Interactions between dietary polyphenols and plant cell wall models

INTERACTIONS BETWEEN DIETARY POLYPHENOLS AND PLANT CELL WALL MODELS Anh Dao Thi Phan B.Eng Food Technology (Can Tho University, Vietnam) MSc Food Science and Technology (University of Copenhagen, Denmark) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 ARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation Abstract The nutritional and health benefits of polyphenols in protecting humans from the risk of chronic diseases (e.g cardiovascular disease, cancers) are now well established from both epidemiological and intervention studies Recently, there has been emerging interest in the interactions between polyphenols and food components, particularly dietary fibres derived from plant cell walls (PCW), in determining the health-promoting effects of polyphenols These interactions may have a major role in controlling the bioaccessibility of polyphenols by modulating the release of polyphenols in the upper digestive tract, or delivering bound polyphenols to the colon, for further release and metabolism by the resident microbiota This project addresses important aspects related to polyphenol bioaccessibility through investigations of: (i) the interactions between diverse polyphenols and cellulose, a main dietary fibre component, and determination of the mechanism behind such interactions; (ii) the effects of environmental factors, that are relevant to food systems and digestion conditions, on the interactions between polyphenols and cellulose; (iii) the binding selectivity of diverse polyphenols to different PCW components (cellulose, hemicellulose, pectin), and identification of factors that influence such binding selectivity; and (iv) the release and metabolism of polyphenols associated with PCW during in vitro gastrointestinal digestion and colonic fermentation A systematic understanding of the interactions between polyphenols and PCWs and the mechanisms behind such interactions has been developed Binding kinetics and binding isotherm studies of diverse polyphenols to cellulose and other PCW components permit quantification of the binding extents/rates, and estimation of the apparent maximum binding capacities and binding affinities of different polyphenols to various PCW components The binding capacities achieved were in the range of 20–60% of the PCW mass, and the apparent maximum binding capacity was predicted to be 30–150% PCW mass, depending on the polyphenol molecules and the PCW components This implies that a high amount of ingested polyphenols might be present in the human digestive tract in non-bioaccessible forms, if the interactions with PCWs are relatively stable under gastric and small intestinal conditions By employing a model system of bacterial cellulose-based composites which have previously been shown to represent important features of the chemical organization and assembly of primary PCWs, this project overcomes the difficulties of working with the complexity of heterogeneous cell wall materials and is able to define the differences in binding behaviours of polyphenols to various PCW i constituents The binding selectivity of diverse polyphenols to different PCW components was determined and quantified, with the binding capacities following the order of Cellulose>Cellulosearabinoxylan>Cellulose-xyloglucan>Cellulose-pectin>Apple cell walls, for the binding of (+/-)catechin and ferulic acid, while Cellulose-pectin composites showed the greatest binding for cyanidin-3-glucoside (Cya-3-glc) In addition, both intrinsic factors (e.g the chemical characteristics of polyphenols, the chemical organization and local microstructure of cell wall components, and the charged