruit starch by cultivar. (A, E) Starch yield; (B, F) Starch-bound protein; (E, G) Total protein; and (D, H) Starch-bound protein/total protein. Black bars represent A. altilis while grey bars represent the hybrids. Error bars are standard errors of the mean. A significant difference ( = 0.05) is indicated using Welch’s t-test within the A. altilis and hybrids group. *Ma’afala protein content is higher than all other cultivars, however, this cannot be statistically determined as all samples are collected from an individual tree (pseudoreplication). 6.3.4 Differences in the starch-bound protein across breadfruit cultivars of A. altilis and hybrids. The starch-bound protein (Figure 6.3B) levels showed a more than 100X range from 0.01 % (Roihaa) to 1.86 % (Ma’afala). Ma’afala performed exceptionally well in all classes where the protein was quantified with 5-100X higher protein content than the next highest performing cultivar (Sewan (Aa✕Am); Figure 6.3 B-D). Yuley and Ulu elise ((Aa✕Am)), and Roihaa (Aa) had little to no starch-bound protein (Figure 6.3B). The Aa cultivars, except for Ma’afala, generally had lower starch-bound protein levels than the hybrids, with the hybrids Sewan and Meinpohnsakar having the next highest levels (Figure 6.3B). Total protein ranged from 2.54 % (Yuley (Aa✕Am) to 9.46% (Ma’afala; Figure 6.4C), and total protein levels were fairly consistent between all the cultivars with the exception of Ma’afala with only a difference of ~1%. Investigation of the ratio of starch-bound to total protein showed greater variability with Ma’afala again the highest (19.7%) and Roihaa (0.4%) the lowest (Figure 6.3D). Roihaa, Otea, and White (Aa), Ulu elise, Midolab, and Yuley (Aa✕Am) had the lowest levels, with Sewan and Meinpohnsakar (Aa✕Am) having the next after Ma’afala (Figure 6.3D). No significant trends 83 were observed for protein content when grouped by A. altilis and hybrids. (Figure 6.3 F-H). 6.3.5 Starch size and structure of different breadfruit cultivars of A. altilis and hybrids. Breadfruit starch was contained in a mixture of a polyhedral, compound, and segmented granules (Figure 6.4). The hybrids (Aa✕Am; Figure 6.4 H-P) contained more membranous clusters of compound starch granules than Aa (Figure 6.4 A-G). Across all cultivars, breadfruit starches contain both small and large granules ranging from 2.04 µm to 17.66 µm (Figure 6.5 A-C). Two general groupings of cultivars were observed with respect to minimum starch granule length, with the Aa generally having a smaller minimum size (Figure 6.5D) with the exception of Puaa and Ma’afala (Aa; Figure 6.5A). Maximum granule size was more evenly split amongst the cultivars (Figure 6.5B), and this was reflected in the A. altilis and hybrids averages that were not significantly different (Figure 6.5E). Roihaa starch granules had the shortest length (2.04 µm; Figure 6.5A) and average length (5.92 µm; Figure 6.7c). Yuley had the largest starch granule size with a length of up to17.66 µm (Figure 6.5B). Otea had the highest mean length of starch granule at 9.81 µm. Hybridization did not have a significant effect on the average granule size (Figure 6.5F). 84 85 Figure 6.4. Scanning electron micrographs of cultivars of breadfruit starches at 5000x magnification. A-G are Artocarpus altilis; H-P are Artocarpus altilis x Artocarpus mariannensis. (A) Cultivar ‘Apu’; (B) Cultivar ‘Ma’afala; (C) Cultivar ‘Otea’; (D) Cultivar ‘Puaa’; (E) Cultivar ‘Roihaa’; (F) Cultivar ‘Ulu Fiti’; (G) Variety ‘White’; (H) Variety ‘Lamae’; (I) Cultivar ‘Lipet’; (J) Cultivar ‘Meindapahk’; (K) Cultivar ‘Meinpohnsakar’; (L) Cultivar ‘Midolab”; (M) Cultivar ‘Sewan’; (N) Variety ‘Ulu elise’; (O) Variety ‘Yap’; (P) Cultivar ‘Yuley’. 86 87 Figure 6.5. Characteristics of starch granules by cultivar and hybridization. (A, D) Minimum (Min) size of granule; (B, E) Maximum (Max) size of granule; and (C, F) Mean size of the granule. Measurements were made at 5000x magnification. Error bars represent the standard error of the mean. For the mean length, the significance is shown with stars comparing between treatments (p ≤ 0.05) with Tukey’s multiple comparisons test. 6.3.6 The swelling power and solubility of different breadfruit cultivars of A. altilis and hybrids. The swelling power varied by 2-3 fold amongst cultivars from 7.8% (Ulu fiti) to 15.6% (Yuley; Figure 6.6A). Ulu fiti, followed by Ma’afala (both Aa) had the lowest swelling power, while the hybrids Yuley, Midolab, Meinpadahk, and Lipet, and the Aa cultivar Otea had the highest levels (Figure 6.6A). No significant difference was observed between the two groups, though Aa cultivars generally had slightly lower swelling power (Figure 6.6C). The solubility of the cultivars was significantly different between the hybrids and Aa (Figure 6.6D) ranging from 0.7 % (Ulu Fiti) to 6.4% (Lemae; Figure 6.6B). Lipet and Lemae (Aa✕Am) were highest followed by Midolab and Ulu elise (Aa✕Am), Roihaa (Aa), Sewan, and Yuley (Aa✕Am); Figure 6.6B). 88 Figure 6.6. Processing potential characteristics of breadfruit starches by cultivar and hybridization. (A, C) Swelling power; (B, D) Solubility. Black bars represent A. altilis while grey bars represent the hybrids. A significant difference ( = 0.05) is indicated using Welch’s t-test within the group of A. altilis and hybrids. 89 6.3.7 Principal component analysis (PCA) identifies Yuley, Ulu fiti, and Ma’afala as distinct cultivars. Unsupervised multivariate analysis by PCA showed that Yuley, Ulu fiti, and Ma’afala formed unique and distinct clusters from the other cultivars (Figure 6.7A) with the first two principal components (PC) capturing 60.8 % of the variance in the data (Figure 6.7A). The loadings plot (Figure 6.7B) showed that protein and swelling power had the strongest influence on the first dimension, while fiber had the strongest influence on the second dimension. Correlation analysis did not identify any significant correlations aside from within classes of parameters. Specifically, total protein and starch-bound protein were strongly correlated as expected, with three distinct correlated clusters: protein, fiber, and a third which included fruit weight, swelling power, and solubility (Figure 6.7C). 90 A 91 B 92 Figure 6.7. Principal component analysis (PCA) and correlation analysis of IDF, SDF, TDF, starch protein, starch bound protein in total protein, total protein, swelling power, solubility, fiber content, fruit whole weight, and edible fruit weight of the 16 cultivars. (A) Principal component (PC) 1 and PC2; (B) Loading plots; and (C) Pearson correlations. C 93 Figure 6.8. Comparison of breadfruit fiber content to other common flours using the USDA database (Source: USDA National Nutrient Database for Standard Reference 28 Software v.2.3.8). 94 6.4 Discussion. There is a need to determine nutritional variability to select elite cultivars for cultivation (Turi et al., 2015) and studies have found morphological and nutritional variability between breadfruit cultivars (Daley et al., 2020; Jones et al., 2013a, 2011a; Liu et al., 2015; Ragone and Cavaletto, 2006; Turi et al., 2015). Prior to this study, limited information was available on starch and flour characteristics across breadfruit cultivars, but these parameters are important for nutrition and industrial applications. We compared the dietary fiber and starch properties in different breadfruit cultivars to identify elite cultivars for starch and flour application. In the current study, Yuley had an exceptional performance in both starch and fiber characteristics with the highest IDF, SDF, TDF, and starch swelling power, significantly higher than most of the cultivars including the commercial cultivar, Ma’afala. The classic definition of dietary fiber is the non-digestible carbohydrate that is contained in foods and has physiological benefits such as reducing risks for developing coronary heart disease, stroke, hypertension, diabetes, obesity, and certain gastrointestinal diseases (Anderson et al., 2009). USDA reported that the median fiber intake for Americans is less than 50 percent of dietary recommendations. Most Americans intake an average of 15 g per day versus the requirement of 25 g per day for women and 38 g per day for men (USDA, 2011). Most of the intake is through the consumption of bread, rolls, buns, and pizza crust made with refined flour; white flour makes up almost half of the grain products. USDA strongly recommends people to consume whole grains, as well as whole grains products to fulfill the requirement of dietary fiber. However, the overwhelming preference for gluten-free products among the public is likely to prevent people from doing so. Breadfruit flour is gluten-free flour and has an average high level of total fiber. On average, breadfruit has a fiber content (11.8 %), which is higher than most common commercial flours, such as wheat (whole grain 10.7 % and white 2.7 %), corn (7.3 %), rice (brown 4.6 % and white 2.6 %), and potato (5.9 %; Figure 5.8). We found that the cultivar Yuley produced more total fiber than 23 other flour types, including the popular gluten-free flour options as soy flour, 95 quinoa, sorghum flour, millet flour (Figure 6.8). Based on our results, 100g of Yuley flour would meet 44.7 - 81 % (21 – 38 g/day) of the total fiber reported in Health Canada’s Adequate Intake (AI) between age 9 to above 70 years old. Yuley is a hybrid cultivar of AaXAm that was originally collected on the island of Yap in the Federated States of Micronesia and it is one of the larger cultivars of breadfruit with an average edible fresh weight of 1671 grams per fruit (Jones et al., 2011a). The cultivar Ulu fiti had the highest starch yield among the cultivars we tested and was in the middle of the range of values previously reported in the literature on unknown cultivars (Akanbi et al., 2009; Rincón, 2005). Starch quality can be assessed using several criteria including starch-bound protein content, swelling power, and solubility, which is associated with the overall structure and size of the starch granules. The ability of flours to absorb water when heated in an aqueous suspension is known as swelling power and is a measure of the swollen sediment weight per g of dry flour, while the percentage of dissolved flour from a heated solution is referred to as solubility (Huang et al., 2019). Though a higher total protein content is considered desirable, lower starch-bound protein