Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2018-06-01 Evaluation of Pigments from a Purple Variety of Atriplex hortensis L. for Use in Food Applications Eva Graciela Vila Roa Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Life Sciences Commons BYU ScholarsArchive Citation Vila Roa, Eva Graciela, "Evaluation of Pigments from a Purple Variety of Atriplex hortensis L. for Use in Food Applications" (2018). All Theses and Dissertations. 7436. https://scholarsarchive.byu.edu/etd/7436 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Evaluation of Pigments from a Purple Variety of Atriplex hortensis L. for Use in Food Applications Eva Graciela Vila Roa A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Michael L. Dunn, Chair Oscar Pike Frost Steele Eric Jellen Department of Nutrition, Dietetics, and Food Science Brigham Young University Copyright © 2018 Eva Graciela Vila Roa All Rights Reserved ABSTRACT Evaluation of Pigments from a Purple Variety of Atriplex hortensis L. for Use in Food Applications Eva Graciela Vila Roa Department of Nutrition, Dietetics, and Food Science, BYU Master of Science Atriplex hortensis L., also known as orach, is a leafy vegetable from the Amaranthaceae family, which has historically been consumed as a potherb, like spinach. The brightly colored leaves are a source of high quality protein, but may also be of interest as a potential source of natural food pigments. An aqueous extraction was obtained from the freshly harvested leaves of the ‘Triple Purple’ variety of A. hortensis. The extract was spray-dried into a powder, and individual pigments were analyzed using HPLC and LC-MS. The powder was also included as a color additive in a typical stabilizer/sweetener preparation and mixed into plain yogurt. Two batches of colored yogurt were held under light and dark conditions and tested for pH and color (L*a*b*) every 15 days. A visual sensory panel was performed on days 0, 45, and 90 to evaluate the color acceptance. A total of three types of betacyanins and six types of anthocyanins were tentatively identified by HPLC and/or LC-MS. Orach pigments in yogurt were not stable under full light exposure. The color of samples exposed to light degraded within days. There were statistically significant differences found in L*a*b* scores in the dark treatment, beyond 30 days; but these modest changes in dark-stored samples were not found to be statistically significant in the consumer sensory panel. The tentative identification of both anthocyanins and betacyanins in orach is a novel finding in botanical research, as the literature indicates that these two pigment classes are mutually exclusive. The application of heat during pigment extraction, spray drying, and yogurt color additive preparation, did not appear to appreciably affect stability of orach pigments, indicating that orach extract could be used as a color in different process applications, if protected from light. Keywords: red color, natural color additive, anthocyanin, betalain, yogurt ACKNOWLEDGEMENTS I would like to express my gratitude to my late father for teaching me to work hard and be the best in everything I do. I also want to thank my mother and sister for their encouragement, love, and support. I would like to give thanks to my husband for being patient and courageous to follow me wherever my dreams take me. I would like to thank Dr. Michael Dunn for being a big support not only in my project, but also in my personal life. Finally, I want to express my gratitude to Dr. Oscar Pike, Dr. Frost Steele and Dr. Eric Jellen for their guidance, assistance, and meaningful feedback. TABLE OF CONTENTS TITLE .............................................................................................................................................. i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii INTRODUCTION .......................................................................................................................... 1 MATERIALS AND METHODS .................................................................................................... 4 Orach Leaf Preparation ............................................................................................................... 4 Pigment Extraction ...................................................................................................................... 4 HPLC Analysis of Color Extract ................................................................................................ 4 Pigment Identification by LC-MS .............................................................................................. 5 Yogurt Color Base Preparation ................................................................................................... 6 Yogurt Preparation ...................................................................................................................... 6 Stability Test ............................................................................................................................... 7 pH ................................................................................................................................................ 7 Colorimeter ................................................................................................................................. 7 Visual Consumer Sensory Testing .............................................................................................. 8 iv Statistical Analysis ...................................................................................................................... 9 RESULTS AND DISCUSSION ................................................................................................... 10 Pigment Identification ............................................................................................................... 10 Color stability in yogurt ............................................................................................................ 12 CONCLUSION ............................................................................................................................. 15 REFERENCES ............................................................................................................................. 16 TABLES ....................................................................................................................................... 18 FIGURES ...................................................................................................................................... 22 APPENDIX A. Review of Literature ........................................................................................... 23 APPENDIX B. Color Base Formulation ..................................................................................... 32 APPENDIX C. Color JMP Output ............................................................................................... 33 APPENDIX D. pH JMP Output ................................................................................................... 55 APPENDIX E. Sensory Panel Questionnaire ............................................................................... 61 APPENDIX F. Sensory Panel JMP Output .................................................................................. 66 APPENDIX G. Disqualification of a second field from the study ............................................... 84 v LIST OF TABLES Table 1 Betalains and anthocyanins reported by Cai et al., 2005, and entered into the Mass Hunter Qualitative Analysis Software to assist with pigment identification in orach extracts.... 18 Table 2 Betalains, anthocyanins, and potential isomers tentatively identified in orach pigment extract. ........................................................................................................................................... 19 Table 3 Color changes in yogurt containing orach extract following refrigerated storage under light (200 Lux) for up to 30 days or in the dark for up to 90 days................................................ 20 Table 4 Total color ( E*ab), chroma ( C*ab), and hue ( H*ab) differences in yogurt colored with orach extract and stored refrigerated in the dark for 90 days................................................ 20 △ △ △ Table 5 Consumer sensory acceptance of yogurts colored with orach extract or carmine (control) at Days 0, 45, and 90 (n=50). .......................................................................................................