nature of cell wall polymers) and extrinsic factors (e.g pH, temperature and salt) are major factors in modulating the binding process in a food system and/or under human digestion conditions, suggesting the simultaneous and complex effects of multiple factors on the binding rates and extents of polyphenols This project observed the incomplete release of polyphenols associated with cellulose and apple cell walls under gastrointestinal digestion conditions, with the most release occurring in the gastric phase as a result of the effects of the acidic environment on polyphenol/PCW interactions There was limited release of bound (+/-)-catechin (2035%) and bound Cya-3-glc (3060%), whereas greater release was observed for the bound ferulic acid (4070%) This indicates that a large proportion of polyphenols are likely to be delivered to the colon for further release and metabolism during the fermentation by colonic microbiota In order to investigate the potential release and metabolism of polyphenols in the large intestine, a 72h in vitro colonic fermentation was conducted for the complexes of polyphenols with apple cell walls (ACW) or bacterial cellulose (BC), using a pig faecal inoculum All ACW substrates were fermented faster than the BC substrates, as evidenced by the significantly (p Cya-3-glc > (+/-)-catechin The released polyphenols were completely metabolized by pig feacal microbiota via various proposed pathways, leading to the formation of a small number of possible metabolites Although the presence of different PCWs did not show an alteration in the metabolic pathways mediated by pig faecal bacteria for the three polyphenols studied, polyphenols associated with PCWs underwentfaster microbial metabolism compared to the control sample without any PCW The results suggest the essential role of PCW in the transportation of polyphenol precursors to the colon, and subsequent enhancement of the microbial metabolism,leading to the production of polyphenol metabolites that might exert health benefits ii 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 iii Publications during candidature: Peer-reviewed papers:  PHAN, A D T., NETZEL, G., WANG, D., FLANAGAN, B M., D’ARCY, B R & GIDLEY, M J 2015 Binding of dietary polyphenols to cellulose: Structural and nutritional aspects Food Chemistry, 171, 388-396 (Incorporated in Chapter 3)  PHAN, A D T., D'ARCY, B R & GIDLEY, M J 2016 Polyphenol-cellulose interactions: effects of pH, temperature and salt International Journal of Food Science and Technology, 51, 203-211 (Incorporated in Chapter 4) Conference abstracts and presentations:  PHAN, A D T., NETZEL, G., D’ARCY, B R & GIDLEY, M J 2014 Adsorption of dietary polyphenols to cellulose: Quantification and mechanisms Proceeding “Polyphenol Communication 2014”, 365-366, 27th International Conference on Polyphenols and 8th Tannin Joint Conference, Nagoya University, Japan, 2-6th September 2014 (Poster presentation)  PHAN, A D T., NETZEL, G., D’ARCY, B R & GIDLEY, M J 2013 The roles of cellulose in controlling the bioaccessibility of dietary polyphenols Nutrition Society of Australia & Nutrition Society of New Zealand Joint Annual Scientific Meeting, Brisbane, 4-6th December 2013 (Oral presentation)  PHAN, A D T., NETZEL, G., D’ARCY, B R & GIDLEY, M J 2013 Interactions between food phenolic compounds and cellulose: mechanism and characterization The 46th AIFST Annual Conference, Brisbane Convention & Exhibition Centre, Brisbane, 14-16th July 2013 (Poster presentation)  PHAN, A D T., NETZEL, G., D’ARCY, B R & GIDLEY, M J 2013 Interactions between food phenolic compounds and cellulose AIFST Food Science Summer School, University of New South Wales, Sydney, 6-8th Feb 