content is generally preferred for starch applications as it indicates higher purity of the starch extract and therefore higher quality for various applications (Nwokocha and Williams, 2011; Raeker et al., 1998). Ma’afala was identified as a high-protein-containing cultivar in this study as in others (Jones et al., 2011a). The protein content of Ma’afala starch in this study was significantly higher than the other cultivars. It is also higher than previously reported values of 0.61% to 1.39% protein in breadfruit starches of unknown cultivars (Nwokocha and Williams, 2011; Rincón, 2005). The high protein content is thought to affect surface charge and rate of hydration which in turn affects the starch swelling and gelatinization (Nwokocha and Williams, 2011). In terms of swelling power and solubility, the starch extracted from Ulu fiti was generally of lower quality and starch extracted from Yuley had the highest swelling power making it the most versatile starch for many applications. The low swelling power and solubility of Ulu fiti seem to 96 be influenced by the starch structure as it had the fewest starch granules in its SEM image. Low solubility was also reported in Huang et al., (2019) and was suggested to be due to the low amylose ratio present in breadfruit starch, which might explain our observations in Ulu fiti. The cultivar Lemae (Aa✕Am) also had high solubility and has previously been recommended as a starch source for its good aroma and texture (Ragone and Cavaletto, 2006). Hybridization between A. altilis and A. marianesis had a significant impact on the starch quality in breadfruit, with the hybrid cultivar Yuley, in particular, demonstrating excellent starch and fiber properties. Hybrids had significantly higher SDF, starch solubility and contained membranous clusters of starches that are more likely to form aggregates or compound starch granules. Compound starches are resistant to enzymatic attack (Bird et al., 2009) and could partially explain the reported low glycemic index of breadfruit when compared to many common staples such as wheat, cassava, yam, and potatoes (Turi et al., 2015). 6.5 Conclusion Yuley, Ulu fiti, and Ma'afala were found to have distinct starch and fiber profiles in multivariate analysis. Hybridization was found to impact starch structure, solubility, and soluble dietary fiber content. The hybrid cultivar Yuley had the highest IDF, SDF, TDF, swelling power, and low starch-bound protein and should be considered as an elite starch/fiber cultivar. Among the Aa cultivars, Ulu fiti was a generally high-yielding but low-quality starch source, with the lowest swelling power and solubility. Ma’afala, as expected, had significantly higher protein content in its starch than the other cultivars and may represent a useful starch source in applications where higher protein content is desirable. This shows the importance of conserving and studying breadfruit cultivar of A. altilis and hybrids diversity. 97 Chapter 7: Quality and consistency of Ma’afala as a source of flour 7.1 Synopsis Research has shown that Ma’afala is an excellent source of nutrition for food security and this has potential economic benefits for the developing world (Liu et al., 2020, 2015). It is, therefore, necessary to compare the nutritional properties of Ma’afala from various environments to fully establish it as the best cultivar suitable for combating global malnutrition. I hypothesize that all Ma’afala trees have similar nutritional content irrespective of their growing conditions. To investigate this, I compared Ma’afala flour characteristics from clones originating from a single maternal tree that was grown in different models. Specifically, I did the following: (1) determined the fruit weight and % edible weight of the clones and maternal tree; (2) determine the starch yield, the protein content of the starch and flour from the clones and maternal tree, and (3) compared the precipitation and temperature of the locations. 7.2 Materials and Methods 7.2.1 Field collections Samples were collected from commercially propagated Ma’afala trees distributed through Global Breadfruit LLC and planted at 6 locations in Hawaii. The locations were selected to represent the wet and dry sides of the islands, irrigated and non-irrigated trees, and different agricultural management practices. 98 Figure 7.1. Map of Ma’afala collection sites. Ornamental Horticultural Model and Orchard Agricultural Model (22°12'39.53"N 159°24'57.70"W ), Dryland Wind Break (21°53'21.10"N 159°25'3.59"W), Sustainable Agroforestry System (22°11'3.79"N 159°20'37.057"W), McBryde Botanic Garden (21°54'29.55"N 159°30'41.75"W), Kahanu Botanic Garden (20°47'55.46"N 156° 2'10.85"W). The locations were mapped and annotated using QGIS.org, 2021. QGIS Geographic Information System. QGIS Association. http://www.qgis.org 7.2.1.1 Ornamental horticulture Samples were harvested from 2 Ma’afala trees planted at Kauai Miniature Golf and Botanical Garden (Kilauea, HI 96754, United States). These trees were planted in a highly manicured public space in co-cultivation with ornamental trees, shrubs, and herbaceous 99 species. They received regular irrigation and fertilizer to promote growth as well as pruning and shaping for the public space. The trees also have regular interactions with tourists and mini-golf players. For this study, it will be referred to as an ornamental model. Figure 7.2. Harvest of breadfruit samples from the trees at the Mini-Golf Course. Photo Credit: Susan Murch 100 7.2.1.2 Breadfruit orchard Samples were harvested from a private plantation of 61 Ma’afala trees near Kilauea, HI (Dave’s Plantation; 22°12'39.53"N 159°24'57.70"W). This plantation is arranged in a traditional orchard style with rows of trees regularly spaced. The fruit is harvested for sale and processing. For this study, it will be referred to as the orchard model. Figure 7.3. Orchard-style Ma’afala plantation. Photo Credit: Susan Murch 7.2.1.3 Sustainable agroforestry farm samples Samples were harvested from 3 Ma’afala trees on a private sustainable agroforestry farm near Kapa’a, Hawaii (Matt’s Farm; 22°11'3.79"N 159°20'37.057"W). The trees were grown 101 on native, uncultivated soil without supplemental irrigation. For this study, it will be referred to as the Agroforestry model. Figure 7.4. Ma’afala trees grown in a sustainable agroforestry model. Photo Credit: Susan Murch 7.2.1.4 Dryland windbreak samples Samples were harvested from 12 Ma’afala trees planted on the dry side of Kauai at Mahaulepu (Mahaulepu; 21°53'21.10"N 159°25'3.59"W). Trees were grown as a windbreak 102 along the cliffside and received only natural rainfall with about 4 to 5 dry months per year. For this study, it will be referred to as the windbreak model. Figure 7.5. Ma’afala trees grown as a windbreak on an exposed cliff. Photo Credit: Susan Murch 103 7.2.1.5 Botanic garden samples Samples were harvested from 2 Ma’afala trees planted at the McBryde Botanic Garden in Kalaheo Hawaii garden (McBryde; 21°54'29.55"N 159°30'41.75"W). Trees received irrigation when young, supplemental water during dry months, regular pruning, leaf clearing, and care. For this study, it will be referred to as common garden model 1. Figure 7.6. Ma’afala tree grown in a botanic garden for display. Photo Credit: Susan Murch 7.2.1.6 Kahanu botanic garden samples Samples were harvested from 5 Ma’afala trees planted at Kahanu Botanic Garden on the southeast side of Maui (Kahanu; 20°47'55.46"N 156° 2'10.85"W). For this study, it will be referred to as the common garden model 2. The Ma’afala clones originated from a single tree at 104 Kahanu that is decades older than the commercial clones and would be referred to as the control model. A 105 Figure 7.7. (B) Ma’afala clones grown in the common garden test model before it started bearing fruit, fruits are shown above (A). Photo Credit: Jan Vozenilek, Copper Sky Productions, Kelowna, BC, Canada 7.2.2 Preparation of breadfruit samples A detailed description of the collection and preparation of the breadfruit flour samples have been previously published (Jones et al., 2013b, 2011a; Liu et al., 2014). The fruit was peeled and cut into quarters longitudinally. The seeds were removed, and half of the fruit was sliced into 2 mm sections using a mandolin vegetable slicer (Benriner, Philadelphia, PA) and dried in a fruit drier (Excalibur Dehydrator™). The dehydrated fruit slices were ground into flour using the finest setting of a commercial Electric Mill Grinder (WonderMill™). 7.2.3 Isolation of breadfruit starch Starch was isolated from breadfruit flour using the optimized method as described in Chapter 3, section 3.2.3. B 106 7.2.4 Protein determination of flour, and starch Determination of total protein content of flours and starch-bound protein was performed using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit, (Thermo Scientific, Montreal, Canada). The procedure of the isolation of protein and BCA assay are fully described in Chapter 3, section 3.2.4.2, and section 3.2.4.3 respectively 7.2.5 Precipitation and temperature observations Record of precipitation and temperature observations of all the Ma’afala locations for 6 months till the date of harvest was obtained from the National Centers for Environmental Information, U.S. Department of Commerce, NOAA, NESDIS. The record of 2019 was used for Kahanu as there was no record for its station for the harvest year. 7.2.6 Experimental design and statistical analysis The models that represent different growing conditions are described above and were assigned code names: ornamental model (M1); orchard model (M2); windbreak model (M3), agroforestry model (M4); common garden 1 (M5A); common garden 2 (M5B), and control model (CM). The experiments on the starch yield, starch protein, and flour protein were conducted on 39 flours from the clones planted in the different growing models with their number of replicates in bracket respectively; M1 (3), M2 (7), M3 (2), M4 (2), M5A (8), and M5B (17) and compared to flour from CM (1). All results were plotted in Prism (v9.0, GraphPad Prism LLC). All significant differences were determined by ANOVA and Tukey multiple comparisons at α = 0.05 using Prism (v9.0, GraphPad Prism LLC). Pearson correlation coefficient analysis was performed to determine the co-linearity among precipitation and protein/starch properties using Excel™. 