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Isolation and Characterization of Nonstarter Lactobacillus Spp ISOLATION AND CHARACTERIZATION OF NONSTARTER LACTOBACILLUS SPP. IN SWISS CHEESE AND ASSESSMENT OF THEIR ROLE ON SWISS CHEESE QUALITY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Nurdan A. Kocaoglu-Vurma, 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 E. Yousef Food Science and Nutrition Graduate Program ABSTRACT Nonstarter Lactobacillus strains affect the quality of many cheese varieties. Though the use of Lactobacillus casei as an adjunct culture is common for Swiss-type cheese manufactured in Switzerland, few published reports exist on adjunct use and none exist for adjunct use in U.S.-manufactured Swiss cheese. High quality Swiss cheeses vary in sensory, chemical, microbiological, and physical characteristics. Determining the compositional characteristics of commercial Swiss cheeses will establish the typical range for each characteristic in cheeses intended for the American market and will complement descriptive sensory and consumer preference studies. The objectives of this study were to isolate and identify nonstarter Lactobacillus strains in high quality commercial Swiss cheeses, to investigate citrate metabolism among nonstarter lactobacilli, to study the effect of nonstarter Lactobacillus strains as adjunct cultures on Swiss cheese characteristics, and to determine chemical, microbiological, and physical characteristics of commercial Swiss-type cheeses. Lactobacilli were selected from six domestic and two European Swiss cheeses with selective medium and the strains from each cheese were genetically typed and speciated. Qualitative and quantitative citrate utilization assays were performed on each strain. The total number of Lactobacillus cells ranged from 4.8 × 104 to 7.1 × 107 CFU/g cheese. Strains belonging to L. casei, L. rhamnosus, and L. fermentum species were most ii frequently encountered. Lactobacillus casei strains predominated in the cheeses originating in Switzerland; whereas, the domestic cheeses contained a wider variety of Lactobacillus species, including different strains of L. casei, L. rhamnosus , L. gasseri, L. delbrüeckii, and L. fermentum. Citrate differential medium was valuable in rapid assessment of citrate utilization of lactobacilli. On this medium, L. helveticus, L. gasseri, and L. delbrüeckii strains did not metabolize citrate, while, L. casei, L. fermentum, and L. rhamnosus strains utilized citrate. Percent relative growth in modified MRS broth with glucose or citrate confirmed that L. delbrüeckii and L. helveticus strains cannot metabolize citrate as the sole carbon source. Among the other strains tested, L. casei strains were strong citrate utilizers followed by L. rhamnosus, L. fermentum, and L. gasseri strains. A putative citP gene fragment from one citrate-utilizing L. casei strain was amplified, cloned, and sequenced. Distribution of the putative citP gene in Swiss cheese nonstarter lactobacilli was determined by Southern hybridization using amplified fragment as a probe. Eight out of 22 strains tested had sequence homology to the probe. Twelve cheeses were manufactured using a commercial starter combination and three previously isolated nonstarter Lactobacillus strains, L. casei A26, L. casei B21, and L. rhamnosus H2. Cheeses were analyzed during ripening for microbial and chemical composition. The use of adjunct cultures diminished high variability in total Lactobacillus counts in cheeses manufactured without adjunct addition. Lactobacillus casei strains were able to utilize all citrate present in cheese before the end of the warm room ripening phase. There were no significant differences among cheeses in regards to protein, fat, moisture, and salt contents. The pH of the mature cheeses ranged from 5.4 to 5.5, and free amino acid concentration ranged from 5 to 7 mmol/kg cheese. Lactic, iii acetic, and propionic acid levels of cheeses were not significantly different. Based on electronic nose and descriptive sensory results, cheeses made with adjunct L. casei strain A26 were more similar to control cheese in development of certain flavor attributes. Fifteen cheeses (4 U.S.-manufactured baby Swiss, 10 U.S.