EFFECTS OF COMPATIBLE SOLUTES ON COLD TOLERANCE OF PROPIONIBACTERIUM FREUDENREICHII AND THE SIGNIFICANCE OF PROPIONIBACTERIUM COLD TOLERANCE IN SWISS CHEESE MANUFACTURING DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Corunda T. Pruitt, M.S. * * * * * The Ohio State University 2005 Dissertation Committee: Dr. W. James Harper, Adviser Approved By Dr. Polly D. Courtney Dr. Mike Mangino ________________________ Adviser Dr. Ahmed Yousef Graduate Program in Food Science and Technology ABSTRACT Propionibacterium freudenriechii is one of the bacteria utilized in Swiss cheese starter cultures. Its carbon dioxide production is responsible for the development of the eyes commonly associated with Swiss cheese. The bacteria must endure high cook temperatures and low storage temperatures during ripening. Many bacteria are capable of synthesizing compatible solutes in response to different stresses. The primary focus of the present study was to determine the growth capabilities and transport of P. freudenreichii strains in the presence of exogenous glycine betaine, proline, and glutamate at low temperatures and evaluate the effects of storage temperature on eye formation in Swiss cheese manufactured with these dairy P. freudenreichii strains differing in low temperature growth abilities. Four P. freudenreichii strains were grown in chemically defined media with the addition of 1 mM or 100 mM of glycine betaine, proline, or glutamate. The cultures were anaerobically incubated at 30, 22, 10, 7.2, and 4°C and spectrophometrically monitored to determine growth capabilities. Strains capable of growing at 10, 7.2, and 4°C were characterized as cold-tolerant and strains not able to grow at these temperatures were characterized as cold-sensitive. Compatible solute transport was assessed using 14C- labeled amino acids added to a cell suspension with a final concentration of 1 mM. The growth rate at the lowest temperature decreased and the lag phase increased for strains ii P843 and P57 and were therefore considered cold sensitive. At the same temperature the growth rate for strains P728 and P873 increased with a decreased lag phase. These strains were considered cold tolerant. All strains transported proline most abundantly at all temperatures with its cryoprotective effects observed at 22°, 10°, and 7.2°C by increased growth rates. Glycine betaine and glutamate were not transported as greatly as proline until a decrease in temperature (7.2°C). All solutes increased the maximum growth rate of all strains at this lower temperature indicated they exert a cryoprotective at this temperature. These strains were also subjected to several freeze-thaw cycles to deterimine if glycine betaine exterts a cryoprotective effect at temperatures below refrigeration. Cells were frozen for 24 hours and thawed 30 minutes at 30°C. After four days a decrease in cell viability was observed as well as after five days. There was a difference between all strains suggesting that freeze-thaw tolerance is strain dependent in propionibacteria. Strain P572 was the most freeze-thaw tolerant with a 1.01-fold reduction after four cycles. Swiss cheese was manufactured with a cold-tolerant strain (P873), a cold- sensitive strain (P572) and an intermediate strain (P196). All cheese blocks experienced identical warm room treatment (21°C for 17 days) but differed in cool ripening storage. The blocks were stored at 0, 4, or 7.2°C. Samples were taken at days 0, 30, 60, and 90. Counts of viable propionibacteria cells remained constant throughout the ripening treatment with a range of 108-9. The twenty most common amino acids were quantified in addition to citrulline during each ripening phase from water soluble cheese extracts. Differecnes were observed among each strain but only differences were observed between strains after 90 days at 4°C and 7.2°C. Total concentrations were higher in iii strains P196 and P873 after these ripening periods. Digital images of day 60 and 90 samples were analyzed for splits. More eyes were seen in cheeses stored for ninety days at 7.2°C. More splits were also seen at this time and temperature. Fewer eyes and splits were observed in cheeses stored for sixty days at 0°C. Cheeses manufactured with strain P196 possessed the most eyes after sixty days at 4°C and cheeses manufactured with strain P873 possessed the most splits after ninety days at 7.2°C. Cold tolerance varies among dairy propionibacteria and the role of glycine betaine, proline, and glutamate as compatible solutes may contribute to cryoprotection of some Propionibacterium strains. Strain selection, ripening temperature and ripening time may have an affect on split occurrence often seen in Swiss cheeses. iv