107 7.3 Results 7.3.1 Fruit morphology of Ma’afala differs across various growing models. The average fruit harvest weight across the different models was from 513.7 g (M3) to 1216.8 g (M1; Figure 7.8A). M1 produced fruit that weighed higher than the control, CM (1156.7g), M5B (1073.4 g), more than twice that of M2 (541.5 g), and M3, and almost twice of M4 (642.1 g). The average fruit harvest weight of both the control model, CM, and its clone grown in the same botanical garden, M5B was similar. The edible fruit weight is the obtained weight after the skin, core and seeds have been removed and are used to produce the flour. The percentage of edible fruit weight of fruit whole weight is expressed as % edible fruit weight and ranged from, 75.2 % (M4) to 82.3 % (CM). The % edible fruit weight obtained from the control model was similar to its clone grown in the same botanical garden, M5B (80.2 %) and M1(80.2 %; Figure 7.8B). The % edible yield gotten from M4 was not much lower than that of M3 (77.0 %). Statistical significance analysis was not done as there was no information on the fruit harvest weight of M5A and the % edible yield per fruit of both M5A and M2. 108 Figure 7.8. Fruit and nutritional characteristics of Ma’afala from the different growth models. Mean with standard error bars of (a) Fruit harvest weight (b) % edible fruit weight (c) Starch yield, (d) Starch-bound protein, (e) Flour protein. Within each group, means without a common superscript differ (p ≤ 0.05) using Tukey's multiple comparisons test. Treatments were breadfruit sourced from different growth models, but all grown from Tree 55 – the Ma’afala mother tree from Kahanu Garden (Maui, Hawaii). 2 clones of Ma’afala grown in MiniGolf Course (22°12'39.53"N 159°24'57.70"W). 62 clones of Ma’afala grown in Dave’s Plantation (22°12'33.26"N 159°24'58.06"W). 12 clones of Ma’afala grown in Mahaulepu (21°53'21.10"N 109 159°25'3.59"W). 3 clones of Ma’afala grown in Matt’s farm (22°11'3.79"N 159°20'37.057"W). 2 clones of Ma’afala grown in McBryde’s garden (21°54'29.55"N 159°30'41.75"W). 5 young clones of Ma’afala grown in Kahanu garden (20°47'55.46"N 156° 2'10.85"W). Figure 7.9. Nutritional properties of Ma’afala from two extreme models. Mean with standard error bars of (a) Fruit harvest weight (b) % edible fruit weight (c) Starch yield, (d) Starch-bound protein, (e) Flour protein of Ma’afala from two growth models. Within each group, means with an asterisk differ (p ≤ 0.05) using Welch’s Unpaired t-test. Treatments were breadfruit sourced from different models, but all grown from Tree 55 – the Ma’afala mother tree from Kahanu Garden (Maui, Hawaii). 2 clones of Ma’afala grown in MiniGolf Course, M1 (22°12'39.53"N 110 159°24'57.70"W). 12 clones of Ma’afala grown in Mahaulepu, M3 (21°53'21.10"N 159°25'3.59"W). 7.3.2 Significant variability in the starch yield of Ma’afala from different growing models The starch yield obtained from the different locations was significantly different with M5A (28.9 %) having the highest yield and was three folds of the control model, CM (9.5 %) which was the lowest (Figure 7.8C). The starch yield from M5A was significantly higher than M2 (13.1 %), M1 (14.8 %), and M5B (18 %) but not M3 (18.8 %) and M4 (20.2 %; Figure 7.8C). The starch yield of the control model, CM was not significantly different from the varying models of all its clones except M5A (Figure 7.8C). 7.3.3 Differences in the starch protein of Ma’afala from different growing models The starch protein (Figure 7.8D) was significantly different ranging from 0.13 % (M4) to 1.86 % (CM). The starch protein from the control model, CM was similar to its clone grown in the same botanical garden, M5B (1.32 %), more than 12 folds of M3 (0.15 %), 6 folds of M1 and M5A (0.31 %), and almost 3 folds of M2 (0.63 %; Figure 7.8D). 7.3.4 Protein content of Ma’afala flour differs significantly across growing models. The flour protein also significantly differed with the model range like that of starch protein from 2.08 % (M4) to 7.60 % (CM; Figure 7.8E). The protein content of the flour from the control model, CM was closest to its clone grown in the same botanical garden, M5B (6.19 %) and they both significantly differed from the flour of M5A (2.81 %) and M4 (2.08 %) but not from M2 (4.51 %, Figure 7.8E). However, only CM and not M5B significantly differs from M3 (3.01 %) and M1 111 (3.42 %; Figure 7.8E). In summary, the flour protein from CM was significantly different from other models except for M5B and M2 (Figure 7.8E). 7.3.5 Climate data of the different growing models of Ma’afala The amount of precipitation was the highest in model CM which was the same location as M5B (0.2 in) and was significantly different from that of M3 (0.08 in) and M5A (0.08 in) which were the lowest (Figure 7.10A). Although not significantly different, M4, M1, and M2 (0.12 in) had a lower amount of precipitation in comparison to CM. The precipitation was strongly positively correlated with the protein content of both starch (0.9) and flour (0.9) and negatively correlated to the starch yield (-0.6). The mean maximum temperature, minimum temperature, and temperature at the time of observation of Kauai island (79.4 F, 69.6 F, 72.3 F) was slightly higher than Maui Island (78.3 F, 67.7 F, 70. 1 F) respectively and did not vary significantly (Figure 7.10B). 112 Figure 7.10. Record of (A) Precipitation and (B) Temperature observations from the National Centers for Environmental Information, U.S. Department of Commerce, NOAA, NESDIS of Ma’afala Locations. The precipitation data were obtained from MiniGolf Course (USC00514561 Station, Kilauea 1134, HI US, 2015/04/13 - 2015/04/30), Dave’s Plantation (USC00514561 Station, Kilauea 1134, HI US, 2015/04/13 – 2015/04/30), Mahaulepu (US1HIKI0020 Station, Koloa 2.1 SSE, HI US, 2015/04/14 – 2015/10/14), Matt’s farm (US1HIKI0021 Station, Anahola 2.8 N, HI US, 2015/04/15 – 2015/10/15), McBryde’s garden (US1HIKI0014 Station, Kalaheo 1.3 SE, HI US, 2015/02/15 – 2015/08/15) and Kahanu garden (USC00513317 Station, Kapalua w. Maui airport 462.4, HI US, 2015\05/25 – 2015/11/25). The temperature data on Kauai was from USC00514561 Station, Kilauea 1134, HI US (2015/04/13 – 2015/04/30) while Maui was from 113 USC00511125 Station, Hana airport 355, HI US (2019\01/01 – 2019/06/30). "At Obs." = Temperature at time of observation 7.4 Discussion Research using the different environmental models was established to be a top priority needed to understand the impact of climate on breadfruit productivity (Lincoln et al., 2018). A review by Needham et al., (2020) suggested the impact of environmental factors on breadfruit nutritional composition. Ma’afala is the first widely available commercial cultivar with more than 30, 000 trees distributed globally and can produce 8.5 t.ha-1 fruits within 8 years (Liu et al., 2014). There was a need to compare the nutritional quality of Ma’afala under different growth conditions to determine variability and consistency in the crop. Age did not affect the nutritional content but rather the growing conditions as indicated by the similarity between the nutritional content of the maternal tree and its younger clones grown in the same model but not its younger clones grown in different models. The nutritional content of the clones grown in a similar model (Botanic Gardens) but different islands were significantly different despite having similar temperatures, this seems to be because of the significant difference in their precipitation. This is similar to the review analysis by Needham et al., (2020) that indicated that precipitation but not temperature appears to affect the nutritional content of breadfruit, although the study reported the limitations of discrepancies in reporting cultivars and unknown farm practices. The ornamental model that received the regular irrigation and fertilizer had the best fruit weight but did not have the best starch yield nor protein content. Although, the neglected windbreak model that receives the lowest rainfall had the lowest fruit weight but surprisingly had comparable starch yield and starch protein content that was not significantly different from other models. This is interesting as breadfruit was found to increase in area and quality in a study that explored its potential in future climate scenarios (Mausio et al., 2020). My results show that 114 breadfruit still had good nutritional quality despite lack of rainfall and is a good plant to be conserved for preventing food insecurity in future climate scenarios. Further studies should investigate other agronomic conditions that might also influence the differences in the nutritional content of these models. The significant difference in the fruit whole weight between the ornamental model and neglected windbreak model reflects the impact of care, rainfall, and fertilizer on the fruit quality. 7.5 Conclusion There were significant differences in starch yield, starch-bound protein content, and flour protein content of Ma’afala cultivar obtained from the different locations. Precipitation was positively correlated to breadfruit nutritional content. The maternal tree had the highest starch and flour protein content which was similar to its clone grown under the same model and in the same location, but clones grown in other locations were variable. These results showed that the location where Ma’afala is grown influences its nutritional quality and future studies should investigate other agronomic conditions that might influence these differences. 115 Chapter 8: Ma’afala (A. altilis) is an excellent source of fiber and starch in comparison to corn, rice, and wheat 8.1 Synopsis Ma’afala is one of the two commonly grown breadfruit cultivars in Samoa that is identified with export potential, the second being Puou, and a study by Molimau-Samasoni et al., (2020) found Ma’afala to have a longer storage life than Puou with possible better export opportunity. It has also been granted “Generally Recognized as Safe” status by the United States Food and Drug Administration. There is limited information on the comparison of the starch from this excellent breadfruit cultivar (Ma’afala) to the widely used traditional starches such as corn, wheat, and rice. I hypothesized that the Ma’afala cultivar is comparable to traditional sources of starch. To investigate this, I compared: (1) Ma’afala to corn, rice, whole wheat, and all-purpose wheat flour for insoluble dietary fiber, soluble dietary fiber, and total dietary fiber and (2) Ma’afala to corn, rice, whole wheat, and all-purpose wheat flour for starch yield, starch protein, structure, swelling, and solubility. 