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I Purple Corn (Zea Mays L.) Cob Anthocyanins

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I Purple Corn (Zea Mays L.) Cob Anthocyanins Purple Corn (Zea mays L.) Cob Anthocyanins: Extraction, Quantification, Spray Drying and Complexation with Proteins DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Fei Lao Graduate Program in Food Science and Technology The Ohio State University 2016 Dissertation Committee: Dr. M. Monica Giusti, Advisor Dr. Sheryl A. Barringer Dr. C. Lynn Knipe Dr. John H. Litchfield i Copyrighted by Fei Lao 2016 i Abstract Interests of applying natural colorants such as anthocyanins as alternatives to synthetic dyes in food have been a market trend in recent years. This study evaluated purple corn cob (PCC) as economic natural source for high quality food-used anthocyanins colorant production. PCC is an anthocyanin-rich plant source. Different from anthocyanins in other fruits and vegetables, production and application of PCC anthocyanins is not as simple and straightforward due to the fiber-rich hard texture of the cob and relatively complicated biomatrix of the corn. Some of the PCC pigments undergoing traditional processing have acidic water solubility issues, which limit their application in most aqueous-based foods. The objective of this study is to optimize preparation of PCC anthocyanins, to produce high quality PCC pigments for more general food application. To achieve this goal, the critical conditions in PCC pigments extraction and spray drying were evaluated, and the key structure (anthocyanin-protein complexation) which was believed to be correlated to water solubility problems was investigated using infrared spectroscopy. Aqueous ethanol with water and ethanol ratio around 1:1 with slightly acid addition (0.01% v/v HCl) was able to efficiently recover PCC anthocyanins and phenolics. The yield was comparable to analytical labolatory used solvents under the same extraction conditions. Spray drying PCC pigments with mild inlet/outlet temperatures (150°C/105°C) and appropriate amount (5%, m/v) of carrier could produce satisfactory ii quality PCC pigment powders with least color degradation and more than 85% of pigment yield. The only hazy reconstituted PCC pigment solution was obtained from spray drying alcoholic PCC extract, which suggested the acidic water insolubility issue was mainly coming from the extraction rather than the heated dehydration process. Anthocyanin-tannin-protein complex was proposed in previous studies to be the major contributor in the insoluble PCC pigment rich particles. Confirmed by the mid infrared (MIR) spectroscopy, the PCC anthocyanins and protein could form complexation in the aqueous matrix. Hydrogen bonding was the major driving force to stabilize the complexation. The anthocyanin:protein molar ratio in the complexation depended on the environmental pH, as hydrophobic interaction and ion chelation might also be involved into complexation when the matrix pH was acidic. A rapid MIR prediction model to quantify protein levels in anthocyanin-rich matrix was also developed based on protein unique Amide signals. In addition, the present study also discussed the advantages and disadvantages of four commonly used anthocyanin quantification methods in PCC pigment analysis. All four spectrophotometric and HPLC approaches outcomes for PCC were linearly correlated (R2≥0.90) to each other. The total anthocyanins methods produced highest values, followed by the pH differential method, HPLC method with intact pigments, and HPLC method with acid hydrolyzed pigments. Overall, our study showed PCC could be used to produce high quality anthocyanin powders ready for food application, as well as provided important insightful understandings on anthocyanin-protein complexation. Information in this study may help iii food companies interested in the transition from synthetic dye to natural colorant using PCC, for cleaner and more consumer-friendly labels. iv Acknowledgments First and earnest, I would like to express my most sincere gratitude to my advisor, Dr. M. Monica Giusti, for offering me the opportunity to be a part of her lab and this amazing program. Her expertise in anthocyanins, endless creativity and curiosity, patient advisement, and positive attitude inspired me to explore more and trained me to be a food scientist. This exciting but challenging research project would not have been accomplished without her continuous encouragement and support. I would like to thank my committee members, Dr. Sheryl A. Barringer, Dr. C. Lynn Knipe, and Dr. John H. Litchfield, for their invaluable suggestions and support. Tons of thanks to Dr. Luis E. Rodriguez-Saona for providing convenient access to experimental facilities and continually guidance to my infrared study. I would also like to thank my fellow lab mates and department peers in the past five years, for their considerate assistance and kindness, both in the lab and in general. Thanks to Chinese Scholarship Council for providing fellowship to my first four years graduate study in the Ohio State University. Thanks to Alicorp S.A.A. (Lima, Peru), Agroindustrial S.A.C (Lima, Peru), Zanaceutica E.I.R.L. (Lima, Peru), and Globenatural International S.A. (Chorrillos-Lima, Peru) for providing funding, purple corn materials, and pigments production information to this research. v Finally, I am eternally grateful to my parents, Wenbiao Lao and Yixin Liu, as well as my dearest friends, for always standing by my side, offering me helping hands and mental support whenever I needed them the most throughout my PhD program. vi Vita June 23, 1988 .................................................Nanning, China 2007 to 2011 ..................................................B.E., Food Science and Engineering, China Agricultural University 2011 to present ..............................................Ph.D, Food Science and Technology, The Ohio State University Publications Lao, F., Giusti, M.M., 2016. Quantification of Purple Corn (Zea mays L.) Anthocyanins Using Spectrophotometric and HPLC Approaches: Method Comparison and Correlation. Food Anal. Methods 9, 1367–1380. Fields of Study Major Field: Food Science and Technology vii Table of Contents Abstract ............................................................................................................................... ii Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii List of Tables .................................................................................................................... xv List of Figures ................................................................................................................. xvii Chapter 1: Introduction ...................................................................................................... 1 Chapter 2: Literature Review .............................................................................................. 5 2.1 Anthocyanin Chemistry............................................................................................. 5 2.1.1 Structure.............................................................................................................. 6 2.1.2 Stability ............................................................................................................. 12 2.2 Purple Corn Pigments and Proteins ......................................................................... 20 2.2.1 Purple corn ........................................................................................................ 20 2.2.2 Purple corn anthocyanins .................................................................................. 22 2.2.3 Purple corn phlobaphenes ................................................................................. 23 2.2.4 Purple corn proteins .......................................................................................... 24 viii 2.2.5 Anthocyanin-Protein Complexation in Purple Corn ....................................... 27 2.3 Anthocyanin-Protein Complexation ........................................................................ 29 2.3.1 Anthocyanic vacuolar inclusion (AVI)............................................................. 30 2.3.2 Hordeumin ....................................................................................................... 33 2.3.3 Insoluble deposits in red wine bottle ............................................................... 34 2.3.4 anthocyanin-whey protein aggregate particles ................................................ 35 2.4 Infrared Technology for Proteins Analysis ............................................................. 36 2.5 Industrial Processing of Food Anthocyanins .......................................................... 39 2.5.1 Extraction.......................................................................................................... 41 2.5.2 Spray drying ..................................................................................................... 46 Chapter 3: Health Benefits of Purple Corn Phenolics ...................................................... 50 3.1 Abstract ................................................................................................................... 50 3.2 Introduction ...........................................................................................................
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