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Development of New Functional Food Traits in Peanuts Kim-Yen Phan

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Development of New Functional Food Traits in Peanuts Kim-Yen Phan Development of New Functional Food Traits in Peanuts Kim-Yen Phan-Thien Thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy School of Chemical Engineering The University of New South Wales Sydney, Australia August, 2012 25,*,1$/,7<67$7(0(17 µ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¶ 6LJQHG«««««««««««««««««««««««««« ««« ϭϴͬϬϭͬϭϯ 'DWH««««««««««««««««« &23<5,*+767$7(0(17 µ, KHUHE\ JUDQW WKH 8QLYHUVLW\ RI 1HZ 6RXWK :DOHV RU LWV DJHQWV WKH ULJKW WR DUFKLYH DQG WR PDNH DYDLODEOH P\ WKHVLV RU GLVVHUWDWLRQ LQ ZKROH RU SDUW LQ WKH 8QLYHUVLW\OLEUDULHVLQDOOIRUPVRIPHGLDQRZRUKHUHDIWHUNQRZQVXEMHFWWRWKH SURYLVLRQVRIWKH&RS\ULJKW$FW,UHWDLQDOOSURSULHWDU\ULJKWVVXFKDVSDWHQW ULJKWV,DOVRUHWDLQWKHULJKWWRXVHLQIXWXUHZRUNV VXFKDVDUWLFOHVRUERRNV DOO RUSDUWRIWKLVWKHVLVRUGLVVHUWDWLRQ ,DOVRDXWKRULVH8QLYHUVLW\0LFURILOPVWRXVHWKHZRUGDEVWUDFWRIP\WKHVLVLQ 'LVVHUWDWLRQ$EVWUDFW,QWHUQDWLRQDO WKLVLVDSSOLFDEOHWRGRFWRUDOWKHVHVRQO\ ,KDYHHLWKHUXVHGQRVXEVWDQWLDOSRUWLRQVRIFRS\ULJKWPDWHULDOLQP\WKHVLVRU, KDYH REWDLQHG SHUPLVVLRQ WR XVH FRS\ULJKW PDWHULDO ZKHUH SHUPLVVLRQ KDV QRW EHHQJUDQWHG,KDYHDSSOLHGZLOODSSO\IRUDSDUWLDOUHVWULFWLRQRIWKHGLJLWDOFRS\RI P\WKHVLVRUGLVVHUWDWLRQ HUWDWWLRL Q 6LJQHG««««««««««««««««««««««««««««« «« ϭϴͬϬϭͬϭϯ 'DWH««««««««««««««««« $87+(17,&,7<67$7(0(17 µ, FHUWLI\ WKDW WKH /LEUDU\ GHSRVLW GLJLWDO FRS\ LV D GLUHFW HTXLYDOHQW RI WKH ILQDO RIILFLDOO\DSSURYHGYHUVLRQRIP\WKHVLV1RHPHQGDWLRQRIFRQWHQWKDVRFFXUUHG DQG LI WKHUH DUH DQ\ PLQRU YDULDWLRQV LQ IRUPDWWLQJ WKH\ DUH WKH UHVXOW RI WKH FRQYHUVLRQWRGLJLWDOIRUPDW¶WDOIRRUPDW¶ 6LJQHG««««««««««««««««««««««««««««« «« 'DWH«««««««««««««««««ϭϴͬϬϭͬϭϯ Acknowledgements I am indebted to my supervisor Dr. N. Alice Lee and my co-supervisor Dr. Graeme C. Wright for their research direction, generous advice, and personal encouragement. They have been excellent mentors, from whom I have learnt a great deal during my PhD study. Teleconferences and trips to Kingaroy to visit Graeme were always stimulating. Alice, being rather more easily accessible, was no stranger to unscheduled knocks on the door and I thank her for her patience and kindness. I received valuable advice from a number of other researchers and technicians. In particular, Dorothy Yu and Rabeya Akter (Elemental and Solid State Analysis Unit, Mark Wainwright Analytical Centre, UNSW) advised on the elemental analysis. Dr. Mirta Golic (Peanut Company of Australia) and Prof. Brynn Hibbert (School of Chemistry, UNSW) helped me with the NIRS analysis. Dr. Victor Wong and I spoke frequently about HPLC. Discussions with Dr. John Earl (Children’s Hospital at Westmead) clarified issues with the ECD and inspired some fun (if futile) sessions rigging up split-flow LC configurations. Lewis Adler and Dr. Leanne Stephenson advised on the HRMS. Dr. Martin Bucknall (Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre, UNSW) was a fount of knowledge on GC-MS, and I look forward to doing some data-mining with his advice. Camillo Taraborrelli has been an exceptionally organised lab manager; the lab wouldn’t run half as well without him. I thank UNSW for enabling my PhD through provision of a University Postgraduate Award and PRSS travel award to attend Pacifichem 2010. The Grains Research and Development Corporation funded travel to the United States to attend the 2010 APRES meeting and generously provided me a one year scholarship during the extension of my PhD enrolment. The Peanut Company of Australia supplied all my peanuts and provided financial support for the research. I thank my friends, old and new, lab mates and random unibar mates, for adding laughs, perspective, and a bit of life balance to the study. Finally, I thank my family, Lai Kuen Wong, Kim-Chi Phan-Thien, and Minh Phan-Thien, for their unconditional love and belief in me. | Acknowledgements 2 Abstract Two categories of functional food traits were researched: dietary minerals and antioxidants. The primary objectives were to (1) characterise diverse peanut phenotypes using established methods or developing and validating analytical methods if necessary; (2) estimate genotypic variation in the functional food trait; and (3) investigate the stability of the functional food trait through studies of genotype-by-environment (G × E) interaction. Essential mineral concentrations in kernels were analysed by inductively coupled plasma- optical emission