8.2 Materials and Methods 8.2.1 Flours The commercial flours included in this study were flour types of yellow corn, rice, whole wheat, all-purpose wheat flour, and breadfruit flour. Yellow corn, rice, organic whole wheat, and all-purpose wheat flours were obtained commercially from Real Canadian Superstore (Kelowna, BC, Canada). Breadfruit flour (Ma’afala) used for the comparison between these traditional sources was obtained from McBryde garden (Kauai, Hawaii). 116 8.2.2 Isolation of starch Starch was isolated from the flours using the optimized method as described in Chapter 3, section 3.2.3. 8.2.3 Total Dietary Fiber (TDF)/Soluble Dietary Fiber (SDF)/ Insoluble Dietary Fiber (IDF) The total dietary fiber assay kit (Sigma-Aldrich, St. Louis, USA) was used for fiber analysis according to manufacturer instructions and following the American Association of Cereal Chemists (AACC) method NO. 32-07 (AACC, 2000). The experiment was conducted in triplicate. The flour samples (1 g) and an empty glass bottle without flour (blank) were homogenized in 50 ml Phosphate buffer (0.025 M monosodium phosphate with 0.075 M disodium phosphate; Sigma-Aldrich, Germany) and was incubated with 100 µL α-amylase solution at 95 ° C pre-heated water bath for 15 min with continuous agitation. After cooling to room temperature, 100 µL protease solution (Fisherbrand, Hampton, USA) was added to the sample, followed by a 30 min incubation at 60 ° C. 10 ml HCl (0.325 M) was added to the samples before they were incubated with 100 µL amyloglucosidase for 30 min at 60 ° C. The samples were filtered through nylon woven filters (mesh opening: 53 µm, open area: 36 %, thickness: 60 µm; Spectra/Mesh®, Spectrum Laboratories, Inc.). After filtration, both the residue and filtrate were used to determine the total dietary fiber (TDF) content. The residue was washed and rinsed with acetone, 78 % ethanol, and 95 % ethanol and oven-dried at 100 ± 5 ° C overnight for insoluble dietary fiber (IDF) calculation, followed by protein and ash correction. The filtrate was adjusted with 4 equal volumes of preheated (60 ° C) of 95 % ethanol and allowed to sit overnight. Soluble dietary fiber (SDF) was precipitated, filtered, and oven-dried at 100 ± 5 ° C overnight. Protein and ash correction was performed for 117 the calculation of SDF content. The IDF, SDF, and TDF were calculated using Equations 6.1, 6.2, and 6.3 respectively as described in Chapter 6. 8.2.4 Total ash analysis Total ash content was determined using the AACC method 08-01 (AACC, 2000) as described in Chapter 6, section 6.2.5. 8.2.5 Protein determination of flour, starch, and fiber Determination of total protein content of flours, starch-bound protein, and the oven-dried fiber residue (SDF and IDF) was performed using a Pierce Bicinchoninic Acid (BCA) Protein Assay Kit, (Thermo Scientific, Montreal, Canada). The procedure of the isolation of protein and BCA assay are fully described in Chapter 3, section 3.2.4.2, and section 3.2.4.3 respectively. 8.2.6 Swelling power and solubility The determination of the swelling power and solubility of the starches was carried out following the procedure described in Chapter 5, section 5.2.9 8.2.7 Scanning electron microscopy (SEM) of starch The starches from the different flours were imaged using SEM with details fully described in Chapter 4, section 4.2.1 8.2.8 Image processing from SEM The size of the starch granules was processed from the SEM images using ImageJ as described in Chapter 6, section 6.2.8. 118 8.2.9 Experimental design and statistical analysis The experiments on the IDF, SDF, TDF, starch protein, swelling power, and solubility were conducted in triplicates for each flour. All results were plotted in Prism (v9.0, GraphPad Prism LLC). Significant differences across cultivars were done using ANOVA and Tukey’s multiple comparisons test with Prism (v9.0, GraphPad Prism LLC). Pearson correlation coefficient analysis was performed to determine the co-linearity of starch-bound protein, minimum starch size, and swelling power in Excel™. 8.3 Results 8.3.1 Breadfruit is a better source of dietary fiber as compared to traditional sources. In general, Ma’afala was a better source of fiber than any of the conventional flours tested (Figure 8.1). Ma’afala (4.9 %) had significantly higher total dietary fiber than yellow corn (2.4 %), rice (2.2 %), whole wheat (3.3 %), and all-purpose wheat (2.7 %; Figure 8.1c). SDF made up the bulk of this difference with Ma’afala having as much as double the amount (4.2%) of SDF as compared to yellow corn (2.1%), rice (2.1 %), whole wheat (2.8 %), and all-purpose wheat flour (2.4%; Figure 8.1b). IDF was also found to be increased for the Ma’afala flour (0.7 %), though not significantly, this still represents a more than three-fold increase in rice (0.2 %), and two-fold as compared to yellow corn (0.3 %) and all-purpose wheat (0.3 %) with the next highest traditional starch source whole wheat which containing 0.5% IDF (Figure 8.1a). 119 Figure 8.1. Mean with standard error bars of (a) Insoluble dietary fiber, (b) Soluble dietary fiber, and (c) Total dietary fiber of breadfruit flour compared with traditional flours. Within each group, significance is shown with stars comparing between treatments (p ≤ 0.05) using breadfruit as control with Dunnett's multiple comparisons test. Treatments were flour types of Yellow corn flour, Rice flour, All-purpose wheat flour, Whole wheat flour, and Breadfruit (Ma’afala) flour. 8.3.2 Breadfruit has higher starch yields as compared to traditional flours. Breadfruit had a higher starch yield than all the commercial flours analyzed (Figure 8.2a). Ma’afala (38 %) had a significantly higher starch yield than yellow corn (16.5 %), rice (7.4 %), or all-purpose wheat (25.4%). Although not significantly different, Ma’afala also had a higher starch yield than whole wheat (31.3 %). 120 Figure 8.2. Mean with standard error bars of (a) Yield, (b) Protein, (c) Swelling power, and (d) Solubility of breadfruit starch compared with traditional starch. Within each group, significance is shown with stars comparing between treatments (p ≤ 0.05) using breadfruit as control with Dunnett's multiple comparisons test. Treatments were starch extracted from flour types of 121 Yellow corn flour, Rice flour, All-purpose wheat flour, Whole wheat flour, and Breadfruit (Ma’afala) flour. 8.3.3 Breadfruit starch is of higher purity than traditional sources with low levels of starch-bound protein. Ma’afala (0.17 %) had a significantly lower starch-bound protein than rice (1.77 %), whole wheat (1.96 %), and all-purpose wheat (1.01%; Figure 8.2b). The starch-bound protein of Ma’afala was higher than that of yellow corn (0.02 %), although there was no significant difference (Figure 8.2b). 8.3.4 The swelling power and solubility of breadfruit in comparison to traditional sources The swelling power of Ma’afala (8.9 %) was significantly higher than yellow corn (7.9 %), rice (4.1 %), whole wheat (6.2 %), and all-purpose wheat (6.7 %; Figure 8.2c). Although yellow corn (3.1 %), whole wheat (8.9 %), and all-purpose wheat (5.8 %) had higher solubility than Ma’afala (0.9 %), there was no significant difference in the solubility of Ma’afala when compared to rice (3 %; Figure 8.2d). 8.3.5 Starch granule structure and size Breadfruit (Figure 8.3E) and rice starch granules (Figure 8.3B) were both polyhedral and segmented granules. Both organic (Figure 8.3D) and all-purpose wheat (Figure 8.3C) starch had a mixture of large, intermediate, and small granules. Corn starch (Figure 8.3A) was comprised of a mixture of rounded and irregular granules. Breadfruit minimum starch size (4.59 µm) was comparable to that of corn (4.53 µm) and less than that of rice (2.37 µm), all-purpose 122 wheat (2.39 µm) and whole wheat (2.42 µm; Figure 8.4). The maximum starch granule size of breadfruit (9.17 µm) was less than rice (13.51 µm) and over two-fold less than all-purpose wheat (26.34 µm), whole wheat (22.36 µm) or corn (19.16 µm; Figure 8.4). A strong correlation (R2 = 0.8) was found between the minimum starch size (Table 8.1) and swelling power (Figure 8.2c) of all the starches tested Figure 8.3. Scanning electron micrographs (SEM) of breadfruit starch compared with traditional starch at 5000x magnification. Treatments were starch extracted from flour types A. Yellow corn flour; B. Rice flour; C. All-purpose wheat flour; D. Whole wheat flour; E. Breadfruit (Ma’afala) flour. 123 Figure 8.4. Characteristics of starch granules by flour type. (a) Minimum (Min) size of granule; (b) Maximum (Max) size of granule; and (c) Mean size of granule. Measurements were made at 5000x magnification. Error bars represent the standard error of the mean. 124 Table 8.1. Size of breadfruit starch in comparison to traditional starch sources at 5000x magnification. Treatments were starch extracted from flour types A. Yellow corn flour; B. Rice flour; C. All-purpose wheat flour; D. Whole wheat flour; E. Breadfruit (Ma’afala) flour and viewed using a scanning electron microscope. Starch type Number of objects counted Minimum (μm) Mean (μm) Maximum (μm) Yellow corn flour 11 4.53 10.33 19.16 Rice flour 11 2.37 5.36 13.51 All-purpose wheat flour 32 2.39 7.15 26.34 Whole wheat flour 31 2.42 7.2 22.36 Breadfruit (Ma'afala) flour 14 4.59 7.07 9.17 8.4 Discussion Ma’afala was found to contain a higher quality of protein than other breadfruit cultivars, wheat, corn, rice, soybean, and yellow pea (Liu et al., 2015). However, there is limited information on the comparison of Ma’afala starch and flour to common starch/flour sources such as rice, corn, and wheat. I hypothesized that the Ma’afala cultivar is comparable to traditional sources of starch. My study showed that Ma’afala a better source of dietary fiber than yellow corn, rice, whole wheat, and all-purpose wheat. Dietary fiber is the non-digestible carbohydrate present in food and it promotes good health ( Anderson et al., 2009, Zhang et al., 2018). The TDF of all the flours falls within the previously reported range of 0.34 – 3.94 g/100 g for cereal grains (Li and Andrews, 2002). This research shows that Ma’afala is of high starch quality as it had the highest starch yield, swelling power, and comparable starch structure. The