i Health eefits of o-utritive food opoets This thesis is presented for the degree of Doctor of Philosophy to The University of Western Australia 2015 Kerry Ivey Bachelor of Science (Nutrition) Curtin University, School of Public Health Postgraduate diploma (Dietetics) Curtin University, School of Public Health Supervisors: Professor Rihard L. Prie, Professor Joatha M Hodgso, Assoiate Professor Deorah Kerr Foreword i Health eefits of o-utritive food opoets Foreword and contents Kerry Ivey Bachelor of Science (Nutrition) Curtin University, School of Public Health Postgraduate diploma (Dietetics) Curtin University, School of Public Health Supervisors: Professor Rihard L. Prie, Professor Joatha M Hodgso, Assoiate Professor Deorah Kerr Foreord and contents ii SUMMARY This thesis is presented as a series of epidemiological, intervention and review papers which aimed to explore the health benefits of non-nutritive food components. Flavonoids and probiotics represent two diverse groups of non-nutritive food components, that are thought to provide benefits to human health. Flavonoid compounds Flavonoids are a diverse group of compounds which share a common flavan nuclear structure. Major classes of flavonoids include flavonols, proanthocyanidins, flavanols, flavones, flavanones and isoflavones. There is strong in vitro and intervention data suggesting health benefits of flavonoid intake. However, data from epidemiological studies are less clear. As such, relationships between flavonoid intake and health outcomes were explored in a population of elderly postmenopausal women. We found that consumption of flavonols from both tea and non-tea sources was associated with reduced risk of atherosclerotic vascular disease mortality. Similarly, consumption of proanthocyanidins was associated with better renal function and lower risk of adverse renal outcomes. The beneficial associations of flavonoids appeared to extend beyond intakes of isolated flavonoid classes. We observed a beneficial association between total-flavonoid intake and reduced risk of all-cause mortality. When exploring our ability to assess flavonoid intake in populations, the two major flavonoid food composition databases, the United States Department of Agriculture and Phenol-Explorer databases, yielded highly correlated intake estimates for total- flavonoids, flavanols , flavanones and anthocyanidins. However, the poorer correlation between flavonol and flavone intake estimates derived from each database was likely Foreord iii due to differences in United States Department of Agriculture (USDA) and Phenol- Explorer (PE) methodologies. Probiotic bacteria Probiotics are defined as live microorganisms, which when administered in adequate amounts, confer a health benefit to the host. Despite a long history of safe use, incorporation of probiotics into current therapeutic guidelines, and strong mechanistic data, there is an identified lack of statistically significant data supporting beneficial effects of probiotics. In order to investigate the health benefits of daily consumption of yoghurt and its probiotics, a 6-week randomised, controlled, double blind, factorial trial in 156 overweight men and women was implemented. We found that supplementation with probiotic yoghurt is more efficacious than probiotic capsules at improving fecal probiotic content. However, this trial, which supplemented with the strains Lactobacillus acidophilus La5 and Bifidobacterium animalis subsp. lactis BB12, did not improve type 2 diabetes and cardiovascular disease risk factors. With the aim of summarising current evidence for the hypocholesterolaemic benefits of probiotic supplementation, a Cochrane approved systematic review and meta-analysis was performed. It was concluded that daily probiotic supplementation improves total cholesterol and low density lipoprotein cholesterol concentrations. Furthermore, in a population based study of elderly postmenopausal women, habitual high probiotic yoghurt consumption was associated with improved common carotid artery intima media thickness; a risk factor for atherosclerotic vascular disease. Foreord i Concluding remark Taken as a whole, the results of the studies presented in this thesis support the concept that the non-nutritive food components, probiotics and flavonoids, may play a role in improving cardio-metabolic health. Foreord ACKNOWLEGDEMENTS There are times in one’s life, where things go right, great minds come together, and friendships are formed. My PhD candidature has been one such time. The problem, however, arises articulating my gratitude to those who have contributed to this time. On major lesson I have learnt during my candidature is to stand on the shoulders of the giants before you. With this in mind I will turn to the great