Dedicated to my parents v ACKNOWLEDGMENTS I would like to acknowledge all of those who have helped me along the way in this long and difficult journey I call advanced education. These people made great contributions, large and small, in helping me fulfill the requirements through their knowledge, time, and support. I thank my past and present advisers, Dr. Polly D. Courtney and Dr. W. James Harper for guiding me through this entire process. I thank them for providing me with an unforgettable experience in a scientific environment of learning, independence, and enjoyment. I would like to extend my appreciation to Dr. Mike Mangino and Dr. Ahmed Yousef for serving on my dissertation committee and offering extensive advice on my project. I would like to all of my lab mates who helped create a tolerable work environment through their laughter and assistance. I would like to express my appreciation to the Swiss Cheese Consortium and The Ohio State University College of Food, Agriculture and Environmental Science for their financial support. Finally I would like to thank my family and friends who supported and encouraged me during some difficult times and the one guy who has my heart. I thank you all and love you. vi VITA July 15, 1977…………………………………………… Born – Dallas, Texas 1995- 1999………………………………………………B.S. Biology Xavier University New Orleans, Louisiana 2000-2002………………………………………………M.S. Biology Jackson State University Jackson, Mississippi 2002-present…………………………………………….Graduate Research Associate The Ohio State University Columbus, Ohio PUBLICATIONS Research Publication 1. Dave, S., M. K. Pangburn, C. Pruitt, and L. S. McDaniel. 2004. Interaction of human factor H with PspC of Streptococcus pneumoniae. Indian Journal of Medical Research 119(Suppl): 66-73. Research Abstract 1. Pruitt, C.T., O. Anggraeni, and P.D. Courtney. 2004. Cold tolerance and compatible solute uptake in dairy Propionibacterium freudenreichii. Abstract 99D-13. Institute of Food Technologists Annual Meeting, Las Vegas, NV. FIELDS OF STUDY Major Field: Food Science and Technology vii TABLE OF CONTENTS Page Abstract……………………………..……………………………………………………..ii Dedication…………………………………………………………………………………v Acknowledgments……………….……………………………………………………….vi Vita…………………………..………………………………………………………..…vii List of Tables………………………………………………………………….………….xi List of Figures………………………………………………….………………………..xiii Chapters: 1. Literature Review…………………………………………………………………1 1.1 Introduction……………………………………………………………………1 1.2 Cheese Manufacture…………………………………………………………...2 1.2.1 Swiss Cheese Manufacture………………………………………….4 1.2.2 Swiss Cheese Eye Formation……………………………………….5 1.2.3 Split Defects………………………………………………………...7 1.3 Compatible Solutes as Osmoprotectants and Cryoprotectants……………9 1.3.1 Listeria monocytogenes....................................................................14 1.3.2 Other Bacteria……………………………………………………...17 1.4 Effects of Membrane Composition on Compatible Solute Transport……….19 1.4.1 Compatible Solute Transport Systems………………………….…20 viii 1.4.1.1 Listeria monocytogenes………………………….………….….21 1.4.1.2 Other Bacteria…………………………….……………….……24 1.5 Conclusion…………………………………………………………….……26 2. Cold Tolerance and Compatible Solute Uptake of Dairy Propionibacterium freudenreichii…………………………………………………………………………..28 2.1 Introduction………………………………………………………………...28 2.2 Materials and Methods…………………………………………….……….31 2.3 Results……………………………………………………………….……..34 2.4 Discussion…………………………………………………………….……38 3. Determination of Freeze-Thaw Resistance in Dairy Propionibacterium freudenreichii 3.1 Introduction………………………………………………………….…….57 . 3.2 Materials and Methods…………………………………………….………59 3.3 Results…………………………………………………………….……….60 3.4 Discussion…………………………………………………………………61 4. The Effects of Storage Temperature on Split Defects in Swiss Cheese Manufactured with Dairy Propionibacterium freudenreichii Differing in Low Temperature Growth Capabilities……………………………………………………………………………66 4.1 Introduction……………………………………………………………….66 4.2 Materials and Methods……………………………………………….…...67 4.3 Results……………………………………………………………….……71 4.4 Discussion………………………………………………………….……..77 Conclusions…………………..………………………………………………………88 ix References………………………………………………………………………………..90 Appendices: Appendix A: Graphs displaying cold tolerance of Propionibacterium strains….98 Appendix B: Bacteria enumeration and digital images of Swiss cheese manufactured with different Propionibacterium strains……………………..…103 x LIST OF TABLES Table Page 2.1 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and the lag phase of P. freudenreichii strain P843 at various temperatures……………………………………………………………………..44 2.2 Effects of glycine betaine, proline, and glutamate on the maximum specific growth rate (µmax) and