starch yield of Ma’afala was higher than 125 the reported value of 17.8 % breadfruit and lower than 82.31% broken rice (Koh and Long, 2012). A cause of these observed disparities could be because the starch was extracted from the fruit and not the flour. The different shapes and sizes of starch granules play an important role in functionality and digestibility (Magallanes-Cruz et al., 2017). Small starch granules are highly susceptible to aggregation while large starch granules show high swelling and high susceptibility to breakage during the pasting process. Interestingly, this phenomenon was observed in this study as the minimum starch size of the traditional flours and breadfruit were strongly positively correlated to their swelling power. This minimum starch size of breadfruit could be the reason for its increased swelling power when compared to other traditional starch sources. The effect of exclusion of small starch granules on swelling power can be further investigated which will be useful for industrial applications that require high starch swelling power. Breadfruit starch granules contained a mixture of spherical aggregates and segmented granules consistent with previous studies (Loos et al., 1981; Nwokocha and Williams, 2011; Rincón, 2005; Tan et al., 2017) and similar to rice starch (Zuo et al., 2009). The spherical aggregates are called compound granule which is typical to rice and oats, they consist of multiple granules, ranging from two to several dozen per amyloplast (Wani et al., 2012; Zhou et al., 1998). Compound starches are known to be resistant to enzymatic attack (Bird et al., 2009) which could be responsible for the reported low glycemic index of breadfruit when compared to many common staples such as wheat, cassava, yam, and potatoes (Turi et al., 2015). The protein content of Ma’afala starch is lower than the reported values of 0.61 % (Rincón, 2005) and 1.39 % by Nwokocha and Williams, (2011). The low protein content is good quality as the high protein content of starch indicates low purity of the starch extract (Nwokocha and Williams, 2011; Raeker et al., 1998). Also, surface charge and rate of hydration may be affected by high protein content which in turn affects the starch swelling and gelatinization (Nwokocha and Williams, 2011). This relationship between protein content and swelling power was seen by the correlation between the two properties of the traditional flours and breadfruit. Therefore, this 126 suggests that the low protein content of breadfruit starch influences its swelling power, giving it better quality than traditional starch sources. Overall, this study demonstrates that the Ma’afala breadfruit cultivar has excellent flour and starch properties in comparison to corn, rice, and wheat which should be exploited for industrial applications. 8.5 Conclusion Ma’afala flour is a better source of starch and increasing dietary fiber intake than yellow corn, rice, all-purpose, and whole wheat flour. Rice starch has preferably been used in infant formula due to its small granules, palatability, hypoallergenic, digestibility, and non-GMO but its limitations include arsenic content, breadfruit starch may represent a safer alternative. 127 Chapter 9: Characterization of glandular trichomes in breadfruit 9.1 Synopsis The development of novel bioproducts requires understanding the underlying physiology, chemistry, and molecular biology of the plant. The site and storage of essential oil and secondary metabolites in many plants is the glandular trichomes (Lange et al., 2000; Wagner et al., 2004). To the best of my knowledge, there has been no detailed study on the glandular trichomes of breadfruit across cultivars. Understanding the differences in the glandular trichomes in breadfruit might provide insight into the difference in secondary metabolite across breadfruit cultivars. I hypothesized that breadfruit contains glandular trichomes. To investigate this, I did the following: (1) Developed a microscope method to view and quantify trichomes on breadfruit leaves; (2) Determined the correlation of length of breadfruit leaf to the number of trichomes; (3) Determined if the number of glandular trichomes varies cultivars, and (3) Developed methods for isolation of trichomes from breadfruit leaves. 9.2 Materials and Methods 9.2.1 Scanning electron microscopy (SEM) of breadfruit glandular trichomes This was based on the method of Pathan et al., (2010) as there is no reported study on a method to view breadfruit glandular trichomes. The breadfruit leaves were immersed in nitrogen-cooled methanol for 20-40seconds and re-immersed in liquid nitrogen for 20seconds. The leaves were freeze-dried immediately for 10hours. Alternate sample preparation was done by just allowing the sample to air-dry without any chemical fixation for the same hours as the freeze-drying. The dried leaves were mounted onto aluminum stubs using double-sided adhesive tape. They were coated with gold using a sputter coater (Cressington sputter coater 128 HR, 208) to 20nm thickness. Samples were viewed using a scanning electron microscope (Tescan Mira3 XMU field emission gun) at an acceleration potential of 10.0kv. The micrographs were captured using automatic image capture software. All variables such as brightness, contrast, beam intensity, were kept constant for each magnification during the microscopy 9.2.2 Count of glandular trichomes The number of glandular trichomes of 3 different lengths each of five breadfruit cultivars was measured. The glandular trichomes were counted manually from the acquired SEM images at 500x magnification. 9.2.3 Isolation of breadfruit glandular trichomes Breadfruit glandular trichomes were extracted from its leaf material using the modified glass bead abrasion method (Lange et al., 2000) with methylcellulose extraction buffer (Lane et al., 2010). 1mm aurinticarboxylic acid, 5mm thiourea, 2mm DTT was added to methylcellulose buffer (200mm sorbitol, 10mm sucrose, 25mm MOPSO, 0.5mm PO4, 10mm sodium bisulfite, 10mm Ascorbic acid, 1mm EDTA, 1%PVP-40, 0.6% methylcellulose at ph 6.6) for RNA preservation. The extraction buffer was added to the breadfruit leaves and soaked for 1 hour. The leaves were abraded using glass beads filled with the extraction buffer. Glandular trichomes were isolated from the mixture by sieving and rinsing with wash buffer (10% glycerol,25 mm PO4 buffer, 1 mm EDTA 2 mm aurinticarboxylicacid, 5 mm thiourea, and 2 mm DTT). 129 9.2.4 Experimental design and statistical analysis SEM imaging of both different sample preparation was done for each of the 5 breadfruit cultivars. The lengths and number of trichomes were conducted in triplicates for each of the 5 breadfruit cultivars. All results were plotted in Prism (v9.0, GraphPad Prism LLC). Significant differences across cultivars were done using ANOVA with Prism (v9.0, GraphPad Prism LLC). Pearson correlation coefficient analysis was performed to determine the co-linearity of length, and the number of glandular trichomes in Excel™. 9.3 Results 9.3.1 Breadfruit contained glandular trichomes and can be viewed without chemical fixation. Breadfruit contained both glandular (Figure 9.1A) and non-glandular trichome (Figure 9.1B; 1C). My result also showed that breadfruit glandular trichomes can be viewed successfully without sample preparation that involves chemical fixation and freeze-drying (Figure 9.2). 130 Figure 9.1. Types of Trichomes in Breadfruit. Glandular (A) and Non-glandular (B, C) trichomes in breadfruit. Figure 9.2. SEM images of Breadfruit glandular trichomes using different sample preparation 131 9.3.2 Number of trichomes did not differ across cultivars The average number of trichomes did not vary significantly but ranged from 4.3 (Puupuu) to 80.7 (Meitehid; Figure 9.3). Meitehid had more than two folds of glandular trichomes of Ma’afala (38.3), five folds of Toneno (12.3), eight folds of T8 (9.7), and 18 folds of Puupuu (Figure 9.3). Figure 9.3. Mean with standard error bars of the number glandular trichomes across breadfruit cultivars. 132 9.3.3 The length of breadfruit leaf was not correlated to its number of trichomes The number of trichomes within all breadfruit cultivars was not correlated to their length (-0.2; Table 9.1). Although when the correlation was compared within each cultivar, only Ma’afala (-0.9) and T8 (-0.5) had their length correlated to the number of trichomes (Table 9.1; Figure 9.4). Figure 9.4. The number of glandular trichomes and the leaf length across breadfruit cultivars. 133 Table 9.1. Correlation coefficients (r) between leaf length and the number of glandular trichomes Correlation between length and number of glandular trichomes Correlation coefficient (cultivars) -0.2 Correlation coefficient (Ma'afala) -0.9 Correlation coefficient (Meitehid) -0.2 Correlation coefficient (Puupuu) -0.1 Correlation coefficient (T8) -0.5 Correlation coefficient (Toneno) -0.3 9.3.4 Breadfruit glandular trichomes were successfully isolated The modified glass bead extraction method successfully extracted glandular trichomes from breadfruit and was confirmed by comparing them to Lavender glandular trichomes using optical microscopy (Figure 9.5). 134 Figure 9.5. Optical Microscopy of glandular trichomes from lavender and breadfruit using the same extraction method. 