minds of the past in order to articulate the contribution my supervisors, collaborators and friends have made to the last three years of my life. The first person I must acknowledge is my primary supervisor, Professor Richard Prince, who has not only inspired me to be a better scientist, but more importantly, a better person. Intelligence plus character-that is the goal of true education Martin Luther King Jr. Educating the mind without educating the heart is no education at all Aristotle If you would be a real seeker after truth, it is necessary that at least once in your life you doubt, as far as possible, all things René Descartes True education does not consist merely in the acquiring of a few facts of science, history, literature, or art, but in the development of character David O. McKay My secondary supervisor was Professor Jonathan Hodsgon, who with his wisdom and sensibility has inspired, encouraged and enabled me to develop my own research path. The mediocre teacher tells. The good teacher explains. The superior teacher demonstrates. The great teacher inspires William Arthur Ward Do not train a child to learn by force or harshness; but direct them to it by what amuses their minds, so that you may be better able to discover with accuracy the peculiar bent of the genius of each Plato My PhD candidature has provided me with the opportunity to collaborate with many talented people from a wide variety of backgrounds. Thank you to the following people for sharing their impressive knowledge, opening my eyes to new fields, and making my learning opportunities immensely pleasurable; Deborah Kerr (supervisor), Wai Lim (renal physiology), Kevin Croft (chemistry), Catherine Rawlinson (metabolomics), and Rongchang Yang (microbiology). Education: that which reveals to the wise, and conceals from the stupid, the vast limits of their knowledge Mark Twain Play is the highest form of research Albert Einstein Educated men are so impressive William Shakespeare The traditional path of many Dietitians is to pursue purely clinical research. The following people have shared their talent, wisdom and time to teach me important lab based skills that will forever make me a better scientist: Catherine Rawlinson (GCMS), Rongchang Yang (DNA extraction and PCR), as well as Jenny Wang, Anthony Buzzai and Lawrence Liew (general lab skills). Give a bowl of rice to a man and you will feed him for a day. Teach him how to grow his own rice and you will save his life Confucius Foreord i Thank you to Helena Moneta, Nancy Lin, Blagica Stojceski, Fiona Edmonds, Jenny Wang, Lawrence Liew and Anthony Buzzai, who have made my PhD candidature an immensely happy and successful experience. The making of friends, who are real friends, is the best token we have of a man's success in life Edward Everett Hale Happiness is the highest good Aristotle Thank you to Benjemin Williams, for understanding me, accepting me and helping me to grow. A friend is one that knows you as you are, understands where you have been, accepts what you have become, and still, gently allows you to grow William Shakespeare Thank you to my parents and brother, for supporting me. There is no school equal to a decent home and no teacher equal to a virtuous parent Mahatma Gandhi And finally, I return once again to my fantastic supervisor Richard, who has become and will continue to be one of my greatest friends. Perhaps the most delightful friendships are those in which there is much agreement, much disputation, and yet more personal liking George Eliot Foreord ii STATEMENT OF CANDIDATE CONTRIBUTION I declare that this thesis contains published work and work prepared for publication, which have been co-authored. None of the material herein has been presented for the purpose of obtaining any other degree. The candidate contributions are detailed below. For all publications arising from this thesis, in conjunction with co-authors, Miss Ivey devised the pre-specified data analysis protocol and undertook the manuscript review. Miss Ivey was solely responsible for implementation of all data analysis, literature review, manuscript preparation and manuscript submission. In regards to the randomised controlled trial presented in Appendix A, Miss Ivey, in collaboration with Professors’ Prince and Hodgson, devised the pre-specified study protocol and reviewed grant submissions. In conjunction with Dr Rongchang Yang (Murdoch University, Perth, Australia), Miss Ivey performed the fecal DNA extractions, Nanodrop, quantitative PCR, and digital PCR. Miss Ivey was solely responsible for the literature review, implementation and management of the trial, direct supervision of staff and students, data analysis, and preparation of grant applications. Student signature Coordinating supervisor signature