Utah State University DigitalCommons@USU All Graduate Theses and Dissertations Graduate Studies 12-2020 Understanding the Microbial Population of Lactococcus lactis. subsp. Strains During Cheese Making and Initial Storage Rhitika Poudel Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Dietetics and Clinical Nutrition Commons Recommended Citation Poudel, Rhitika, "Understanding the Microbial Population of Lactococcus lactis. subsp. Strains During Cheese Making and Initial Storage" (2020). All Graduate Theses and Dissertations. 7938. https://digitalcommons.usu.edu/etd/7938 This Thesis is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. UNDERSTANDING THE MICROBIAL POPULATION OF Lactococcus lactis. subsp. STRAINS DURING CHEESEMAKING AND INITIAL STORAGE By Rhitika Poudel A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Nutrition and Food Sciences Approved: ______________________ __________________ Donald McMahon, Ph. D. Craig J. Oberg, Ph.D. Major Professor Committee Member ______________________ __________________ Michael Lefevre, Ph.D. Randall Thunell, Ph.D. Committee Member Committee Member D. Richard Cutler, Ph.D. Interim Vice Provost of Graduate Studies UTAH STATE UNIVERSITY Logan, Utah 2020 ii Copyright © Rhitika Poudel 2020 All Rights Reserved Any materials in this thesis can be used by the Western Dairy Center, the BUILD Dairy program, and Donald McMahon. iii ABSTRACT Understanding the Microbial Population of Lactococcus lactis. subsp. strains During Cheese Making and Initial Storage by Rhitika Poudel, Master of Science Utah State University, 2020 Major Professor: Dr. Donald J. McMahon Department: Nutrition, Dietetics and Food Sciences Traditionally, starter cultures for cheddar cheese are combinations of Lactococcus lactis subsp. lactis and cremoris. Our goal was to compare their growth and survival during cheesemaking and after salting and pressing. Cheddar cheese was made in duplicate using two strains of lactis (600-M1, E36) and cremoris (B36, G61) using 520- kg of pasteurized milk and 31°C set and 38°C cook temperatures. Milled curd was salted with 2.0, 2.4, 2.8, 3.2 and 3.6% salt, pressed for 3 h and stored at 6°C and sampled after 6 d. Starter culture numbers were enumerated by plating on both M17 and Reddy’s agar. Flow cytometry event numbers were divided into three groups based upon bacterial cells permeability to Sybr Green and propidium iodide: living-nonpermeable, living- semipermeable, and dead-permeable cells. Cheese make time (set-to-mill time) varied from 210 to 393 min depending on the strain. The two cremoris strains were different from each other in terms of acid production, increase in cell numbers during cheese manufacture and cheese make time. Acid production rates during cheesemaking (with cook temperature of 38°C) was more dependent on the individual strain than whether it was a lactis or cremoris strain. The cheese made with lactis strains contained ~4 times (~ iv 0.5 log) more bacterial cells than those made using cremoris. None of the four strains tested were influenced by the amount of salt added to the curd (at least in the range of cheese S/M of 3.5 to 5.5%). Higher pH in cheeses with higher salting levels was attributed to the higher salt cheeses having up to 20 g/kg less moisture content. Based on flow cytometry, ~5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 5 to 15% of the cells were designated as being alive but live- semi permeable, with cremoris strains having the higher number of such cells. Live- nonpermeable cells were up to one log higher than plate count numbers depending on the strain. We concluded that using only plate counting does not fully describe the fate and activity of starter culture during cheese storage, and that flow cytometry provides additional information on microbiology of cheese that aids further understanding of the role of starter cultures in cheese aging and flavor development. (91 pages) v PUBLIC ABSTRACT Understanding the Microbial Population of Lactococcus lactis. subsp. strains During Cheese Making and Initial Storage Rhitika Poudel Cheddar cheese is manufactured by fermenting milk with lactic acid bacteria added as Lactococcus lactis. The two major subspecies used are lactis and cremoris. Starter culture for this study was prepared and donated by Vivolac Cultures Corporation, Greenfield, Indiana. Cheddar cheese was made in duplicate using two strains of lactis and cremoris. Milled curd was salted with 2.0, 2.4, 2.8, 3.2 and 3.6% salt, pressed for 3 h and stored at 6°C. The curd was sampled during cheese making and after 6d storage. Two different methods of enumeration were employed. One method involved plate counting which only enumerates those bacteria that can reproduce and form visible colonies. The other method was flow cytometry (FC) that counts the number of cells based upon their combination with fluorescent dyes, which could be divided into three groups: live, live- semipermeable, and dead cells. None of the four strains tested were influenced by the amount of salt added to the curd. Acid production rates during cheesemaking (with cook temperature of 38°C) was more dependent on the individual strain than whether it was a lactis or cremoris strain. Based on flow cytometry, ~5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 5 to 15% of the cells were designated as being alive but live- semi permeable ,with cremoris strains having the higher percentage of such cells. Thus, it can be said that plate count does not fully describe the fate and activity of starter culture during cheese storage, and that flow cytometry provides additional information on vi microbiology of cheese and can help track living cells during cheese storage and flavor development. vii Dedicated to my family for their endless love, support and sacrifices. I wouldn’t be where I am without you. viii ACKNOWLEDGMENTS First and foremost, I wish to express my sincere gratitude to my advisor, Professor Donald J. McMahon for giving me the opportunity to work and allowing me to grow as a research scientist. This journey would not have been possible without his support. I would like to thank him for the continuous motivation, patience and immense knowledge. His guidance has helped me in all the time of research and writing of this thesis. I would like to thank my committee members Prof. Craig J. Oberg, Prof. Michael Lefevre, and Dr. Randall Thunell for their time, support and expertise. I would like to extend my thanks to Dr. Eric Bastian for his constant encouragement and insightful comments through BUILD dairy programs. Thanks to Tara B. Johnson and Kim Rasmussen for their kind support. Also, thanks to my fellow lab mates, Ireland Green, Brynli Tattersall and Sophie Overbeck for their cooperation. I would also like to thank Sapana Lohani and all my friends who in one way or another shared their support either morally or physically. Special thanks to Kshitij Parajuli and Medha Khatiwada for their constant support and guidance and for all the love and care for the last 3 years. Special mention to darling Samaira. Lastly, I would like to thank my family, my parents, Ram Chandra Poudel and Sudha Sharma, for always believing in me and giving me strength. I want to thank my brother Rhishav Poudel for always loving me, my sister Rhiju Poudel, for being my rock when I need it the most and always being supportive of my decision, my brother in law ix Shiv Pandey, for his advice and making sure I never lost track of my dreams and my eight month old nephew Ahvaan for spreading light in our lives. Rhitika Poudel x CONTENTS LIST OF FIGURES .......................................................................................................... xii LIST OF TABLES ........................................................................................................... xiv LIST OF ABBREVIATIONS .......................................................................................... xvi INTRODUCTION ...............................................................................................................1 HYPOTHESIS AND OBJECTIVES ...................................................................................3 LITERATURE REVIEW ....................................................................................................4 Cheddar Cheese Manufacture ...................................................................................4 Starter Culture ...........................................................................................................4 Flow Cytometry .........................................................................................................5 Live and Dead Staining .............................................................................................8 Metabolically Active Non-Culturable (MANC) Cells ............................................11 MATERIALS AND METHODS .......................................................................................13 Starter Cultures ........................................................................................................13 Cheese Manufacture ................................................................................................13