135 9.4 Discussion Breadfruit is enriched in terpenes with insecticidal activity (Glover, 2015; Jones et al., s2012), used to treat a wide range of diseases (Navarro et al., 2007; Ragone, 1997) and its genome stated to have expanded pathways of plant-host interaction (Sahu et al., 2020). However, glandular trichomes which are known to be the site of synthesis of these secondary metabolites such as terpenes (Lange et al., 2000; Wagner et al., 2004) have not been previously characterized in breadfruit. I hypothesized that breadfruit contains glandular trichomes and can be viewed using a scanning electron microscope without sample preparation. This study showed that breadfruit contained both glandular and non-glandular trichomes. Soft plant tissues such as glandular trichomes usually require chemical fixation, careful dehydration, and dried before viewing through SEM (Bhattacharya et al., 2020; Bomblies et al., 2008) as simple air-drying can cause collapse and shrinkage (Nation, 1983). The chemical fixation involves corrosive chemicals like isoamyl acetate, glutaraldehyde, and osmium tetraoxide. The use of chemicals such as hexamethyldisilazane (HMDS) or critical-point drying (CPD) are methods used to dry plant samples for SEM and a recent study by Bhattacharya et al., (2020) found HMDS to produce results comparable to the CPD method. CPD is preferred for drying plant tissues but its limitation includes longer sample preparation time and costs while HDMS has safety concerns as it is corrosive (Bhattacharya et al., 2020; Livingston et al., 2020; Shively and Miller, 2009). My study shows that there was no distortion in the glandular trichomes that were viewed without chemical fixation and just air-dried. The results were identical to the other half samples that were chemically fixed in nitrogen-methanol and freeze-dried. This will be of interest to research that involves the study of glandular trichomes as it does not require expensive equipment, devoid of toxic chemicals or chemical-induced structural change and cuts down sample preparation time. 136 Trichomes are important in plants and their density has been correlated with rust resistance of beans (Zaiter et al., 2019), necrotized leaf area (Rennberger et al., 2017), and mechanism of plant defense (Valverde et al., 2001). The number of trichomes varied across cultivars but not significantly unlike the study of Rennberger et al., (2017) that found trichomes to differ among five different cucurbits. Valverde et al., (2001) suggested that a possible explanation of the correlation between leaf shape and trichome size is that the trichomes inhibit herbivory until leaves are fully expanded. Although there was no correlation between the length of all cultivar leaves and its number of trichomes, the fully expanded leaves in this study had low trichome density. This is interesting as breadfruit has been earlier described to have insecticidal activity and the differences in the trichome density could offer a varying amount of insecticidal activity. Further investigations on the metabolites contained in the leaves or glandular trichomes of the young and expanded leaves could lead to the development of novel terpenes. 9.5 Conclusion Breadfruit cultivars have different trichome densities, although they do not differ significantly. The difference in the secondary metabolites of young and fully expanded leaves should be investigated for novel terpenes. 137 Chapter 10: Summary I hypothesized that breadfruit is a crop with the potential in developing novel bioproducts and breadfruit-based food products. I established three objectives necessary to investigate this hypothesis. 10.1 Objective 1 Breadfruit has the potential to combat global infant malnutrition, I hypothesized that breadfruit-based-infant formula will meet the world health organization’s standard (WHO). My results showed that breadfruit flour can serve as a key ingredient for a WHO standard infant formula. 10.2 Objective 2 To investigate the starch characteristics across breadfruit cultivars and in comparison, to traditional sources of starch, I optimized the starch extraction method for breadfruit flour. My studies determined that 100g of flour was optimal for starch yield as the starch extracted from this mass was significantly seven times more than the 20g mass of flour. The physical appearance of flour across cultivars varied upon slurry which led to the hypothesis that pasting and textural properties were different across cultivars. This was found to be true however, there was a limitation in the experimental design as there was no replicate analysis for each cultivar, and a specific scale was not designed for the physical appearance. Future studies should investigate the correlation of the physical appearance of the slurred flour to its starch yield, pasting, and textural properties. This is potentially useful as a cue in selecting cultivars for excellent starch yield, pasting, and textural properties based on slurring the flour in water which will be both cost and time-efficient. The breadfruit cultivars had a similar polygonal shape and size of starch granules except for Aveloloa. This was interesting because Aveloloa is traditionally known for its sweetness and reserved for high chiefs. 138 There are estimated to be >300 unique cultivars of breadfruit including both diploids and triploids but data on the molecular structure and composition of breadfruit starch across cultivars of A. altilis and hybrids is limited. Starch quality can be assessed using several criteria including starch-bound protein content, swelling power, and solubility, which is associated with the overall structure and size of the starch granules. My results showed that the starch yield and starch protein were not significantly different across A. altilis and hybrids but varied across cultivars. The starch granules were different across A. altilis and hybrids with the hybrids appearing to have more membranous cluster types. Hybridization did not only impact the starch structure of the hybrids, but it also influenced the solubility and soluble dietary fiber. Further study and growth of hybrid cultivars will improve our understanding of starch evolution and has advantages for commercial starch production. Across cultivars, Yuley, Ulu fiti, and Ma'afala had distinct starch and fiber profiles. I proposed that Yuley should be considered as an elite cultivar due to its high fiber content, swelling power, and low-starch protein. Ulu fiti had a good starch yield but with low quality in terms of swelling power and solubility. Ma’afala was significantly enriched in starch protein and should be considered for applications requiring high protein. Literature has found Ma’afala flour to be of excellent commercial value, I hypothesized that all Ma’afala trees have similar nutritional content irrespective of their growing conditions. My studies revealed that the starch yield, starch protein, and flour protein varied significantly with precipitation being a strong influence. However, the starch yield and starch protein of Ma’afala trees grown in a neglected windbreak model were surprisingly not significantly different from other growing models. This is interesting as it suggests that breadfruit might retain its good nutritional qualities under harsh climate conditions. To establish Ma’afala as a commercial starch cultivar, I compared the fiber and starch characteristics of Ma’afala to corn, rice, or wheat starch (traditional sources). My studies showed that Ma’afala (breadfruit) had better insoluble, soluble, total fiber content, starch yield, and swelling power than all the analyzed traditional sources which are of industrial value. 139 10.3 Objective 3 Glandular trichomes are the major site and storage of many secondary metabolites and essential oil, I hypothesized that breadfruit contains glandular trichomes. My studies showed that breadfruit contains both glandular and non-glandular trichomes. Breadfruit glandular trichomes were comparable to that of Lavender trichomes as observed by an optical microscope. I developed a method to view glandular trichomes using a scanning electron microscope which is time-efficient, free of toxic chemicals or structural damage, and might also be suitable in studying other plants. Another interesting observation from this study was that fully expanded leaves had a low number of trichomes. There might be a difference in the secondary metabolites between the young and expanded leaves and future investigations can be done to determine these differences. 10.4 Conclusion Overall, the starch extraction method from breadfruit flour was optimized. Breadfruit was determined to be a source of high-quality starch with Yuley cultivar being the best in fiber and starch applications. The commercial cultivar, Ma’afala is a better source of fiber and starch than traditional sources (corn, rice, or wheat). Precipitation was found to influence the amount of protein and starch gotten from Ma’afala trees grown across different growing conditions. Literature has shown breadfruit to be gluten-free, non-GMO, more digestible than wheat flour, non-toxic in chemical and histological analysis, and granted “Generally Recognized as Safe” by FDA. Breadfruit starch can be used as a key ingredient for infant formula as my results show its starch to be of high quality with advantages over the usually preferred corn or rice starch. Breadfruit flour is also suited for infant formula as my results determined that a breadfruit-based infant formula will meet the WHO’s nutritional standards for infants. 140 Breadfruit contained glandular trichomes that were comparable to lavender and a new method was developed to view breadfruit trichomes with SEM. Overall, this research can act as a basis for developing an infant formula from breadfruit starch/flour and other breadfruit-based food products. 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Product Name Ingredient list Similac Total Comfort Corn Maltodextrin, Whey Protein Hydrolysate, High Oleic Safflower Oil, Sucrose, Soy Oil, Coconut Oil, Calcium Citrate, Potassium Phosphate, Calcium Phosphate, M. Alpina Oil*, Potassium Hydroxide, Sodium Chloride, Magnesium Chloride, Ascorbic Acid, C. Cohnii Oil**, Potassium Chloride, Choline Chloride, Calcium Hydroxide, Taurine, Choline Bitartrate, M-Inositol, Ferrous Sulphate, Cytidine 5'-Monophosphate, Zinc Sulphate, Disodium Guanosine 5'-Monophosphate, Ascorbyl Palmitate, D-Α-Tocopheryl Acetate, L-Carnitine, Disodium Uridine 5'-Monophosphate, Adenosine 5'-Monophosphate, Tocopherols, Niacinamide, Calcium Pantothenate, Potassium Citrate, Cupric Sulphate, Vitamin A Palmitate, Riboflavin, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Manganese Sulphate, Folic Acid, Potassium Iodide, Phylloquinone, Sodium Selenate, Biotin, Vitamin D3, Cyanocobalamin. (Contain Milk and Not Suitable For Infants With Galactosemia) 158 Similac Step 2 Skim Milk, Lactose, High Oleic Sunflower Oil, Soy Oil, Coconut Oil, Whey Protein Concentrate, Potassium Citrate, Whey Protein Hydrolysate, Tricalcium Phosphate, Calcium Carbonate, Soy Lecithin, Ascorbic Acid, Choline Bitartrate, Magnesium Chloride, Potassium Chloride, Sodium Chloride, Ferrous Sulphate, Taurine, L-Tryptophan, M-Inositol, Cytidine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate, Zinc Sulphate, Adenosine 5'-Monophosphate, D-Α-Tocopheryl Acetate, Mixed Tocopherols, Niacinamide, L-Carnitine, Calcium D-Pantothenate, Vitamin A Palmitate, Copper Sulphate, Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Manganese