spectroscopy (ICP-OES) and ICP-mass spectrometry (ICP-MS) with the use of a dynamic reaction cell (DRC) after preparation of samples by microwave-assisted closed acid digestion. Antioxidant capacity was assessed using ABTS•+, DPPH•, Folin-Ciocalteu total phenolics, and ORAC assays adapted to a 96-well microplate format for high-throughput analysis. The phytochemical profile was quantitatively analysed by high performance liquid chromatography (HPLC) with the use of a photodiode array (PDA) detector, after ultrasound- and enzyme-assisted extraction and solid phase extraction to purify and concentrate the extracts. Genotypic variation for essential minerals and antioxidant capacity was estimated by analysis of 32 full-season maturity and 24 ultra-early maturity genotypes from the Australian Peanut Breeding Program (APBP). The studies established useful levels of variation of more than 10% relative standard deviation (RSD) among the genotypes in concentrations of most of the tested essential minerals, and of more than 20% RSD in antioxidant capacity, although only the ORAC assay distinguished statistically significant differences between genotypes. Studies of G × E interaction affecting the essential mineral and antioxidant capacity traits revealed that genotype, environment, and G × E interaction all significantly affected trait expression. The results confirmed that there was substantial genetic control of essential mineral concentrations and antioxidant capacity in peanut kernels, but that it will be important to characterise environmental interaction to enable plant/seed selection in the APBP and potentially manipulate the interaction through agronomic or postharvest management. The essential minerals data were used to develop approximately predictive calibrations for Ca, K, Mg, and P by near-infrared reflectance spectroscopy (NIRS) of sufficient accuracy to be useful as plant/seed selection tools in plant breeding. Techniques that enable high-throughput, non-destructive, time/cost-effective analysis of trait segregation are valuable due to the extremely large number of samples that are generated in breeding programs. | Abstract 3 Five peanut genotypes with diverse antioxidant capacity phenotypes were quantitatively profiled for p-coumaric acid, salicylic acid, resveratrol, and daidzein. The co-eluting compounds, caffeic/vanillic acid and ferulic/sinapic acid, were quantified on caffeic acid equivalent and ferulic acid equivalent bases, respectively. The HPLC analysis established significant genotypic differences in phytochemical concentrations and also the importance of the bound (e.g., conjugated and matrix-embedded) fraction. Fractions of the HPLC eluate were evaluated by ORAC assay to evaluate relative contributions to antioxidant capacity, and allowed identification of a number of unknown compounds that made important contributions to antioxidant capacity. HPLC analysis of kernels subjected to various roasting treatments (150 °C, 0-70 min and 160 °C, 0-32.5 min) showed that ferulic/sinapic acid concentrations declined with roasting duration, but that most other tested analytes were relatively thermo-stable. | Abstract 4 Table of contents Acknowledgements ........................................................................................................................2 Abstract ..........................................................................................................................................3 Table of contents ...........................................................................................................................5 List of abbreviations .......................................................................................................................8 List of tables ................................................................................................................................ 11 List of figures ............................................................................................................................... 15 1 Introduction ........................................................................................................................ 18 2 Development and validation of analytical methodology .................................................... 21 2.1 Validation of elemental analysis by ICP-OES and ICP-MS ........................................... 21 2.1.1 Objectives ...........................................................................................................
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