EFFECTS OF THERMAL PROCESSING ON ISOMERIZATION AND BIO ACCESSIBILITY OF L YCOPENE PRECURSORS A Thesis Presented in Partial Fulfillment of the Requirements for The Degree Master of Science in the Graduate School of The Ohio State University By Marjory Renita, B.S. ***** The Ohio State University 2005 Master's Examination Committee: Approved by Dr. Steven J. Schwartz, Advisor Dr. Joshua A. Bomser Dr. Mark L. Failla Advisor Graduate Program in Food Science ABSTRACT Consumption of dietary carotenoids has been associated with the prevention of several chronic age-related diseases. For example, ingestion of cooked tomato products is inversely correlated with the risk of prostate cancer. Processing to produce tomato- based products also has been shown to enhance the bioavailability of lycopene, the most abundant carotenoid in many varieties of tomatoes. Plant breeders are developing unique varieties to deliver increased concentrations of carotenoids in food products. The characteristics and regulation of carotenoid biosynthesis in tomato fruit continues to be investigated extensively. Tangerine, one of the distinct variety of tomatoes, contains high levels of the lycopene precursors: (15Z)- phytoene, (15,9'Z)-phytofluene, (9,9'Z)-ζ -carotene, and (7,9,9'Z)-neurosporene, compared to typical red tomatoes. Either (9,9'Z)-ζ-carotene or (7,7',9,9'Z )-lycopene are also relatively abundant in tangerine tomato. These carotenoids are predominantly in the (Z)-configuration in contrast to the (E)-configuration of lycopene in red tomatoes. Recent evidence has shown that thermal processing may increase isomerization of carotenoids such as β-carotene or lutein, whereas lycopene is relatively stable. The predominant form of these carotenoids in nature is the (E)-configuration, while thermal processing may induce (Z)-isomerization. The effect of thermal processing to the ii tangerine tomatoes, especially on the (Z)-lycopene precursors present in this variety have not been studied. Human clinical studies have reported significant amount of the (Z)-lycopene isomers in plasma or serum levels and 70-80% in benign and malignant prostate tissues, in contrast to the significant amount of (E)-lycopene in the dietary tomato products. The (Z)-lycopene isomers have been proposed to be more bioavailable than the (E)- configuration. Greater absorption of these isomers observed in clinical trials coincide with results evaluating the bioaccesibility of (Z)-lycopene isomers using in vitro digestion coupled with Caco-2 cells. However, there is minimal reports that have been reported on the bioacessibility and bioavailability of the (Z)-lycopene precursors. The aim of this research is to examine the effects of thermal processing on isomerization and bioaccesibility of the lycopene precursors: phytoene, phytofluene, ζ- carotene, and neurosporene, from tangerine tomatoes. Rapid analytical methods such as high performance liquid chromatography coupled with photodiode array detector (HPLC-PDA), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), and nuclear magnetic resonance (NMR) will be used for separation, identification, and quatification of the carotenoids including their isomers. In vitro digestion coupled with Caco 2 cell cultures will be employed to determine the bioaccessibility (digestive stability, micellarization, and cell uptake) of the lycopene precursors. In this study, we observed that the lycopene precursors were relatively stable during thermal processing and no significant degradation was noted. Better extractability and increased isomerization towards the (E)-configuration were found. Processing was also associated with increases in the bioaccessibility of the lycopene precursors during in iii vitro digestion and Caco-2 cell uptake. Overall, this study demonstrated that thermal processing may induce better extractability and isomerization of the lycopene precursors: phytoene, phytofluene, ζ-carotene, and neurosporene. These carotenoids and their isomers are readily digested, absorbed, and distributed in the human body. Futher clinical trials will be required to examine and confirm the bioavailabilty of the lycopene precursors and their isomers. iv Dedicated to my beloved parents and sister v ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Steven J. Schwartz, for his great support, encouragement, advise, patience, and faith on me, which made this research and thesis possible. I am also grateful for all of the support and guidance from my committee members, Dr. Mark Failla and Dr. Josh Bomser, both from the nutrition department. I am very delighted being as a member of Haas Chair Lab under Dr. Schwartz's supervision. I thank all of my previous and present lab members, for their great support and teaching me in depth of using the analytical equipments. It is also a great experience to have the possibility of collaborative studies with other departments to broaden my knowledge. I also appreciate the great help and mentor from Dr. Failla and his lab for digestion and cell culture work. Finally, I am indebted to my parents for their great support and encouragement for me to study abroad and finish my education. vi VITA January 27, 1982 ................... Born, Surabaya, Indonesia June 1999 ............................... Graduated from St.Ursula High School, Jakarta, Indonesia June 2003 ....................... :....... B.S. Food Science and Technology, The Ohio State University, Columbus, OH 2003-Present.. ........................ Graduate Research Associate, Food Science and Nutrition, The Ohio State University, Columbus, OH PUBLICATIONS Research Publications 1. Lee, J. H., Renita, M., St Martin, S. K., Schwartz, S. J., and Vodovotz, Y. "Isoflavone characterization and antioxidant activity of Ohio soybeans." Journal of Agricultural and Food Chemistry, 52, 9, 2647-2651, (2004). Peer-reviewed Abstracts and Posters 1. Renita, M., Schwartz, S. J. "Thermal processing increases isomerization of lycopene precursors from tangerine tomatoes." IFT Annual Meeting. July 16-20, 2005. New Orleans, LO. 2. Renita, M. Failla, M. L., Schwartz, S. J. "In vitro digestion and Caco-2 cell uptake oflycopene precursors from tangerine tomatoes." International Symposium on Carotenoids. July 17-22, 2005. Edinburgh, Scotland. FIELD OF STUDY Major Field: Food Science and Nutrition Vil TABLE OF CONTENTS ABSTRACT ·------------------------------------------------------------------------------------------------------------------11 DEDICATION ·-------------------------------------------------------------------------------------------------------------- v ACKNOWLEDGMENTS -------------------------------------------------------------------------------------------- v1 VITA ------------------------------------------------------------------------------------------------------------------------------ Vll TABLE OF CONTENTS _______________________________________________________________________________________________ vm LIST OF TABLES --------------------------------------------------------------------------------------------------------x LIST OF FIGURES ·------------------------------------------------------------------------------------------------------ Xl CHAPTER 1: REVIEW OF THE LITERATURE _____________________________________________________ 1 1.1. Carotenoids _____________________________________________________________________________________________ 1 1.1.1. Structure, classifications, and nomenclature _____________________________ 2 1.1.2. Carotenoids in food products _____________________________________________________ 5 1.1.3. Health benefits of carotenoids ____________________________________________________ 5 1.2 Biosynthesis of carotenoids ___________________________________________________________________ 8 1.2.1. Biosynthesis of carotenoids in choloroplast______________________________ 8 1.2.2. Tomato as a model for the study of carotenoid biosynthesis ___ 14 1.2.3_ Recent advances of carotenoid biosynthesis studies _________________ 16 1.3. Analytical methods ________________________________________________________________________________ 18 1.3 .1. Isolation methods _______________________________________________________________________ _19 1.3.2. High performance liquid chromatography (HPLC) _________________ 20 1.3.3. Identifications using several analytical techniques __________________ 21 1.4. Bioaccessibility and bioavailability of carotenoids _____________________________ 27 1.4_ l, Mechanisms of carotenoid bioacessibility, uptake, and absorption ·---------------------------------------------------------------------------------28 1.4.2. In vitro digestion and Caco-2 cell cultures ________________________________ 31 1.4.3. Methods assessing bioavailability in clinical studies _______________ 34 Vlll 1.5. Effects of thermal processing on carotenoids ______________________________________ 35 1.5.1. Degradation and structural changes ___________________________________________ 35 1.5.2. Thermal processing effects on bioavailability of carotenoids __ 37 1.6. Lycopene precursors ______________________________________________________________________________ 39 1.6.1. Lycopene precursors in food products _______________________________________ 39 1.6.2. Lycopene precursors in vivo ______________________________________________________ 41 1.6.3. Lycopene precursors in cancer cells __________________________________________ 43 1. 7. Hypotheses and objectives ____________________________________________________________________ 44 1.8. List of references