Sulphate, Β-Carotene, Folic Acid, Potassium Iodide, Phylloquinone, Biotin, Sodium Selenate, Vitamin D3, Cyanocobalamin. Similac Step 1 Skim Milk, Lactose, High Oleic Sunflower Oil, Soy Oil, Coconut Oil, Whey Protein Concentrate, Potassium Citrate, Whey Protein Hydrolysate, Calcium Carbonate, Soy Lecithin, Ascorbic Acid, Choline Bitartrate, Magnesium Chloride, Sodium Chloride, Potassium Chloride, Ferrous Sulphate, Taurine, L-Tryptophan, M-Inositol, Cytidine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Guanosine 5'-Monophosphate, 159 Disodium Uridine 5'-Monophosphate, Zinc Sulphate, Adenosine 5'-Monophosphate, D-Α-Tocopheryl Acetate, Mixed Tocopherols, Niacinamide, L-Carnitine, D-Calcium Pantothenate, Vitamin A Palmitate, Cupric Sulphate, Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Manganese Sulphate, ß-Carotene, Folic Acid, Potassium Iodide, Phylloquinone, Biotin, Sodium Selenate, Vitamin D3, Cyanocobalamin. Similac simply nurture Skim Milk, Lactose, High Oleic Sunflower Oil, Soy Oil, Coconut Oil, Galactooligosaccharides (Gos), Whey Protein Concentrate, Whey Protein Hydrolysate, M. Alpina Oil (A Source Of Ara), Soy Lecithin, C. Cohnii Oil (A Source Of Dha), Taurine, L-Tryptophan, M-Inositol, Cytidine 5’-Monophosphate, Ascorbyl Palmitate, Disodium Guanosine 5’-Monophosphate, Disodium Uridine 5’-Monophosphate, Adenosine 5’-Monophosphate, Mixed Tocopherols, L-Carnitine, Lutein, Minerals (Potassium Citrate, Calcium Carbonate, Potassium Hydroxide, Magnesium Chloride, Sodium Chloride, Calcium Chloride, Ferrous Sulphate, Zinc Sulphate, Copper Sulphate, Manganese Sulphate, Potassium Iodide, Sodium Selenate), Vitamins (Ascorbic Acid, Choline Bitartrate, D-Alpha Tocopheryl Acetate, Niacinamide, Calcium D-Pantothenate, Vitamin A Palmitate, Thiamine Hydrochloride, Pyridoxine 160 Hydrochloride, Riboflavin, Beta-Carotene, Folic Acid, Phylloquinone, D-Biotin, Vitamin D3, Cyanocobalamin). As Needed: Tricalcium Phosphate, Dry Skim Milk. Similac Sensitive Lactose Sensitivity Corn Syrup, Sucrose, Milk Protein Concentrate, High Oleic Vegetable Oil (Safflower Or Sunflower), Coconut Oil, Soy Oil, Potassium Citrate, Sodium Citrate, Potassium Chloride, Calcium Phosphate, Whey Protein Hydrolysate, Ascorbic Acid, Magnesium Phosphate, Choline Chloride, Magnesium Chloride, Choline Bitartrate, Ferrous Sulphate, Taurine, M-Inositol, Cytidine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Guanosine 5'-Monophosphate, D-Α-Tocopheryl Acetate, Disodium Uridine 5'-Monophosphate, L-Carnitine, Zinc Sulphate, Adenosine 5'-Monophosphate, Mixed Tocopherols, Niacinamide, Potassium Hydroxide, Calcium D-Pantothenate, Cupric Sulphate, Vitamin A Palmitate, Thiamine Hydrochloride, Riboflavin, Pyridoxine Hydrochloride, Potassium Iodide, Folic Acid, Manganese Sulphate, Phylloquinone, Biotin, 161 Sodium Selenite, Vitamin D3, Cyanocobalamin(Contain Milk And Not Suitable For Infants With Galactosemia). Similac Neosure Skim Milk, Corn Syrup Solids, Lactose, Soy Oil, High Oleic Safflower Oil, Medium Chain Triglycerides (Modified Coconut And/Or Palm Kernel Oils), Whey Protein Concentrate, Coconut Oil, Whey Protein Hydrolysate, Calcium Phosphate, Ascorbic Acid, M. Alpina Oil*, Calcium Carbonate, M-Inositol, C. Cohnii Oil**, Potassium Citrate, Magnesium Chloride, Potassium Chloride, Taurine, Ferrous Sulphate, Choline Bitartrate, Choline Chloride, L-Carnitine, Ascorbyl Palmitate, Cytidine 5'-Monophosphate, D-Α-Tocopheryl Acetate, Disodium Guanosine 5'-Monophosphate, Zinc Sulphate, Disodium Uridine 5'-Monophosphate, Niacinamide, Adenosine 5'-Monophosphate, Mixed Tocopherols, Calcium D-Pantothenate, Cupric Sulphate, Thiamine Chloride Hydrochloride, Vitamin A Palmitate, Riboflavin, Pyridoxine Hydrochloride, Β-Carotene, Folic Acid, Manganese Sulphate, Phylloquinone, Biotin, Sodium Selenate, Vitamin D3, Cyanocobalamin 162 Similac Lower iron Skim Milk, Lactose, High Oleic Sunflower Oil, Soy Oil, Coconut Oil, Whey Protein Concentrate, Potassium Citrate, Whey Protein Hydrolysate, Calcium Carbonate, Soy Lecithin, Ascorbic Acid, Choline Bitartrate, Magnesium Chloride, Sodium Chloride, Potassium Chloride, Taurine, L-Tryptophan, M-Inositol, Ferrous Sulphate, Cytidine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate, Zinc Sulphate, Adenosine 5'-Monophosphate, D-Α-Tocopheryl Acetate, Mixed Tocopherols, Niacinamide, L-Carnitine, D-Calcium Pantothenate, Vitamin A Palmitate, Cupric Sulphate, Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Manganese Sulphate, ß-Carotene, Folic Acid, Potassium Iodide, Phylloquinone, Biotin, Sodium Selenate, Vitamin D3, Cyanocobalamin. Similac Isomil with DHA Corn Syrup, Soy Protein Isolate, High Oleic Sunflower Oil, Sucrose, Coconut Oil, Soy Oil, Calcium Phosphate, Potassium Citrate, Soy Lecithin, Sodium Chloride, Potassium Chloride, Magnesium Chloride, M. Alpina Oil*, C. Cohnii Oil**, L-Methionine, M-Inositol, Ascorbic Acid, Choline Chloride, Taurine, Ascorbyl Palmitate, L-Tryptophan, Zinc Sulphate, Ferrous Sulphate, L-Carnitine, Mixed Tocopherols, Niacinamide, Calcium D-Pantothenate, D-Α-Tocopheryl Acetate, Vitamin A 163 Palmitate, Riboflavin, Thiamine Hydrochloride, Cupric Sulphate, Pyridoxine Hydrochloride, Potassium Iodide, Folic Acid, Phylloquinone, Sodium Selenite, Biotin, Vitamin D3, Cyanocobalamin Similac Alimentum Corn Maltodextrin, Casein Hydrolysate (Derived From Milk), High Oleic Safflower Oil, Medium Chain Triglycerides (Modified Coconut And/Or Palm Kernel Oils), Sucrose, Soy Oil, Modified Corn Starch, Calcium Citrate, Calcium Phosphate, Potassium Citrate, Xanthan Gum, Potassium Phosphate, M. Alpina Oil*, L-Tyrosine, Magnesium Chloride, L-Cystine Hydrochloride, Sodium Chloride, Ascorbic Acid, C. Cohnii Oil**, L-Tryptophan, Choline Chloride, Ferrous Sulphate, Potassium Chloride, Taurine, M-Inositol, Dl-Α-Tocopheryl Acetate, Ascorbyl Palmitate, Zinc Sulphate, L-Carnitine, Mixed Tocopherols, Niacinamide, Calcium Pantothenate, Cupric Sulphate, Vitamin A Palmitate, Riboflavin, Thiamine Chloride Hydrochloride, Pyridoxine Hydrochloride, Folic Acid, Potassium Iodide, Phylloquinone, Biotin, Sodium Selenite, Vitamin D3, Cyanocobalamin. M. Alpina Oil (Source of Arachidonic Acid (ARA), C. Cohnii Oil (Source of Docosahexaenoic Acid (DHA). 164 Similac Advance Step 2 Lactose, Dry Skim Milk, High Oleic Safflower Or Sunflower Oil, Soy Oil, Coconut Oil, Galactooligosaccharides (GOS), Whey Protein Concentrate, Tricalcium Phosphate, Whey Protein Hydrolysate, Potassium Citrate, M. Alpina Oil*, Calcium Carbonate, Potassium Hydroxide, C. Cohnii Oil**, Soy Lecithin, Magnesium Chloride, Sodium Chloride, Choline Bitartrate, Ascorbic Acid, L-Tryptophan, Ferrous Sulphate, Taurine, M-Inositol, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Uridine 5'-Monophosphate, Zinc Sulphate, D-Α-Tocopheryl Acetate, Adenosine 5'-Monophosphate, L-Carnitine, Mixed Tocopherols, Niacinamide, Calcium D-Pantothenate, Cupric Sulphate, Vitamin A Palmitate, Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Lutein, Folic Acid, Manganese Sulphate, Β-Carotene, Phylloquinone, Biotin, Sodium Selenite, Potassium Iodide, Vitamin D3, Cyanocobalamin 165 Similac Advance Step 1 Lactose, Dry Skim Milk, High Oleic Vegetable Oil (Safflower Or Sunflower), Soy Oil, Coconut Oil, Galactooligosaccharides (Gos), Whey Protein Concentrate, Whey Protein Hydrolysate, Potassium Citrate, Calcium Carbonate, M. Alpina Oil*, Tricalcium Phosphate, Potassium Hydroxide, C. Cohnii Oil**, Soy Lecithin, Magnesium Chloride, Sodium Chloride, Choline Bitartrate, Ascorbic Acid, L-Tryptophan, Ferrous Sulphate, Taurine, M-Inositol, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Ascorbyl Palmitate, Disodium Uridine 5'-Monophosphate, Zinc Sulphate, D-Α-Tocopheryl Acetate, Adenosine 5'-Monophosphate, L-Carnitine, Mixed Tocopherols, Niacinamide, Calcium D-Pantothenate, Copper Sulphate, Vitamin A Palmitate, Thiamine Hydrochloride, Pyridoxine Hydrochloride, Riboflavin, Lutein, Folic Acid, Manganese Sulphate, Β-Carotene, Phylloquinone, Biotin, Sodium Selenite, Potassium Iodide, Vitamin D3, Cyanocobalamin. 166 Puramino A+ Corn Syrup Solids, Amino Acids (L-Aspartic Acid, L-Leucine, L-Lysine Hydrochloride, L-Proline, L-Alanine, L-Valine, Monosodium Glutamate, L-Isoleucine, L-Serine, L-Threonine, L-Tyrosine, L-Arginine, L-Phenylalanine, Glycine, L-Cystine, L-Histidine, L-Tryptophan, L-Methionine), High Oleic Sunflower Oil, Medium Chain Triglycerides (Fractionated Coconut Oil), Soy Oil, Modified Tapioca Starch, Mortierella Alpina Oil, Crypthecodinium Cohnii Oil*, Taurine, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, Calcium Citrate, Calcium Hydroxide, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Phosphate, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Sodium Citrate, Sodium Iodide, Sodium Selenite And Zinc Sulfate, Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1.*A source of DHA 167 Pregestimil A+ Corn Syrup Solids, Hydrolyzed Casein* (From Milk), Medium Chain Triglycerides (Fractionated Coconut Oil), Modified Corn Starch, Soy Oil, Corn Oil, High Oleic Vegetable Oil (Safflower And/Or Sunflower Oil), Mortierella Alpina Oil**, L-Cystine, Crypthecodinium Cohnii Oil†, L-Tyrosine, L-Tryptophan, Taurine, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide).(Calcium Citrate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Oxide, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Sodium Citrate, Sodium Iodide, Sodium Selenite And Zinc Sulfate).(Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1).