2013 (Poster presentation) iv Publications included in this thesis PHAN, A D T., NETZEL, G., WANG, D., FLANAGAN, B M., D’ARCY, B R & GIDLEY, M J 2015 Binding of dietary polyphenols to cellulose: Structural and nutritional aspects Food Chemistry, 171, 388-396 (Incorporated as Chapter 3) Contributor Statement of contribution Anh Dao Thi Phan (Candidate) Designed experiments (80%) Performed binding experiments (100%) Statistical analysis of data (100%) Wrote paper (90%) Gabi Netzel Designed experiments (5%) Edited paper (10%) Dongjie Wang Performed SEM microscopy (100%) Bernadine Flanagan Performed NMR experiment (100%) Analysed NMR data (100%) Edited paper (5%) Bruce D’Arcy Designed experiments (5%) Edited paper (10%) Michael J Gidley Designed experiments (10%) Wrote paper (10%) Edited paper (75%) v PHAN, A D T., D'ARCY, B R & GIDLEY, M J 2016 Polyphenol-cellulose interactions: effects of pH, temperature and salt International Journal of Food Science and Technology, 51, 203-211 (Incorporated as Chapter 4) Contributor Statement of contribution Anh Dao Thi Phan (Candidate) Designed experiment (80%) Performed experiments (100%) Analysed data (90%) Wrote paper (90%) Bruce D’Arcy Designed experiment (5%) Edited paper (30%) Michael J Gidley Designed experiment (15%) Interpreted data (10%) Wrote paper (10%) Edited paper (70%) vi Contributions by others to the thesis Advice on project conception, experimental design, and the interpretation of research data as well as critical revision of thesis chapters were contributed by my advisory team: Professor Mike Gidley, Dr Bruce D’Arcy, and Dr Gabi Netzel In addition, Dr Barbara Williams was involved during these processes for Chapter Non-routine technical work involving various chemical analyses, SEM microscopy, and NMR study were contributed by other peoples as detailed in the following table: Contributor Dr Barbara Williams Statement of contribution Consulted for experimental design in Chapter Consulted for statistical analysis in Chapter Proof-reading and correction for Chapter Dr Gabi Netzel Assisted UPLC-PDA and UPLC-EIS-MS/MS analysis in Chapter Dr Dongjie Wang Obtained SEM images: Figure 3.1 (Chapter 3) and Figure 5.1 (Chapter 5) Dr Bernadine Flanagan Conducted NMR studies: Figure 3.2, Table 3.1 (Chapter 3), and Figure 5.5 (Chapter 5) Barbara Gorham Assisted in preparation and lab work for the in vitro colonic fermentation experiments (Chapter 6) Dr Deirdre Mikkelsen Assisted in lab work for the in vitro colonic fermentation experiments (Chapter 6) Dagong Zhang Analysed SCFA and NH+4 production: incorporated in Table 6.2 Peter Isherwood (Chapter 6) Dr Cherrie Beahan Analysed mono-saccharides and uronic acids in apple cell wall Dr Roshan Cheetamun extracts: incorporated in Table 5.1 (Chapter 5) and Figure 6.2 (Chapter 6) Dr David Appleton Determined protein content in apple cell wall extracts: incorporated in Table 5.1 (Chapter 5) Statement of parts of the thesis submitted to qualify for the award of another degree None vii Acknowledgements Up to now, this PhD project is the most challenging and exciting journey of my life I have achieved much during the year-PhD study, not only in knowledge of food science and nutrition, but also in understanding myself in face of great challenges I would like to take this opportunity to thank all the people who have given me invaluable support, substantial encouragement, and for cheering me up First of all, I am grateful to my supervisors Professor Mike Gidley, Dr Bruce D’Arcy, and Dr Gabi Netzel for their excellent supervision, invaluable support and discussion throughout the research and thesis writing I have sincerest appreciation for you always having time to discuss and