Characterization of Propionibacteria in Swiss Raw Milk by Biochemical and Molecular-Biological Methods
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Research Collection Doctoral Thesis Characterization of propionibacteria in Swiss raw milk by biochemical and molecular-biological methods Author(s): Fessler, Denise Sophie Publication Date: 1997 Permanent Link: https://doi.org/10.3929/ethz-a-001855109 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETH No. 12328 Characterisation of propionibacteria in Swiss raw milk by biochemical and molecular-biological methods A thesis submitted to the Swiss Federal Institute of Technology (ETH), Zurich for the degree of Doctor of Technical Sciences presented by DENISE SOPHIE FESSLER Dipl. Lm.-lng. ETH born September 25th, 1969 citizen of Walzenhausen (AR) accepted on the recommendation of Prof. Dr. Z. Puhan, examiner Dr. M. G. Casey, co-examiner Dr. S. Lortal, co-examiner Zurich 1997 To my parents Acknowledgements I would like to express my sincere gratitude to Prof. Dr. Z. Puhan for giving me the chance to carry out this thesis and for his supervision. Very special and warm thanks belong to Dr. M. G. Casey for supporting me. He gave me invaluable suggestions and always had an open ear for my problems. Without his patience and confidence this thesis would not have been possible. I thank Dr. Sylvie Lortal for taking over the external examination of this work. I would like to extend my appreciation to the whole Department of Bio¬ chemistry, FAM. I thank Dr. M. Furst, Dr. J. Jimeno, Dr. A. Baer and Dr. J. Meyer for their backing and Mrs. M.-T. Raemy, Mrs. 1. Ryba, Mrs. N. Loosli, Mr. J. Gruskovnjak and Mr. A. Spahni for creating the friendly working environment. I wish also to thank Dr. C. Steffen, manager of the FAM for providing me with the working place and all the employees of the FAM, who gave me their support. I thank Dr. E. Wehrli for the analysis with the Raster Electron Micro¬ scope. Particular thanks belong to my family and all my friends for their unfailing encouragement and friendship. Abbreviations ATCC American Type Culture Collection, Rockville, Md. bp base pair CNRZ Centre National de recherches Zootechniques, Jouy- en-Josas, France cont. continued dist. distilled DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH FAM Federal Dairy Research Institute, Switzerland FAMb Biochemistry, Federal Dairy Research Institute, Switzerland Fig. Figure freud. freudenreichii kb kilo base Lb. Lactobacillus MIBD/SICL Milchwirtschaftliche Inspektions- und Beratungsdienste Dairy Inspection and Consulting Services min minute P. Propionibacterium °SH acidity sherm. shermanii sp. species subsp. subspecies T type strain not analysed Contents Summary / Zusammenfassung / Resume 1 1. Introduction and presentation of the problem 8 2. Literature 10 2.1. History and taxonomy of the genus Propionibacterium 10 2.2. Factors affecting growth of propionibacteria 14 2.2.1. Temperature 14 2.2.2. pH 14 2.2.3. Effect of sodium chloride 14 2.3. Metabolism 15 2.3.1. Propionic acid fermentation 15 2.3.2. Metabolism of aspartate 15 2.3.3. Proteinase and peptidase activity 17 2.3.4. Lipase and esterase activity 17 2.4. Some other features of propionibacteria 18 2.4.1. Plasmids 18 2.4.2. Bacteriophages 18 2.4.3. Bacteriocins 19 2.4.4. Interactions with lactic acid bacteria 19 2.5. Propionibacteria in practice 20 2.5.1. Natural habitat 20 .^ 2.5.2. Use of propionibacteria 21 2.5.3. Propionibacteria as probiotics 22 2.5.4. Propionibacteria in cheese manufacture 23 2.5.4.1. Swiss-type cheese 23 2.5.4.2. "Brown spots" defect 23 2.5.4.3. Split defect 24 2.6. Methods for enumeration, identification, differentiation and classification of propionibacteria 25 2.6.1. Isolation and enumeration 25 2.6.2. Identification and classification based on biochemical methods 26 2.6.3. Identification and classification based on genomic criteria 27 2.6.4. Identification and classification by other methods 28 3. Material and methods 30 3.1. Growth media for propionibacteria 30 3.1.1. Yeast extract lactate broth (YEL) and yeast extract lactate agar (YELA) 30 3.1.2. Minimum growth medium 30 3.1.3. MF95C 30 3.1.4. Peptone whey 31 3.1.5. Dilution solution 31 3.2. Media for other bacteria 31 3.3. Microorganisms 31 3.3.1. Propionibacteria 31 3.3.2. Other bacteria 33 3.4. Sugar fermentation 33 3.5. Growth curves 33 3.6. Survival rate at cheese manufacture conditions 33 3.7. Analysis of the soluble cell free protein extract 34 3.7.1. Preparation of protein extracts 34 3.7.2. Protein gel electrophoresis and staining of gels 34 3.8. Purification of genomic DNA 35 3.9. Restriction enzyme analysis 36 3.10. Ribotyping 