*Modified to be better tolerated in milk allergic babies **A source of ARA †A source of DHA 168 Nutramigen® A+® with LGG® Corn Syrup Solids, Hydrolyzed Casein# (From Milk), Palm Olein Oil, Coconut Oil, Soy Oil, High Oleic Sunflower Oil, Modified Corn Starch, Mortierella Alpina Oil, L-Cystine, Crypthecodinium Cohnii Oil¶, L-Tyrosine, L-Tryptophan, Taurine, Lactobacillus Rhamnosus Gg, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide),(Calcium Citrate, Calcium Hydroxide, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Oxide, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Sodium Citrate, Sodium Iodide, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). #Modified to be better tolerated in milk allergic babies, ¶ A source of DHA, LGG® is a registered trademark of Valio ltd. 169 Nestlé® Good Start® 1 Baby Formula Partially Hydrolyzed Reduced Minerals Whey Protein Concentrate‡‡, Lactose‡‡, Corn Maltodextrin‡, Palm Olein‡‡, Soybean Oil‡, Coconut Oil‡‡, High Oleic Safflower Oil‡‡ Or High Oleic Sunflower Oil‡‡, Mortierella Alpina Oil*,‡‡, Crypthecodinium Cohnii Oil**,‡‡, Taurine‡‡, L-Carnitine‡‡, Nucleotides (Cytidine-5'-Monophosphate‡‡, Disodium Uridine 5’-Monophosphate‡‡, Adenosine 5’- Monophosphate‡‡, Disodium Guanosine 5’-Monophosphate‡‡), Bifidobacterium Lactis (Bb-12)‡‡, Trypsin‡‡, Soy Lecithin‡, Ascorbyl Palmitate‡‡, Mixed Tocopherols‡, Vitamins (Vitamin A Acetate‡‡, Vitamin D3 ‡‡, Dl-Alpha-Tocopheryl Acetate‡‡, Phylloquinone‡‡, Sodium Ascorbate‡‡, Thiamine Mononitrate‡‡, Riboflavin‡, Niacinamide‡‡, Calcium Pantothenate‡‡, Pyridoxine Hydrochloride‡‡, Biotin‡, Folic Acid ‡‡, Vitamin B12‡, Choline Bitartrate‡‡, Inositol‡‡), Minerals (Calcium Chloride‡‡, Tricalcium Phosphate‡‡, Potassium Phosphate‡‡ , Magnesium Chloride‡‡, Potassium Citrate‡‡, Sodium Citrate‡‡, Ferrous Sulphate‡‡, Zinc Sulphate‡‡, Manganese Sulphate‡‡,Copper Sulphate‡‡, Potassium Iodide‡‡, Sodium Selenate‡‡).* A Source of Arachidonic Acid (ARA), ** A Source of Docosahexaenoic Acid (DHA), ‡ From non-genetically 170 engineered (GE) sources, ‡‡ As always, only available from non-GE sources. Gerber® Good Start® A2 Powder Infant Formula Lactose, Vegetable Oils (Palm Olein, Soy, Coconut, High-Oleic Safflower Or High-Oleic Sunflower), Whey Protein Concentrate (Reduced In Minerals), Nonfat Dry Milk*, And Less Than 2% Of: Soy Lecithin, Potassium Citrate, Calcium Citrate, Corn Maltodextrin, Potassium Chloride, 2'-O-Fucosyllactose, C. Cohnii Oil, M. Alpina Oil, Calcium Phosphate, Choline Bitartrate, Sodium Ascorbate, Sodium Chloride, Taurine, Magnesium Chloride, Lactobacillus Reuteri (Dsm 17938) Cultures, Potassium Hydroxide, Nucleotides (Cytidine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate, Adenosine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate), Inositol, Ferrous Sulfate, Alpha-Tocopheryl Acetate, Mixed Tocopherols, Ascorbyl Palmitate, L-Histidine, Zinc Sulfate, Niacinamide, 171 Calcium Pantothenate, L-Carnitine, Copper Sulfate, Vitamin A Acetate, Riboflavin, Thiamine Mononitrate, Pyridoxine Hydrochloride, Manganese Sulfate, Citric Acid, Potassium Iodide, Folic Acid, Phylloquinone, Biotin, Sodium Selenate, Vitamin D3, Vitamin B12. *A2 Beta (Β)-Casein Milk Protein From A2 Milk, 2'-O-Fucosyllactose (2’-Fl): A type of Prebiotic, C. Cohnii Oil: A source of DHA, M. Alpina Oil: A source of ARA Enfamil Lower Iron Infant Formula Powder- Lactose, Modified Milk Ingredients, Palm Olein Oil, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Corn Syrup Solids, Maltodextrin, Taurine, Nucleotides (Adenosine 5'-Monophosphate, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate), L-Carnitine, (May Contain Potassium Hydroxide), (Calcium Carbonate, Cupric Sulfate, Ferrous Sulfate, Magnesium Oxide, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). 172 Enfamil Infant Formula Concentrate- Modified Milk Ingredients, Water, Lactose, Palm Olein Oil, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Mono And Diglycerides, Soy Lecithin, Carrageenan, Maltodextrin, Taurine, Corn Syrup Solids, Nucleotides (Adenosine 5'-Monophosphate, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate), (May Contain Potassium Hydroxide), (Calcium Carbonate, Calcium Chloride, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Manganese Sulfate, Potassium Citrate, Sodium Chloride, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Sodium Ascorbate, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1) Enfamil A+® Infant Formula Powder- Lactose, Modified Milk Ingredients, Palm Olein Oil, Coconut Oil, Soy Oil, High Oleic Sunflower Oil, Polydextrose, Galactooligosaccharide Solids (Gos), Soy Lecithin, Corn Syrup Solids, Mortierella Alpina Oil, Maltodextrin, Crypthecodinium Cohnii Oil*, Taurine, Nucleotides (Adenosine 5'-Monophosphate, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate), 173 L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide),(Calcium Carbonate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Oxide, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Sodium Chloride, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3, And Vitamin K1), *A source of DHA, Ingredients are subject to change. Consult actual product labels for most current information. Enfamil A+® Infant Formula Ready to feed- Water, Modified Milk Ingredients, Lactose, Palm Olein Oil, Soyoil, Coconut Oil, High Oleic Sunflower Oil, Polydextrose, Galactooligosaccharide Solids (Gos), Mono And Diglycerides, Soy Lecithin, Mortierella Alpina Oil, Carrageenan, Crypthecodinium Cohnii Oil*, Taurine, Maltodextrin, Nucleotides (Adenosine 5'-Monophosphate,Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate,Corn Syrup Solids, L-Carnitine, 174 Disodiumuridine 5'-Monophosphate), Ascorbyl Palmitate, Mixed Tocopherols, (May contain Potassium Hydroxide)(Calcium Carbonate, Calcium Chloride, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Phosphate, Manganese Sulfate, Potassium Citrate, Potassium Iodide, Sodium Chloride, Sodium Citrate, Sodium Selenite And Zinc Sulfate) (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Sodium Ascorbate, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). *A source of DHA" Enfamil A+ Soy Water, Corn Syrup Solids, Soy Protein Isolate, Palm Olein Oil, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Soy Lecithin, Mono- And Diglycerides, Mortierella Alpina Oil‡, L-Methionine, Crypthecodinium Cohnii Oil§, Taurine, Carrageenan, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide), Minerals (Calcium Carbonate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Chloride, Magnesium Phosphate, Potassium Chloride, Potassium Citrate, Potassium Iodide, Sodium Chloride, Sodium Selenite And Zinc Sulfate), Vitamins (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, 175 Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). Enfamil A+ Lactose Free Water, Corn Syrup Solids, Milk Protein Isolate, Palm Olein Oil, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Mortierella Alpina Oil*, Mono And Diglycerides, Soy Lecithin, Crypthecodinium Cohnii Oil**, Carrageenan, Maltodextrin, Taurine, Nucleotides (Adenosine 5'-Monophosphate, Cytidine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate), L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide), (Calcium Carbonate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Phosphate, Manganese Sulfate, Potassium Chloride, Potassium Citrate, Potassium Iodide, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, 176 Vitamin D3 And Vitamin K1)., *A source of ARA **A source of DHA. Enfamil A+ Gentlease Corn Syrup Solids, Partially Hydrolyzed Modified Milk Ingredients (Soy), Palm Olein Oil, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Mortierella Alpina Oil*, Lactose, Crypthecodinium Cohnii Oil**, Taurine, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide),(Calcium Carbonate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Phosphate, Manganese Sulfate, Potassium Chloride, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3, and Vitamin K1),*A source of ARA **A source of DHA Enfamil A+ for frequent Spit Up Milk Ingredients, Rice Starch, Palm Olein Oil, Lactose, Maltodextrin, Coconut Oil, Soy Oil, High Oleic 177 Sunflower Oil, Polydextrose, Galactooligosaccharide Solids (Gos), Mortierella Alpina Oil#, Corn Syrup Solids, Crypthecodinium Cohnii Oil**, Taurine, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide), (Calcium Carbonate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Manganese Sulfate, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). *A source of ARA **A source of DHA, Ingredients are subject to change. Consult actual product labels for most current information. Enfamil A+ EnfaCare Water, Maltodextrin, Modified Milk Ingredients, Lactose, High Oleic Vegetable Oil (Sunflower And/Or Safflower Oil), Soy Oil, Medium Chain Triglycerides (Fractionated Coconut Oil), Coconut Oil, Mono- And Diglycerides, Mortierella Alpina Oil*, Soy Lecithin, Crypthecodinium Cohnii Oil**, Carrageenan, Corn Syrup Solids, Taurine, Nucleotides (Cytidine 5'-Monophosphate, Disodium Uridine 5'-Monophosphate, Adenosine 5'-Monophosphate, Disodium Guanosine 5'-Monophosphate), L-Carnitine, Ascorbyl Palmitate, 178 Mixed Tocopherols, (May Contain Potassium Hydroxide), (Calcium Chloride, Calcium Citrate, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Magnesium Chloride, Manganese Sulfate, Potassium Citrate, Potassium Iodide, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Sodium Ascorbate, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). *A source of ARA **A source of DHA Enfamil A+ 2 Infant Formula Milk Ingredients, Corn Syrup Solids, Palm Olein Oil, Lactose, Coconut Oil, Soy Oil, Higholeic Sunflower Oil, Polydextrose, Galactooligosaccharide Solids (Gos), Soy Lecithin, Glucose, Mortierella Alpina Oil*, Crypthecodinium Cohnii Oil**, Taurine, L-Carnitine, Ascorbyl Palmitate, Mixed Tocopherols, (May Contain Potassium Hydroxide), (Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Manganese Sulfate, Potassium Chloride, Sodium Chloride, Sodium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl- Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine 179 Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1), * A source of ARA **A source of DHA, Ingredients are subject to change. Consult actual product labels for most current information. Enfamil 2 Infant Formula Milk Ingredients, Corn Syrup Solids, Palm Olein Oil, Lactose, Ascorbyl Palmitate, Soy Oil, Coconut Oil, High Oleic Sunflower Oil, Soy Lecithin, Taurine, (Calcium Chloride, Calcium Phosphate, Cupric Sulfate, Ferrous Sulfate, Manganese Sulfate, Potassium Citrate, Sodium Selenite And Zinc Sulfate), (Ascorbic Acid, Biotin, Calcium D-Pantothenate, Choline Chloride, Dl-Α-Tocopheryl Acetate, Folic Acid, Inositol, Niacinamide, Pyridoxine Hydrochloride, Riboflavin, Thiamine Hydrochloride, Vitamin A Palmitate, Vitamin B12, Vitamin D3 And Vitamin K1). 180 Appendix B: Supplemental figures for chapter 9 Appendix Figure B1. Image of breadfruit A. Young leaf; B. SEM image of glandular trichomes. Appendix Figure B2: Image of breadfruit A. Fully expanded leaf; B. SEM image of glandular trichomes 181 Appendix Figure B3: SEM images of Ma’afala glandular trichomes. A. Young leaf; B. Mature leaf
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.)
Development of novel bioproducts from breadfruit
Text