University of Kentucky UKnowledge Theses and Dissertations--Plant and Soil Sciences Plant and Soil Sciences 2017 ASSESSMENT OF OIL QUANTIFICATION METHODS IN SOYBEAN AND CHIA SEEDS AND CHARACTERIZATION OF OIL AND PROTEIN IN MUTANT CHIA (SALVIA HISPANICA L.) SEEDS Ahmed N. Al-Bakri University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/ETD.2017.359 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Al-Bakri, Ahmed N., "ASSESSMENT OF OIL QUANTIFICATION METHODS IN SOYBEAN AND CHIA SEEDS AND CHARACTERIZATION OF OIL AND PROTEIN IN MUTANT CHIA (SALVIA HISPANICA L.) SEEDS" (2017). Theses and Dissertations--Plant and Soil Sciences. 93. https://uknowledge.uky.edu/pss_etds/93 This Master's Thesis is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Plant and Soil Sciences by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Ahmed N. Al-Bakri, Student Dr. David Hildebrand, Major Professor Dr. Mark Coyne, Director of Graduate Studies ASSESSMENT OF OIL QUANTIFICATION METHODS IN SOYBEAN AND CHIA SEEDS AND CHARACTERIZATION OF OIL AND PROTEIN IN MUTANT CHIA (SALVIA HISPANICA L.) SEEDS THESIS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Agriculture, Food and Environment at the University of Kentucky By Ahmed Nabeel Abdullah Al-Bakri Lexington, Kentucky Director: Dr. David Hildebrand, Professor of Plant and Soil Science Lexington, Kentucky 2017 Copyright © Ahmed Nabeel Abdullah Al-Bakri 2017 ABSTRACT ASSESSMENT OF OIL QUANTIFICATION METHODS IN SOYBEAN AND CHIA SEEDS AND CHARACTERIZATION OF OIL AND PROTEIN IN MUTANT CHIA (SALVIA HISPANICA L.) SEEDS This thesis includes two main parts: I. Evaluation of techniques for oil (total lipid) quantification of chia and soybean seeds. This study evaluated 10 different methods of seed oil quantification, including some methods that have not been applied to oilseeds before. The main aim of this study was to find one or more techniques that are easy, inexpensive, safe and fast with a small amount of ground seeds. The Soxhlet method was used as a standard to compared between techniques of oil quantification. The oil extraction by the Soxhlet method was evaluated with two solvents petroleum ether and acetone. There is not a statistically significant difference between petroleum ether and acetone solvents. No significant differences for the amount of oil recovered via the Soxhlet method were found between Medium Moisture Content (MMC >10%) and Low Moisture Content (LMC < 4.0 %). The Folch technique provided higher percentages of oil extraction than Bligh and Dyer and hexane-isopropanol techniques. There is not a statistically significant difference (P =0.0844) between Soxhlet method and Folch method but less than the Soxhlet method. A supercritical fluid extraction (HCH) method provided lower yield of oil extraction compared with the Soxhlet method for three varieties of bias samples. A Direct Transesterification (DT) method with LMC and MMC provided a statistically significant difference than the Soxhlet method. The DT with LMC provided higher yield than DT with MMC between samples but lower than the Soxhlet method. A Double Direct Transesterification (DDT) following Griffiths protocol provided more accurate results with the stir bar technique than sonication technique. 11 oilseeds bias samples (10 bias samples of soybean and one of chia) covering an oil content range of 15.4 to 32.6% showed, there is a significant difference between the Soxhlet and DDTG method and high oil quantification found with DDTG. A Double Direct Transesterification following Qiao et al. (2015) provided similar oil extraction to the Griffiths et al. (2010) method and also there is a significant difference between the Soxhlet and DDTQ method and higher oil recovered with DDTQ. The Bead Beating Extraction (BBE) protocol showed, there is a statistically significant difference ( P <0.001) than the Soxhlet method. The BBE provided high oil quantification comparing with the Soxhlet method. The BBE provided the best results since it is the easiest, cheapest and fastest oil quantification method. A Nile red fluorescence technique yield no clear results. II. Characterization of oil and protein in mutant chia (Salvia hispanica L.) seeds. The objectives of this study were to (1) measure heritability in chia plants of levels of oil and protein content and seed yield and (2) Compare differences between two locations over two years for oil and protein percentages in chia and seeds yield. A population of 180 M3 mutant individual chia plants was harvested and for which forty M4 chia seeds were planted based on seed composition characteristics, with six plants representing each characteristic (high and lower oil, protein, and density and high yield). The forty M4 chia seeds were planted, with two replications for two locations have been chosen in Kentucky one on Spindletop farm and the other in Quicksand farm. The M5 progeny seeds from plants grown in Spindletop and Quicksand contained significantly (P <0.05) more protein than did seeds from the M4 parents. There were significant differences (P <0.05) between locations where Quicksand obtain higher oil content than Spindletop. There was no significant difference between M5 parents seeds and M4 progeny seeds and higher yield kg/ha at Quicksand than Spindletop. KEYWORDS: Soxhlet, Bias Samples, Fatty Acid Composition, Omega-3, Seed content. Ahmed Al-Bakri Author signature July 18, 2017 Date ASSESSMENT OF OIL QUANTIFICATION METHODS IN SOYBEAN AND CHIA SEEDS AND CHARACTERIZATION OF OIL AND PROTEIN IN MUTANT CHIA (SALVIA HISPANICA L.) SEEDS BY Ahmed Nabeel Abdullah Al-Bakri David Hildebrand Director of Thesis Mark Coyne Director of Graduate Studies July 18 2017 Date This work is dedicated to my mother and my sister ACKNOWLEDGMENTS I would never be able to achieve this success without the guidance of my advisor, my co- advisor, the prayer and the support of my mother, and support of my friends. I would like to thank and express my gratitude to my advisor, Dr. David Hildebrand, Co- Advisor Dr. Tim Phillips for their help, support, guidance, and patience. I would like to thank and express my gratitude to The Higher Committee of Education Development (HCED) in Iraq for supporting me during my study at the University of Kentucky. I would also like to thank my mother, sister, friends continuous support and help. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iii TABLE OF CONTENTS ................................................................................................... iv LIST OF TABLES ........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix CHAPTER 1: Literature Review ........................................................................................ 1 Assessment of Oil Quantification Methods in soybean and chia seeds .......................... 1 Oilseeds Production .................................................................................................... 1 Organic Solvents Used ................................................................................................ 5 Factors ......................................................................................................................... 7 Main Extraction Methods of Total Lipid Quantification ................................................ 8 Soxhlet Extraction Method (Soxhlet, 1879) ............................................................... 8 Folch et al. (1957) and Bligh and Dyer (1959) ..........................................................