37 3.11. Restriction analysis of 23S rRNA 37 3.11.1. Preparation of DNA extracts 37 3.11.2. Polymerase chain reaction and restriction analysis 38 3.12. RAPD 38 3.13. Plasmids 40 3.14. Emmental cheese 41 3.14.1. Model Emmental cheese manufacture 41 3.14.2. Analysis and sensoric tests performed on model cheeses 43 3.14.3. Emmental cheese manufacture 44 4. Results and discussion 46 4.1. Propionibacterial flora in Swiss raw milk from lowlands and alps 46 4.1.1. Introduction 46 4.1.2. Reproducibility of protein profiles and restriction analysis profiles 46 4.1.3. Propionibacterium species in lowland milk 47 4.1.4. Propionibacteria from alps 52 4.1.5. Classification of P. rubrum 53 4.1.6. P. freudenreichii subspecies 53 4.1.7. Differentiation between strains 54 4.1.7.1. Plasmid profile 55 4.1.7.2. RAPD 56 4.2. Propionibacteria used for Emmental cheese manufacture in Switzerland 60 4.2.1. Introduction 60 4.2.2. Protein profiles, plasmids and RAPD of commercial strains 60 4.2.3. Detection of propionibacteria of commercial cultures 63 4.3. Propionibacteria and cheese faults 66 4.3.1. Introduction 66 4.3.2. Brown spots 66 4.3.2.1. Emmental 66 4.3.2.2. Sbrinz 67 4.3.2.3. Appenzell 69 4.3.2.4. Raclette 70 4.3.2.5. Conclusions 70 4.3.3. Split defect 71 4.3.3.1. Introduction 71 4.3.3.2. Sbrinz 71 4.3.3.3. Gruyere 72 4.3.3.4. Conclusions 72 4.4. Model Emmental cheese manufacture with selected wild Propionibacterium strains 73 4.4.1. Introduction 73 4.4.2. Selection of strains 73 4.4.3. Quality of model Emmental cheese 75 4.4.4. Influence of subspecies 78 4.4.5. Chemical and microbiological analysis 79 4.4.6. Sensorics of model Emmental cheeses 82 4.4.7. Raster electron microscopy 83 4.5. Emmental cheese manufacture with three wild Propionibacterium strains 87 4.5.1. Introduction 87 4.5.2. Quality of cheeses 87 5. Conclusion 90 6. Bibliography 94 7. Annex 102 Summary / Zusammenfassung / Ftesumfe £1; Summary Characterisation of propionibacteria in Swiss raw milk by biochemi¬ cal and molecular-biological methods For the manufacture of Emmental cheese in Switzerland raw milk is used. The formation of eyes, however, is achieved by addition of culture with selected strains of propionibacteria. The wild propionibacteria in raw milk and cheese have until now not been systematically analysed. How¬ ever, it is known that they are responsible for defects such as brown spots and split defect in various hard or semi-hard raw milk cheeses and that technological factors during cheese manufacture may influence the occurrence of these defects. The commercially available P-culture, a mixture of P. freudenreichii strains, has been used for many years and a new culture, Prop 96, is also commercially available since 1996. The strains included in these cultures have until now not been analysed by molecular-biological methods. In the present study the propionibacterial flora in Swiss raw milk was in¬ vestigated and propionibacteria were classified by protein profile analy¬ sis, restriction profile analysis of the 23S rRNA gene, plasmid content and RAPD profiles. Propionibacteria isolated from various cheese types with defects were differentiated and classified, and compared with the propionibacteria strains isolated from raw milk. The propionibacterial flora in Swiss raw milk was found to be extraordi¬ narily rich. All four dairy Propionibacterium sp. were found in lowland raw milk, 71% were P. freudenreichii, 19% P. jensenii, 8% P. acidipropionici and 2% P. thoenii. In alpine raw milk P. acidipropionici was not found, P. freudenreichii made up 55%, P. jensenii 15% and P. thoenii 30% of the total. Among the 278 P. freudenreichii strains 219 (79%) different strains were identified by RAPD at the strain level. For the other species strain diversity was even higher. Only 30% of all analysed strains carried plasmids. Commercial cultures were analysed by RAPD and the presence of the strains making up the P-culture was looked for in Emmental cheese, in order to see, if all the strains grew in the cheese. Of the six strains com¬ prised in the P-culture, four strains were found to be identical, meaning that this culture contains only three different strains. One of these strains -2- Summary / Zusammenfassung / Resume could not be detected in commercially available premium grade Emmen¬ tal and one was only occasionally present. Consequently, it is possible that only one of the P-culture strains is responsible for the good cheese quality. The two strains in Prop 96 were found to be identical, but differ¬ ent from the P-culture strains. In brown spots of Emmental and Sbrinz cheeses various strains of P. freudenreichii were detected by RAPD.