encourage me, not only during the study I had to deal with, but also with difficulties in my student life Your critical comments on my reports, your questions about research data, and your suggestions have resulted in significant contributions to my thesis Many thanks for your inspiration, and for developing my interests in plant cell wall and polyphenol research I specially thank Dr Gabi Netzel and Dr Barbara Williams for your invaluable support during the in vitro colonic fermentation and polyphenol analysis This experiment could not have been carried out without your help and useful advice I would like to express my deep thanks to Dr Bernadine Flanagan and Dr Dongjie Wang for your help and explanation about NMR, SEM and bacterial cellulose production Many thanks to Dr Deirdre Mikkelsen and Dr Nima Gunness for your help and encouragements during my PhD study I wish to thank Mr John Gorham for patiently teaching me confocal imagingtechniques, and Mrs Barbara Gorham for your great assistance in the fermentation experiment I would like to extend my thanks to all CoE - UQ staff, past and present PhD students for the fortnightly meeting/discussion, your help and friendship Many thanks go to all the CNAFS/SAFS staff and students for your cooperation and contribution to a pleasant working environment I sincerely thank all my Vietnamese friends, who are always giving me helps, and spending time with me and sharing with me happiness and sadness during my study life in Australia Special thanks to my colleagues and ex-teachers at Food Technology Department, Cantho University, Vietnam for your support during my PhD journey Without the financial support provided by The Vietnamese Government Scholarship (VIEDMOET), The University of Queensland, and The ARC Centre of Excellent in Plant Cell Walls, my PhD study would never have started viii I am very grateful to my parents, my parents in law for their endless love and unlimited support for the whole of my life I would like to thank my older brother, my sister in law, and my nephews for their taking care our parents, creating a warm and happy atmosphere in our family during my time of being far away from home Last but not least, I would like to thank my beloved husband, Xuan Minh, for your endless love, understanding, encouragement, and support during my life I really appreciate all that you have done for me Thanks to my little daughter for forgiving me for leaving you behind for three years, and for giving me a strong motivation for completion of the PhD ix Cat-Met ACW (72h) 0.025 0.012 ACW-Cat (72h) 0.020 Cat-Met 4.404 4.548 1.534 0.005 0.000 3.426 3.779 0.987 1.098 1.295 0.002 1.866 2.00 AU 4.397 4.542 1.00 0.015 0.010 3.410 0.004 2.413 0.006 1.932 2.019 0.008 0.9070.983 1.098 1.290 1.444 AU 0.010 3.794 0.014 2.239 0.016 2.577 2.569 Microbial metabolism of (+/-)-catechin at 72 h 0.000 0.00 3.00 4.00 5.00 0.00 Minutes 1.00 2.00 3.00 4.00 5.00 Minutes 0.016 BC (72h) Cat-Met 0.040 1.852 2.568 0.018 0.014 0.012 4.395 4.540 3.776 3.406 3.007 0.000 0.000 0.00 1.00 2.00 3.00 4.00 0.00 5.00 1.00 2.00 3.00 (+/-)-Catechin 1.862 1.067 0.10 0.010 0.12 Cont-3-poly without inoculum (72h) 2 0.012 4.00 5.00 Minutes Minutes 0.008 2.567 2.229 2.116 1.523 4.540 0.020 0.010 3.774 0.002 3.408 0.004 2.412 0.006 1.756 1.931 0.897 0.981 1.082 1.292 1.506 0.008 0.896 0.974 1.080 1.283 AU 4.396 0.030 0.010 AU BC-Cat (72h) Cat-Met Cat-Met Cont-3-poly (72h) 0.08 3.426 3.787 1.766 1.290 2.245 2.432 2.574 2.638 0.02 1.981 2.104 0.002 0.04 1.461 1.512 1.673 0.004 0.06 0.680 0.808 0.989 AU AU Cat-Met 0.006 0.000 0.00 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.4: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of (+/-)-catechin, compared