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Mitigation of Undesirable Flavor in Kefir Intended for Adjuvant Treatment of Clostridioides Difficile Infection THESIS Presented

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Mitigation of Undesirable Flavor in Kefir Intended for Adjuvant Treatment of Clostridioides Difficile Infection THESIS Presented Mitigation of Undesirable Flavor in Kefir Intended for Adjuvant Treatment of Clostridioides difficile Infection THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Megan Kathleen Kesler Graduate Program in Food Science and Technology The Ohio State University 2019 Master's Examination Committee: Valente B. Alvarez, Advisor Rafael Jiménez-Flores Ahmed Yousef Copyrighted by Megan Kathleen Kesler 2019 Abstract Clostridioides difficle infection (CDI) is the most common health-care associated infection in the United States. Annually, CDI infects nearly half a million patients, kills 29,000 and costs the healthcare system an estimated US$6.3 billion. Although this pathogen can be treated with antibiotics, in 25% of cases, the bacterium does not respond to antibiotics and the patient develops a recurrent infection. Due to the hardy nature of the bacteria causing the recurrent CDI, additional, more costly treatment methods such as fecal microbiota transplantation must be employed. Alternatively, consumption of kefir, a commercially available fermented dairy beverage with live probiotic cultures, was recently found to be effective in curing recurrent CDI when consumed alongside the prescribed antibiotic treatment. Unfortunately, kefir has limited patient acceptance due to its strong acidic and fruity flavors resulting from the fermentation process. To combat this, kefir may be further processed using freeze-drying or vacuum evaporation to remove undesirable volatile organic compounds (VOCs) that greatly contribute to flavor, but heat during processing must be minimized to avoid reducing the population of beneficial microorganisms. In this study, effect of vacuum evaporation and freeze-drying on kefir VOC concentration, microbial viability and activity, and sensory quality was assessed. Commercially manufactured kefir was subjected to either a vacuum evaporation or freeze-drying treatment, or was not further processed (control). Loss of volatile compounds was monitored at the ppb level using selected-ion flow-tube mass ii spectrometry (SIFT-MS); kefir acceptability was evaluated by untrained panelists using a 9-point hedonic scale, and differences among treatments was determined by comparing mean scores; microbial viability was assessed using selective media for enumeration of Lactobacillus spp. and Lactococcus spp.; and microbial activity was assessed by measuring fermentation rate of milk inoculated with treated and untreated (control) kefir. Vacuum evaporation and lyophilization treatments significantly diminished VOC content in the kefir for 26 out of 27 compounds (P < 0.05). Cumulative VOC concentration for all 27 compounds was reduced by approximately 62% after both vacuum evaporation and freeze-drying treatments. Despite the considerable difference in kefir VOC content, vacuum evaporation and lyophilization treatments did not significantly improve the liking of commercial kefir in sensory tests. The concentrations of Lactobacillus spp. and Lactococcus spp. present in the kefir were significantly reduced by freeze-drying and vacuum evaporation treatments. Although significant, the population of lactic acid bacteria (LAB) in the kefir samples was still considered sufficient for probiotic products. Fermentation rates were slightly reduced after the experimental treatments, with lyophilization having a more significant effect. iii Acknowledgments It is with immense gratitude that I acknowledge the support and help of my advisor, Dr. Valente Alvarez. His guidance and encouragement throughout the project were vital to my success. It was an honor and a pleasure to be mentored by him. I would also like to thank Dr. Rafael Jiménez-Flores for encouraging me to think like a scientist and for treating me as an extended part of his workgroup, Dr. Ahmed Yousef for his time and helpful suggestions, and Dr. Cory Hussain for bringing this project to our group and for his continuing support. I am indebted to Dr. Hardy Castada for his time and assistance, and for his help in developing my methodology. Thank you also to Dr. Israel García-Cano, Dr. Diana Rocha-Mendoza, and Dr. Joana Ortega-Anaya for the many times they helped me in the laboratory. My deepest gratitude to Heather Bell, Steven Simmons, Gary Wenneker, Matt Papic, Stelios Sarantis and Tori Dong for their assistance, moral support, and friendship. My research would not have been possible without their help, and my time in the department would not have been as bright without their company. I will miss working with them. Thank you to all of my friends, old and new, for your encouragement and for the fun times that lifted my spirits when I needed it most. I would especially like to thank Elliot Dhuey for helping me navigate graduate school every step of the way. iv A special thank you to my husband, Ryan, for his unwavering love and support, and for all of the sacrifices he made so that I could achieve this degree. He tirelessly cheered me on and never let me give up. I could not have done it without him by my side. Last but not least, I would like to thank my family. It is because of them that I am where I am today. v Vita June 2012 .......................................................Minnetonka High School May 2017 .......................................................B.S. Food Science and Technology, University of Wisconsin - Madison August 2017 to present .................................Graduate Research Associate, Department of Food Science and Technology, The Ohio State University Fields of Study Major Field: Food Science and Technology vi Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ..................................................................................................................................... vi List of Tables ...................................................................................................................... x List of Figures .................................................................................................................... xi Chapter 1: Introduction ...................................................................................................... 1 Chapter 2: Literature Review .............................................................................................. 4 2.1 Kefir .......................................................................................................................... 4 2.1.1 Introduction to Fermented Milk Products .......................................................... 4 2.1.2 Origin and History .............................................................................................. 5 2.1.3 Definition and Composition ............................................................................... 6 2.1.4 Manufacture ........................................................................................................ 9 2.1.5 Microflora ......................................................................................................... 11 2.1.6 Sensory Characteristics..................................................................................... 13 2.1.7 Therapeutic Properties ...................................................................................... 17 vii 2.2 Clostridioides difficile Infection.............................................................................. 19 2.2.1 Background ....................................................................................................... 19 2.2.2 Treatment Methods ........................................................................................... 21 2.2.3 Kefir and Clostridioides difficile Infection ....................................................... 23 2.3 Kefir Post-Manufacture Processing ........................................................................ 24 2.3.1 Application of Vacuum Drying Technologies for Removal of VOCs ............. 25 2.3.2 Lyophilization ................................................................................................... 25 2.3.3 Vacuum Evaporation ........................................................................................ 28 2.4 Methods of Analyses: Selected-Ion Flow-Tube Mass Spectrometry ...................... 29 Chapter 3: Materials and Methods .................................................................................... 31 3.1 Stock Solution Preparation ...................................................................................... 31 3.2 Sample Preparation ................................................................................................. 31 3.2.1 Control Samples ............................................................................................... 31 3.2.2 Vacuum Evaporation ........................................................................................ 31 3.2.3 Lyophilization ................................................................................................... 32 3.3 Methods of Analysis................................................................................................ 34 3.3.1 Moisture Analysis ............................................................................................. 34 3.3.2 Selected-Ion
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