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Storage Stability of Rebaudioside a in Various Buffer Solutions By Storage Stability of Rebaudioside A in Various Buffer Solutions by Qianyun Gong A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August 4, 2012 Keywords: rebaudioside A, stevia, stevioside, stability, kinetics Copyright 2012 by Qianyun Gong Approved by Leonard N. Bell, Chair, Professor, Department of Poultry Science Shelly McKee, Associate Professor, Department of Poultry Science Tung-shi Huang, Associate Professor, Department of Poultry Science Abstract Rebaudioside A is a non-caloric high intensity sweetener extracted from Stevia rebaudiana. For it to be used in the food industry, rebaudioside A needs to be stable during processing and storage. Kinetic data on its long term stability as affected by solution composition are lacking. The primary objective of this study was to evaluate the storage stability of rebaudioside A in various buffer solutions as a function of pH, buffer type, buffer concentration and temperature. The effect of light exposure on rebaudioside A stability was also evaluated. Rebaudioside A solutions were prepared in 0.02 and 0.1 M phosphate and citrate buffers at pH 3, 5 and 7. Duplicate samples were stored at 20, 30 and 40 °C. Some samples were stored at room temperature under light or dark conditions. Aliquots were removed nine times for approximately nine months. The concentrations of rebaudioside A were analyzed and pseudo- first-order rate constants with 95% confidence intervals were calculated for the loss of rebaudioside A. In phosphate buffer, the degradation of rebaudioside A was generally faster at higher pH values. The pH effect on rebaudioside A stability was generally reversed in citrate buffer. Rebaudioside A broke down significantly faster in phosphate buffer than in citrate buffer at pH 5 and 7; degradation rates were similar at pH 3. Higher buffer concentrations promoted faster degradation. Rebaudioside A degradation was accelerated by the elevation of temperature. The exposure of light did not have an obvious effect in phosphate buffer at pH 7 while it lowered the stability of rebaudioside A in citrate buffer at pH 3. ii For optimum stability of beverages containing rebaudioside A, lower temperatures and lower buffer concentrations are preferred. If the product has a pH value of 5 or 7, citrate buffer is more preferred than phosphate. Dark environments help stabilize rebaudioside A in beverage at pH 3. iii Acknowledgments The author would like to express tremendous gratitude to Dr. Leonard Bell for his help and support throughout these two years. He dedicated numerous time for conducting the experiment and developing of this thesis. She also wants to express appreciation to her thesis committee, Dr. Shelly McKee and Dr. Tung-shi Huang, for review and recommendations of the thesis as well as their continuous advice and support. Then the author would like to thank her family and friends for loving and supporting her to chase her dream for further study in food science. iv Table of Contents Abstract ......................................................................................................................................... ii Acknowledgments........................................................................................................................ iv List of Tables .............................................................................................................................. vii List of Figures ............................................................................................................................ viii Chapter 1 Introduction ................................................................................................................. 1 Chapter 2 Literature Review ......................................................................................................... 2 Introduction ...................................................................................................................... 2 Extraction methods ......................................................................................................... 3 Analysis methods .............................................................................................................. 4 Metabolism ....................................................................................................................... 7 Toxicity study ................................................................................................................... 8 Health effects .................................................................................................................. 11 Food uses and approvals ................................................................................................. 13 Stability ........................................................................................................................... 14 Objective ......................................................................................................................... 17 Chapter 3 Material and Methods................................................................................................. 18 Reagents .......................................................................................................................... 18 Sample Preparation ......................................................................................................... 18 Sampling Procedure ........................................................................................................ 20 v Sample Analysis.............................................................................................................. 20 Data Analysis .................................................................................................................. 22 Chapter 4 Results and Discussion ............................................................................................... 23 Effect of pH..................................................................................................................... 28 Effect of buffer type and concentration .......................................................................... 29 Effect of temperature ...................................................................................................... 31 Effect of light .................................................................................................................. 33 Comparison to other sweeteners ..................................................................................... 34 Chapter 5 Summary and Conclusion .......................................................................................... 35 References ................................................................................................................................. 36 Appendix ................................................................................................................................... 44 vi List of Tables Table 4.1 Pseudo-first rate constants (d-1) with confidence limits for rebaudioside A stored at 20°C ........................................................................................................................................... 26 Table 4.2 Pseudo-first rate constants (d-1) with confidence limits for rebaudioside A stored at 30°C ........................................................................................................................................... 26 Table 4.3 Pseudo-first rate constants (d-1) with confidence limits for rebaudioside A stored at 40°C ........................................................................................................................................... 27 Table 4.4 Time for 10% rebaudioside A concentration decrease (days) .................................... 27 Table 4.5 Rate constants (d-1) calculated from the data presented by Prakash and others (2012) ..................................................................................................................................................... 29 Table 4.6 Activation Energy (kcal/mol) for rebaudioside A degradation in solution ................ 32 Table 4.7 Pseudo-first rate constants with 95% confidence limits (d-1) for rebaudioside A stored under light or dark at room temperature ..................................................................................... 33 Table 4.8 Predicted rebaudioside A loss (%) in 0.1 M buffer solutions after 1 week at room temperature ................................................................................................................................ 34 vii List of Figures Figure 2.1 Structures of rebaudioside A and stevioside .............................................................. 2 Figure 2.2 Degradation of rebaudioside A at pH 2.6 and 100 °C modeled using pseudo zero order kinetics. Data from Chang and Cook (1983) .............................................................................. 15 Figure 2.3 Degradation of rebaudioside A at pH 2.6 and 100 °C modeled using pseudo first order kinetics. Data from Chang and Cook (1983) .............................................................................. 15 Figure 3.1 Chromatograph of rebaudioside A in 0.1 M phosphate buffer at pH 7 and room temperature after 285 days of dark storage (Rebaudioside A eluted at 7.56 min)...................... 21 Figure 4.1 Degradation of rebaudioside A in different buffer solutions
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