to the controls at 72 h 161 Appendix 3: Detection of possible metabolites from the metabolism of ferulic acid in different substrates during 72 h incubation with a pig faecal inoculum at 280 nm Microbial metabolism of ferulic acid at h 0.005 1.984 ACW (0h) 0.0020 Ferulic acid ACW-FA (0h) 4.26 0.004 0.002 0.001 0.0000 1.387 0.753 1.38 0.0005 4.668 AU AU 4.243 0.74 0.0010 0.003 4.66 0.0015 0.000 -0.0005 -0.001 -0.0010 1.0 2.0 3.0 4.0 0.00 5.0 1.00 2.00 Minutes 1.983 BC (0h) 4.262 0.0020 4.00 5.00 Ferulic acid BC-FA (0h) -0.0005 4.249 1.378 0.002 0.0000 1.626 0.004 0.754 AU 0.006 4.018 1.384 0.573 0.750 AU 0.0005 0.008 4.665 0.0015 0.0010 3.00 Minutes 4.671 0.0 0.000 0.00 1.00 2.00 3.00 4.00 5.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 0.014 Cont-3-poly without inoculum (0h) 0.015 1.561 Ferulic acid 0.016 1.984 Minutes Cont-3-poly (0h) Ferulic acid 0.012 0.006 AU 9 AU 0.008 0.897 0.010 0.010 0.000 0.00 2.219 1.384 0.002 4.273 0.005 0.004 0.000 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.5: Representative UPLC chromatograms of all ferulic acid substrates and the controls at h 162 ACW (6h) 7 Microbial metabolism of ferulic acid at h 0.006 0.0025 FA-Met ACW-FA (6h) 0.005 0.0020 0.004 4 8 8 4 5 0.001 0.0000 0.000 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 0.00 5.00 1.00 1.50 0.009 BC (6h) 0.0025 0.50 2.00 0.00 FA-Met 0.003 0.002 3 0.0005 8 0.0010 1 AU AU 0.0015 0.008 3.00 3.50 4.00 4.50 5.00 BC-FA (6h) FA-Met 0.007 0.0020 2.50 Minutes 0.006 0.0015 0.005 AU AU FA-Met 9 0.003 0.001 5 0.002 8 3 8 0.0005 1 0.004 0.0010 0.0000 0.000 -0.0005 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 0.00 5.00 0.50 1.00 1.50 2.00 2.50 Minutes 0.012 FA-Met 0.012 8 Cont-3-poly without inoculum (6h) Ferulic acid 0.014 Minutes 3.00 3.50 4.00 4.50 5.00 Cont-3-poly (6h) Ferulic acid 0.010 0.010 0.008 0.000 4 6 0.002 0.002 0.00 0.004 0.004 2 FA-Met 0.006 0.006 AU AU 0.008 0.000 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 Figure A.6: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of ferulic acid, compared to the controls at h 163 0.0030 ACW (12h) Microbial metabolism of ferulic acid at 12 h 0.0030 0.0025 ACW-FA (12h) FA-Met 0.0025 FA-Met 0.0020 0.0020 0.0010 0.0005 0.0015 8 0.0000 1 0.0005 2 8 0.0010 6 0 AU AU 0.0015 0.0000 -0.0005 -0.0005 1.00 2.00 3.00 4.00 5.00 0.00 1.00 2.00 Minutes FA-Met 0.005 0.0020 AU 94 87 BC-FA (12h) FA-Met 0.003 2.466 2.594 0.002 1.545 72 57 0.0005 43 25 AU 0.004 0.0010 5.00 1.089 0.0025 1.830 0.006 0.0015 4.00 0.007 BC (12h) 0.0030 3.00 Minutes 0.001 0.0000 3.479 0.00 0.000 -0.0005 0.00 1.00 2.00 3.00 4.00 0.00 5.00 1.00 2.00 0.008 FA-Met Cont-3-poly (12h) 2.320 2.475 2.595 0.002 0.002 1.538 1.687 0.004 0.004 0.696 0.848 1.034 AU 0.916 AU 0.006 0.000 0.000 0.00 5.00 1.908 0.008 FA-Met 1.830 0.010 1.333 Cont-3-poly without inoculum (12h) 0.010 0.006 4.00 1.090 Ferulic acid 0.012 3.00 Minutes Minutes 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.7: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of ferulic acid, compared to the controls at 12 h 164 0.014 0.025 ACW (72h) 2.569 0.016 1.758 Microbial metabolism of ferulic acid at 72 h FA-Met 2.569 0.020 0.012 3.779 3.410 2.415 2.015 0.984 1.088 1.290 1.508 0.005 4.395 4.541 AU 0.010 3.779 2.00 3.410 1.00 0.002 2.413 0.004 1.932 2.019 0.006 4.397 4.542 0.015 0.008 0.9070.983 1.098 1.290 1.444 AU 0.010 ACW-FA (72h) 0.000 0.000 0.00 3.00 4.00 0.00 5.00 1.00 2.00 3.00 4.00 5.00 Minutes Minutes 0.018 0.050 BC-FA (72h) 1.758 BC (72h) 2.568 0.016 FA-Met 0.014 0.040 4.396 0.012 0.000 3.410 1.932 1.075 1.293 1.509 0.010 0.000 2.00 3.00 4.00 5.00 0.00 1.00 2.00 3.00 Minutes 1.862 1.067 Cont-3-poly (72h) 2.245 2.432 2.574 2.638 1.766 1.981 2.104 0.02 