Phytonutrient bioaccessibility and metabolism in vitro and in vivo Dorrain Low Yanwen B.App.Sci. (Food Science and Nutrition) (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 Queensland Alliance for Agriculture and Food Innovation, Centre for Nutrition and Food Sciences. i Abstract Mangoes and bananas, two of Queensland’s major tropical crops are sources of bioactive phytonutrients linked to dietary health based on antioxidant, cardio-protective and vasodilatory properties. Bioaccessibility of macro-, micro- and phytonutrients from whole foods is first determined by mastication followed by gastrointestinal liberation from food matrices (cell wall and membrane). Intact plant cell walls effectively encapsulate cellular components e.g. starch, lipid, protein, carotenoids and polyphenols, physically preventing entry of the mammalian digestive system, which is unable to enzymatically break down plant cell walls. Once bioaccessible, ‘free’ nutrients are available for absorption, while the unreleased fraction travels to the colon for fermentation. Nutritional recommendations are usually based on extracted contents of raw plant material; therefore true dietary concentrations have not been well established. This thesis focuses on studying the effects of sequential digestive processing of mango (Kensington Pride) and banana (Cavendish) flesh in the mouth, stomach, small intestine and colon using in vitro and in vivo approaches. In vitro nutrient bioaccessibility studies often favour mechanical processing over mastication. Investigating the effect of mastication (Chapter 3) in mango demonstrated microstructural changes, conferring a range of chewed particle sizes (large particle clusters to cell fragments). Actions not replicable with a cutting blade i.e. compression, squashing and formation of bolus were observed in these masticated particles, collectively enhancing encapsulation of carotenoids. Teeth cutting or slicing also occurred simultaneously, rupturing cell walls and releasing cellular contents. Whilst there was a (small) particle size effect on bioaccessibility, this may be secondary for soft (mango) tissues, in contrast to previous reports that a single robust carrot cell wall appeared to be sufficient to prevent bioaccessibility. There was incomplete carotenoid bioaccessibility from solid chewed mango particles (20-50%) and puree (65-75%) after simulated gastrointestinal conditions. Presence of intact mango cells and vascular fibres after in vitro digestion indicated acidic hydrolysis or other in vitro digestion conditions did not have a major role in breaking down cell walls, and established that unreleased and/or unabsorbed nutrients are expected to survive to the colon because they were shown not to be liberated from the food matrix. Mango and banana cell structures which survived in vivo mastication and in vitro gastrointestinal digestion (Chapter 4 part A), were fermented in vitro with porcine faecal ii inoculum for 48 h. This involved fermentation of non-fibrous cell walls, thereby releasing the effectively encapsulated cell contents for possible metabolism by microbiota. Cumulative gas, short chain fatty acids and ammonia production were greater in mango than banana fermentation. Microscopic and spectroscopic analysis showed that this was due to a major fermentation-resistant starch fraction in banana, which was absent in mango. This study demonstrated distinctive differences in fermentation kinetics between banana and mango, due to preferential degradation of (parenchyma) fleshy cells over resistant starch in banana, and over thick cellulosic vascular fibres in mango. Upon disintegration of fruit matrices, phenolic compounds were consequently exposed to intensive faecal-microbiota metabolism beyond those of human endogenous enzymes. UPLC-PDA and UHPLC-Q-ToF-MS profiles (Chapter 4 Part B) revealed degradation of intact polyphenols within 8-24 h and concomitant formation of intermediate catabolites within 4-8 h. The time available for nutrient digestion and fermentation in the gastrointestinal tract is determined by the passage rate of intestinal contents, which has often been overlooked in in vivo digestibility studies. The influence of dried mango puree (containing pectin as a component of edible pulp) and purified apple pectin on passage time, was examined using pigs as a human model (Chapter 5 Part A). Mango puree and purified apple pectin delayed gastric fractional outflow to the small intestine, reflecting an increased water holding capacity (and corresponding lower dry matter content) of these diets. This increased water holding capacity however reduced overall retention time, leading to a faster passage rate in the small intestine and colon. These results provided insights to the dynamic movement of digesta, which were found on average, to be of 3 h-stomach, 0.3 h-duodenum, 0.5-1.6 h-jejunum, 0.8 h-ileum, 2.3 h-caecum, 7 h-proximal colon, 4.8 h-mid colon and 3.7 h-distal colon. This is important information for comparing the results of in vitro bioaccessibility/metabolism studies (where time is a variable) to in vivo situations where residence time/passage rate is determined by interactions of food with animals and/or humans. Using samples from the same experiment, the possibility of monitoring mango polyphenol metabolism and uptake along the digestive tract and into the blood stream was assessed. Although it was possible to identify classes of compounds undergoing distinctive digestive processing, the complexity of chromatographic profiles and corresponding mass spectra precluded detailed molecular identification. iii This study has evaluated aspects of digestive processing of two archetypal fruits to illustrate the importance of combining in vitro and in vivo studies to achieve a more complete perspective, and has thereby contributed to the process of defining mechanisms of fruits and vegetables contribution to health and well-being as suggested by many epidemiological studies. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. v Publications during candidature Submitted peer reviewed paper: Low, D., D'Arcy, B., & Gidley, M. (2015). Mastication effects on carotenoid bioaccessibility from mango fruit tissue. Food Research International, 67, 238-246. Conference abstracts Dorrain Low, Barbara Williams, Gabriele Netzel, Bruce D’Arcy & Michael Gidley. Polyphenol fermentation and metabolism in vitro. 26th International Conference on Polyphenols and 8th Tannin Joint Conference, Nagoya University, Japan, 2-6th September 2014 (Poster). Dorrain Low, Barbara Williams, Gabriele Netzel, Bruce D’Arcy & Michael Gidley. Overview of phytonutrients bioaccessibility in vitro. 6th International Symposium on Fruit and Vegetables for Human Health, International Horticulture Congress, Brisbane Convention and Exhibition Centre, Brisbane, 17-22nd August 2014 (Oral). Dorrain Low, Bruce D’Arcy & Michael Gidley. Mastication influences carotenoid release in vitro. Nutrition Society of Australia & Nutrition Society of New Zealand Joint Annual Scientific Meeting, Brisbane Convention & Exhibition Centre, Brisbane, 4-6th December 2013 (Oral and poster). Dorrain Low, Barbara Williams, Bruce D’Arcy & Michael Gidley. Polyphenols bioaccessibility in vitro and in vivo: a pig model. ARC CoE in Plant Cell Walls internode meeting, University of Melbourne, Victoria, October 15-16th 2013 (Oral). Dorrain Low, Bruce D’Arcy & Michael Gidley. Carotenoid release from KP mangoes in vitro. 46th AIFST Annual Conference, Brisbane Convention & Exhibition Centre, Brisbane, 14-16th July 2013 (Oral). Dorrain Low, Bruce D’Arcy & Michael Gidley. Carotenoid release from KP mangoes in vitro. AIFST Summer School, University of Sydney, NSW, 6-8th Feb 2013 (Oral). Dorrain Low, Bruce D’Arcy & Michael Gidley. Phytonutrient bioaccessibility of tropical fruits in vitro and in vivo. ARC CoE in Plant Cell Walls Internode meeting, The University of Queensland, Brisbane, 24-25th October 2012 (Oral). vi Publications included