Real-Time Release of Volatile and Non-Volatile Components from Chewing Gum Using a Mechanical Chewing Device A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY Andrea Jean Krause IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Advisor Dr. Gary Reineccius November, 2010 © Andrea Jean Krause, 2010 ii Acknowledgements First, I wish to thank my advisor, Dr. Gary Reineccius for the opportunity to pursue this degree and for his immense patience and contagious positive attitude. Gary has impressed upon me several life lessons that I have tried to remember throughout my PhD. and plan to carry forward for the balance of my life. 1.) There are few things in life so important that you can’t stop to get an ice cream on the way there. And 2.) 87% of the things you worry about don’t happen (also 75% of all statistics are made up!). Your emphasis on independent problem solving was difficult for me at the beginning, but I have come to value it as much as any tangible thing I will take from the University of Minnesota. Additionally, the many special people who have helped me in the lab and who have been there to commiserate over coffee (tea for me!) and chocolate: Debbie Paetznick, Josephine Charve, Jean-Paul Schirle-Keller, Marlene Moskowitz, Ntina Karametsi, and Lu Bing. Thanks to Smita Raithore, Smaro Kokkinidou, and Dr. Devin Peterson for their invaluable help with the LC-MS and other gum-related problems. All those who finished before me who served as valuable inspiration: Mike Mortenson, Segolene Leclercq, Savitha Krishnan, Sarah Schramke, and Jian Zhang. Thank you to my committee members for their project-related input. I would like to acknowledge Dr. Lulu Henson for her assistance and expertise, which was such as asset during the course of this project. Of course I would not be where I am today without the guidance I received during my Master’s degree at NC State from Dr. MaryAnne Drake. She taught me the i importance of hard work and discipline and, of course, that flavor rules! Thank you to Dr. Richard Hartel at the University of Wisconsin for his guidance during my undergraduate years and who is still happy to hear from me after all of these years. Thank you to my wonderful family, who has never questioned my perhaps unconventional journey. (Don’t worry, Mom and Dad…I am getting a “real” job now!) My sister and brother-in-law and sweet nephews and nieces for the great visits and pictures that adorn my desk and refrigerator. And to the Viazis family for accepting me into their family as their daughter and for their long-distance support. I am also grateful for all of the friends too numerous to list who have made my time in the Twin Cities filled with great memories. I’m going to miss all of you dearly. Lastly, my husband Stelios Viazis, who I don’t thank nearly enough for putting up with me day in and day out. He has helped me to believe in myself, do the things I never thought possible (i.e. attempt this degree), and stayed by my side throughout the last five plus years. Ειµαι πολυ τυχ ερη. Σ’αγαπω, αγαπη µου! Here’s to the next step…. ii Abstract To date, the majority of research on chewing gum has been conducted using human subjects in conjunction with time-intensity sensory analysis and/or real-time mass spectrometry techniques (proton transfer reaction mass spectrometry [PTR-MS] or atmospheric pressure chemical ionization [API-MS]). The disadvantages of human subjects include their tremendous variability (salivary flow rate, masticatory force, mouth volume, mastication rate, respiration rate and others), low throughput of samples, and necessary training and compensation. For these reasons, it is desirable to fabricate a chewing device to simulate human mastication. Using this device, formulation and ingredient effects could be elucidated without convolution by inter-individual differences. The trade-off, however, is a lack of end-user perception, a potentially large capital investment, and difficulty replicating the conditions associated with human mastication. In the work presented herein, we have developed such a chewing device to be used as a screening tool for ingredients and formulation effects in chewing gum. The device simplifies the chewing process so a more basic understanding of the release of volatile and non-volatile components from chewing gum can be achieved. Following the construction of the chewing device, suitable methodology was developed to examine the release of volatile aroma compounds into the air (using PTR- MS or API-MS). Aroma compounds extracted in the simulated saliva were evaluated using GC-FID. Non-volatile compounds (polyols and high potency sweeteners) extracted into simulated saliva were measured using HPLC-MS and UPLC-MS. iii Conducting a mass balance of chewing gum components validated the device and methodology. Analysis of chewing gum after simulated mastication revealed that a large portion of the aroma compounds remained in the bolus after 21 min of simulated mastication; only a small portion were found in the air and simulated saliva; the amount depended on the properties of the aroma compound. The water-soluble compounds, however, were almost entirely depleted from the chewing gum after 21 min. In the second study, the chewing device was used to evaluate ingredients designed to delay the release of acesulfame-K (Ace-K), a high potency sweetener commonly used in chewing gum, from chewing gum during mastication. A response surface experimental design was used to optimize the entrapment of Ace-K in polyvinyl acetate (PVA). Three parameters were examined (particle size of Ace-K, total particle size, and core to matrix ratio) at three levels. Embodiments were incorporated into chewing gum containing no high-potency sweetener. Release was examined using the chewing device connected to a fraction collector sampling at 1 min intervals. Ace-K concentrations (mg/ml) were evaluated using UPLC-MS. Statistical analysis revealed the optimum conditions for delayed release (from 11-21 min) to be smaller Ace-K particles with larger total particle size. The third study examined how differences in gum hardness affected the release of volatile aroma compounds and Ace-K. Three chewing gum formulations contained different levels of glycerin (3%, 6% or 9%). Static headspace was used as a measure of the effect of the matrix on volatility. Samples were masticated in the chewing device and the release of volatile aroma compounds was measured using an API-MS. Ace-K release iv was quantified at 1 min intervals using UPLC-MS. There were no significant differences in maximum intensity between the three chewing gums for the three volatile compounds (ethyl butyrate, isoamyl acetate, and limonene). Vapor pressure also was not significantly different between the three samples. These results potentially indicate that differences in perception between gums of differing textures are due to consumer perception and/or mastication rate and not differences in flavor release caused by the matrix or resistance to mass transfer. v Table of Contents Acknowledgements ..........................................................................................................i Abstract ............................................................................................................................iii Table of Contents ............................................................................................................vi List of Equations ..............................................................................................................ix List of Figures .................................................................................................................x List of Tables ...................................................................................................................xiii Thesis Flow Chart ............................................................................................................xiv Chapter 1: Literature Review .......................................................................................1 1.1 Introduction .........................................................................................................2 1.2 Sensory Perception .............................................................................................3 1.2.1. Taste ........................................................................................................3 1.2.1.1 Saliva ................................................................................................4 1.2.1.1.2 Chewing Gum and Saliva ..............................................................5 1.2.2 Olfactory System ......................................................................................6 1.2.3. Taste and Smell Interaction .....................................................................7 1.2.4 Chemesthesis ............................................................................................8 1.2.5 Other Contributors to Flavor Perception ..................................................9 1.3 Chewing Gum .....................................................................................................10 1.3.1 Gum base ..................................................................................................11 1.3.2 Sweeteners ................................................................................................13 1.3.2.1 Caloric ............................................................................................13