1.461 1.512 1.673 0.04 1.290 0.004 0.06 0.680 0.808 0.989 AU 0.006 5.00 FA-Met 0.08 0.008 AU FA-Met 0.10 0.010 0.12 Cont-3-poly without inoculum (72h) 2 Ferulic acid 0.012 4.00 Minutes 3.787 1.00 3.426 0.00 0.002 4.397 4.540 4.540 3.408 0.020 3.774 0.002 2.412 0.004 1.756 1.931 0.006 2.569 AU 0.030 0.008 0.897 0.981 1.082 1.292 1.506 AU 0.010 0.000 0.00 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.8: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of ferulic acid, compared to the controls at 72 h 165 Appendix 4: Detection of possible metabolites from the metabolism of Cya-3-glc in different substrates during 72 h incubation with a pig faecal inoculum at 280 nm ACW (0h) 0.008 4.26 0.0020 ACW-Cyan (0h) Cya-3-glc 0.006 0.74 4.66 0.0015 AU AU 0.0010 1.562 Microbial metabolism of Cya-3-glc at h 0.004 0.0000 1.381 1.38 0.0005 0.002 -0.0005 0.000 -0.0010 0.0 1.0 2.0 3.0 4.0 5.0 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Minutes 1.561 BC (0h) 4.262 0.0020 4.263 1.382 0.002 0.0000 0.683 0.750 AU 4.018 0.0005 0.006 0.004 1.384 0.573 0.750 AU 0.0010 BC-Cyan (0h) 4.665 0.0015 Cya-3-glc 0.000 -0.0005 0.00 1.00 2.00 3.00 4.00 5.00 0.00 1.00 2.00 3.00 Minutes 0.015 Cya-3-glc 5.00 Cont-3-poly (0h) 1.984 0.014 1.561 Cont-3-poly without inoculum (0h) Cya-3-glc 0.016 4.00 Minutes 0.012 0.006 AU 9 AU 0.008 0.897 0.010 0.010 0.000 0.00 2.219 1.384 0.002 4.273 0.005 0.004 0.000 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.9: Representative UPLC chromatograms of all Cya-3-glc substrates and the controls at h and 280 nm 166 0.0030 Cyan-Met 5 8 7 1 1 2 4 5 0.0005 0.0000 Cyan-Met 0.0015 0.0010 3 0.0005 8 0.0010 1 AU AU 0.0015 0.0020 Cyan-Met 3 0.0025 0.0020 ACW-Cyan (6h) 7 ACW (6h) 0.0025 0 7 Microbial metabolism of Cya-3-glc at h 0.0000 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 0.00 0.50 BC (6h) 0.0025 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 0.0025 4.00 4.50 5.00 BC-Cyan (6h) 0.00 Cyan-Met 0.0020 8 1 1 2 0.0000 0.0000 2 1 0.0005 3 1 8 0.0005 0.0010 6 Cyan-Met AU AU 0.0015 0.0010 Cyan-Met 0.0015 0.0020 -0.0005 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.00 Minutes 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.012 0.012 0.010 Cont-3-poly (6h) Cont-3-poly without inoculum (6h) Cya-3-glc 0.014 8 Minutes Cat-Met 0.010 0.008 0.000 9 4 6 6 0.002 0.002 2 8 0.004 0.004 0.00 0.006 9 AU AU 0.006 Cyan-Met 0.008 0.000 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 Figure A.10: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at h and 280 nm 167 Microbial metabolism of Cya-3-glc at 12 h 0.694 0.0020 0.0000 2.596 0.842 0.0010 1 0.0005 2 8 0.0010 0.0015 0.0005 2.317 2.479 AU AU 0.0015 Cyan-Met 1.547 1.691 0.0020 1.333 1.035 Cyan-Met 0.0025 ACW-Cyan (12h) Cyan-Met 0.0030 2.053 ACW (12h) 0.0025 1.147 0.0030 0.0000 -0.0005 0.00 2.00 3.00 4.00 1.00 2.00 3.00 5.00 BC (12h) 1.33 1.03 Cyan-Met 0.0015 2.31 2.03 1.68 0.0005 1.88 1.54 1.15 0.0010 0.0000 -0.0005 0.00 Cyan-Met 2.59 AU 87 72 57 0.0005 43 0.0010 0.69 0.0020 94 0.0020 0.50 Cyan-Met 0.0025 0.0015 5.00 BC-Cyan (12h) 0.0030 0.0025 25 AU 0.0030 4.00 Minutes Minutes 3.48 1.00 2.47 0.00 0.0000 1.00 2.00 3.00 4.00 0.00 5.00 1.00 2.00 3.00 4.00 5.00 Minutes Minutes 1.830 1.908 0.004 0.004 0.002 0.002 0.000 0.000 0.00 1.538 1.687 Cyan-Met 0.696 0.848 1.034 AU 0.916 AU 0.006 2.320 2.475 2.595 0.008 0.008 0.006 Cont-3-poly (12h) 1.090 Cyan-Met Cya-3-glc 0.010 Cyan-Met 0.010 1.333 Cont-3-poly without inoculum (12h) 0.012 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.11: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at 12 h and 280 nm 168 1.165 Cyan-Met 0.020 0.699 0.882 AU 4.397 4.542 3.779 2.00 3.410 1.00 0.002 2.413 0.004 1.932 2.019 0.006 0.9070.983 