-manufactured Swiss, and one European Emmental) were analyzed for characteristics including protein, fat, moisture, salt, pH, short chain free fatty acids, and total free amino acids. Lactobacillus spp., Streptococcus thermophilus, and Propionibacterium spp. were enumerated. Physical characteristics such as hardness, springiness, and meltability were assessed. An electronic nose was used to evaluate the volatile flavor compounds. The values for compositional characteristics ranged from 22.9 to 26.3% for protein, 46.3 to 55.1% for fat in dry matter, 36.4 to 41.8% for moisture, and 0.7 to 3.4% for salt in moisture. The pH values ranged from 5.37 to 5.80 and the free amino acid levels ranged from 2.32 to 10.48 mmol/kg. The Emmental cheese had the highest acetic acid and propionic acid levels. Bacterial counts varied widely: 5 to 8 log CFU/g Lactobacillus spp., 3 to 8 log CFU/g S. thermophilus, and 4 to 8 log CFU/g Propionibacterium spp. The cheeses with higher numbers of Propionibacterium spp. had higher propionic acid levels. Baby Swiss cheeses were softer, on average, than the Swiss cheeses. Meltability, measured by melted diameter, ranged from 18 to 40 mm. The Emmental cheese had the lowest meltability. The electronic nose evaluation differentiated the cheeses into three groups, with the baby Swiss cheeses grouping together. Understanding the occurrence, types, and metabolic capabilities of nonstarter Lactobacillus in Swiss cheese will allow further studies of their role in cheese ripening and their effect on Propionibacterium fermentation. Characterization of nonstarter iv strains from high quality cheeses may lead to new adjunct cultures specific for Swiss cheese. Chemical, microbiological, and physical characterization of Swiss cheeses, combined with sensory evaluation results may allow manufacturers to predict the acceptability of their cheese. v Dedicated to Mustafa, Arın Ozan, and my parents for their unconditional love, support, and inspiration vi ACKNOWLEDGMENTS I would like to thank my adviser Dr. Polly D. Courtney for her excellent guidance and support throughout my graduate studies at the Ohio State University. She is an excellent professor, a perfect adviser, and a wonderful person. I am grateful for the opportunity to learn from her. I would like to express my sincere gratitude to Dr. J.W. Harper, my primary adviser, since October 2004, for providing me the opportunity to work in an outstanding research environment. I would like to thank Dr. Ahmed E. Yousef and Dr. Mike Mangino for their helpful guidance. Throughout this study, I received exceptional collaboration from a number of people. I want to thank Cheryl Wick for sharing her expertise in cheese making and analyses; Gary Wenneker, for his exceptional technical support and assistance in pilot scale cheese production; Dr. Seyhun Yurdugul, Dr. Nurcan Koca, and Dr. Josephine Kuo for their contributions in compositional analyses of commercial cheese samples; and Dr. MaryAnne Drake, for conducting the descriptive sensory analysis at North Carolina State University. I would also like to thank all the members of our laboratory group; Julie Jenkins, Olga Anggraeni, Patcharee Limpatsian, Hyun Chung, Rory McCarthy, Corunda Pruitt, vii Jennifer Kaiser, Chris Wolf, and Dr. Karen Fligner. My deep appreciation extends to Maria Ruhlman and Joy Waite for their suggestions and friendship. I would particularly like to thank my husband Mustafa, simply, for everything. Thanks for always being there for me. Cheese-making and growth curve experiments wouldn’t be as enjoyable without your help, support, and presence. I would like to thank my parents, Nuran and Hakkı Kocaoglu, and my brothers Tayfun Kocaoglu and Dr. Argun Kocaoglu for their unconditional love, support, and inspiration. I also would like to thank Swiss Cheese Consortium, the Center for Innovative Food Technology, and the OARDC Research Enhancement Competitive Grants Program for their financial support. viii VITA July 2, 1973………………………..……………......Born – Istanbul, Turkey July, 1995 ….……………………………………….B.Eng. Food Engineering Istanbul Technical University Istanbul, Turkey 1995 – 1996………………………………………....Technical Sales Engineer Hemel S.A. Istanbul, Turkey 1996 – 1999…………………………………………M.S. Food Science University of California, Davis 1999 – 2000…………………………………………Product Manager Hemakim Ltd. Istanbul, Turkey 2000 – 2001................................................................Teaching/Research Associate Istanbul Technical University Istanbul, Turkey 2001 – present…………………..………..................Graduate Research Associate The Ohio State University Columbus, Ohio FIELDS OF STUDY Major Field: Food Science and Nutrition ix TABLE OF CONTENTS Page Abstract......................................................................................................................ii Dedication..................................................................................................................vi Acknowledgments......................................................................................................vii Vita.............................................................................................................................ix
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