1.098 1.290 1.444 AU 0.008 0.010 Cyan-Met 4.317 4.470 0.030 0.010 3.388 0.012 ACW-Cyan (72h) Cyan-Met 3.686 0.040 ACW (72h) 1.589 1.753 1.906 2.042 2.163 2.277 2.473 2.630 0.014 1.064 0.016 1.368 2.569 Microbial metabolism of Cya-3-glc at 72 h 0.000 0.000 0.00 3.00 4.00 5.00 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 Minutes 0.018 Cyan-Met 0.809 4.540 3.408 0.005 3.774 0.002 2.412 0.004 1.756 1.931 0.010 0.006 4.397 4.543 1.078 1.284 1.503 1.664 1.764 1.932 1.973 2.096 2.228 2.415 0.008 0.897 0.981 1.082 1.292 1.506 AU AU 4.396 0.015 3.780 1.822 0.012 0.010 BC-Cyan (72h) Cyan-Met 3.413 0.020 0.014 2.570 0.982 BC (72h) 2.568 0.016 0.000 0.000 0.00 0.00 1.00 2.00 3.00 4.00 1.00 2.00 3.00 5.00 4.00 5.00 Minutes Cyan-Met Cyan-Met 3.426 3.787 2.245 2.432 2.574 2.638 1.981 2.104 1.461 1.512 1.673 0.02 0.000 0.00 Cyan-Met 0.04 1.290 0.004 0.06 0.680 0.808 0.989 AU AU 0.006 1.766 0.08 0.008 0.002 Cont-3-poly (72h) 0.10 1.067 Cya-3-glc 0.010 0.12 Cont-3-poly without inoculum (72h) 2 0.012 1.862 Minutes 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.12: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at 72 h and 280 nm 169 Appendix 5: Detection of possible metabolites from the metabolism of Cya-3-glc in different substrates during 72 h incubation with a pig faecal inoculum at 499 nm ACW (0h) 0.0010 1.559 Microbial metabolism of Cya-3-glc at h 0.010 ACW-Cyan (0h) Cya-3-glc 0.008 0.0005 AU AU 0.006 0.0000 0.004 -0.0005 0.002 -0.0010 0.000 -0.0015 0.00 -0.002 1.00 2.00 3.00 4.00 0.00 5.00 1.00 2.00 3.00 4.00 5.00 Minutes Minutes BC (0h) 1.560 0.0010 0.008 BC-Cyan (0h) Cya-3-glc 0.0005 0.006 AU AU 0.0000 -0.0005 0.004 0.002 2.215 -0.0010 0.000 -0.0015 0.00 1.00 2.00 3.00 4.00 -0.002 5.00 0.00 Minutes 1.00 2.00 3.00 4.00 5.00 Minutes 0.018 Cya-3-glc 0.016 Cont-3-poly without inoculum (0h) 1.561 0.020 Cont-3-poly (0h) Cya-3-glc 0.015 0.014 0.010 0.010 AU AU 0.012 0.008 0.006 0.005 2.221 0.004 0.002 0.000 0.000 -0.002 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.13: Representative UPLC chromatograms of all Cya-3-glc substrates and the controls at h and 499 nm 170 Microbial metabolism of Cya-3-glc at h 0.0014 0.0012 Cya-3-glc 0.0012 0.0010 ACW-Cyan (6h) 6 ACW (6h) 0.0014 Cyan-Met 0.0010 0.0008 0.0008 0.0006 0.0006 0.0004 0.0004 0.0002 AU AU 0.0002 0.0000 0.0000 -0.0002 -0.0002 -0.0004 -0.0004 -0.0006 -0.0006 -0.0008 -0.0008 -0.0010 -0.0010 -0.0012 -0.0012 -0.0014 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 0.0016 0.0012 BC (6h) 0.0012 BC-Cyan (6h) 0.0010 0.0010 0.0008 0.0008 0.0006 0.0006 Cyan-Met 0.0004 Cya-3-glc 0.0004 0.0002 AU AU 0.0002 0.0000 0.0000 6 0.0014 -0.0002 -0.0002 -0.0004 -0.0004 -0.0006 -0.0006 -0.0008 -0.0008 -0.0010 -0.0010 -0.0012 -0.0012 -0.0014 -0.0014 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 0.00 0.50 1.00 1.50 2.00 Minutes Cya-3-glc 3.50 4.00 4.50 5.00 0.006 Cont-3-poly without inoculum (6h) 1.617 0.014 3.00 Cya-3-glc 0.005 Cont-3-poly (6h) 0.016 2.50 Minutes 0.012 0.004 0.010 0.003 AU AU 0.008 0.006 0.004 Cyan-Met 0.002 0.001 0.002 0.000 0.000 -0.001 -0.002 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 0.00 0.50 1.00 1.50 2.00 2.50 Minutes 3.00 3.50 4.00 4.50 5.00 Figure A.14: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at h and 499 nm 171 Microbial metabolism of Cya-3-glc at 12 h 0.0008 ACW (12h) ACW-Cyan (12h) 0.0006 0.0000 -0.0002 AU AU 0.0000 -0.0002 -0.0004 -0.0004 -0.0006 -0.0006 Cyan-Met Cyan-Met 2.319 0.0002 1.687 0.0004 0.0002 2.057 0.0004 0.864 0.0006 -0.0008 -0.0008 -0.0010 -0.0010 -0.0012 -0.0012 -0.0014 -0.0014 0.00 0.00 1.00 2.00 3.00 4.00 1.00 2.00 5.00 3.00 4.00 5.00 Minutes Minutes BC (12h) BC-Cyan (12h) 0.0006 0.0005 0.0004 Cyan-Met 0.0002 1.70 AU AU -0.0002 -0.0005 -0.0004 2.32 Cyan-Met 0.0000 2.04 0.0000 -0.0006 -0.0010 -0.0008 -0.0010 0.00 1.00 2.00 3.00 4.00 5.00 Minutes 0.00 1.00 2.00 3.00 4.00 5.00 0.010 1.602 Cont-3-poly without inoculum (12h) 0.0008 0.0006 0.008 Cya-3-glc 0.859 Minutes Cont-3-poly (12h) Cyan-Met 0.0004 Cyan-Met -0.0002 -0.0004 0.002 2.308 AU AU 0.0000 0.004 2.043 1.695 0.0002 0.006 -0.0006 -0.0008 0.000 -0.0010 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 -0.0012 0.00 1.00 2.00 3.00 4.00 5.00 Minutes Figure A.15: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at 12 h and 499 nm 172 Microbial metabolism of Cya-3-glc at 72 h 0.0010 1.977 2.621 0.008 ACW (72h) Cyan-Met ACW-Cyan (72h) 0.006 -0.0010 2.225 0.002 -0.0005 0.000 1.00 2.00 3.00 4.00 5.00 0.00 1.00 2.00 Minutes 0.008 5.00 BC-Cyan (72h) Cyan-Met 2.228 0.002 -0.0005 1.844 0.004 0.819 AU 3.960 0.0000 AU 4.00 0.006 0.0005 0.000 -0.0010 0.00 1.980 BC (72h) 2.627 0.0010 3.00 Minutes 2.633 -0.0015 0.00 1.840 AU 3.961 AU 0.004 0.0000 2.627 0.808 0.0005 1.00 2.00 3.00 4.00 0.00 5.00 1.00 2.00 3.00 0.008 Cont-3-poly without inoculum (72h) 5.00 Cont-3-poly (72h) 2.240 1.623 0.010 Cya-3-glc 4.00 Minutes Minutes 0.020 Cyan-Met 1.984 0.015 AU AU 0.006 0.010 0.000 0.000 0.00 0.00 1.00 2.00 3.00 Minutes 4.00 5.00 1.00 2.636 0.005 1.849 0.002 0.811 0.004 2.00 3.00 4.00 5.00 Minutes Figure A.16: Representative UPLC chromatograms show the formation/degradation of possible metabolites from the microbial metabolism of Cya-3-glc, compared to the controls at 72 h and 499 nm 173 Appendix 6: UV-Vis spectra of possible metabolites of the three polyphenol precursors by gut microbiota, recorded at 280 nm Cat-Met 0.005 Cat-Met 1.318 Peak 0.005 Cat-Met 1.909 Peak 0.040 0.004 0.004 0.003 A U 0.024 AU AU 0.003 0.002 0.002 0.016 0.001 0.001 0.008 275.00 300.00 325.00 350.00 0.000 250.00 275.00 300.00 325.00 322.2 0.000 250.00 350.00 275.00 300.00 nm nm FA-MET 0.00220 272.9 0.032 272.3 280.3 0.000 250.00 2.232 Peak FA-MET 1.082 Peak 0.0080 325.00 350.00 nm FA-Met 1.819 Peak 0.040 1.758 Peak 280.3 0.0064 0.00132 0.0048 280.3 0.032 0.024 AU AU 0.00088 0.0032 0.016 0.00044 0.0016 0.008 0.00000 250.00 275.00 300.00 325.00 0.0000 250.00 350.00 275.00 300.00 325.00 350.00 0.000 250.00 275.00 nm nm Cyan-Met 0.0030 272.3 AU 0.00176 0.0030 325.00 350.00 nm Cyan-Met 0.693 Peak 300.00 Cyan-Met 1.035 Peak 0.006 1.392 Peak 260.6 279.0 0.005 0.0024 0.0024 309.2 270.4 0.004 0.0018 AU AU 293.8 AU 0.0018 0.0012 0.003 0.0012 0.002 0.0006 0.0000 250.00 0.0006 275.00 300.00 nm 325.00 350.00 0.0000 250.00 0.001 0.000 275.00 300.00 325.00 350.00 260.00 280.00 nm 300.00 320.00 340.00 nm Figure A.17: UV spectra of possible metabolites scanned from 250 nm – 350 nm 174 Appendix 7: UV-Vis spectra of possible metabolites of Cya-3-glc by gut microbiota, recorded at 499 nm Cyan-Met 0.0035 Cyan-Met 1.669 Peak 0.0140 2.008 Peak 295.6 0.0112 0.0021 0.0084 485.7 AU AU 0.0028 0.0014 490.7 0.0056 0.0028 0.0007 279.6 313.5 346.4 0.0000 250.00 300.00 373.1 350.00 400.00 nm 450.00 500.00 0.0000 250.00 300.00 350.00 400.00 450.00 500.00 nm Figure A.18: UV-VIS spectra of possible Cya-3-glc metabolites scanned from 250 nm – 500 nm 175 ... expressed as [A] µg adsorbed polyphenols per mg dry weight of plant cell wall models, and [B] µg adsorbed polyphenols per mg cellulose of different plant cell wall models, for different exposure... Primary plant cell walls: molecular components, structure and functions 2.1.1 Plant cell wall structure and functions 2.1.2 Molecular components of primary plant cell walls ... associated with polyphenol intake 22 2.4 Interactions between polyphenols and plant cell walls 23 2.4.1 The role of plant cell walls in controlling the bioaccessibility of phenolic