Lactobacillus casei group: identification, characterization and genetic evaluation of the stress response A PhD dissertation presented by Federica Ginaldi to the University of Udine for the degree of PhD in the subject of Food Science (Cycle XXVIII) Department of Agricultural, Food, Environmental and Animal Sciences UNIVERSITY OF UDINE Italy March 2016 Coordinator: Prof. Mara Lucia Stecchini, Department of Food Science University of Udine, Italy Supervisors: Prof. Lucilla Iacunin, PhD Department of Food Science University of Udine, Italy Reviewers: Prof. Maret Du Toit, PhD Department of Viticulture and Oenology University of Stellenbosch, South Africa Dr. Anna Reale, PhD Institute of Food Sciences National Research Council, Avellino, Italy I declare that my PhD thesis has been amended to address all the Referee's comments. To my parents Table of contents Summary ........................................................................................ 7 1. Stress response in Lactobacillus casei group ............................. 9 1.1 Introduction ........................................................................... 9 1.2 Lactobacillus casei group ..................................................... 9 1.3 Identification of species belonging to L. casei group ......... 11 1.4 Technological and probiotic utilization of L. casei group strains ........................................................................................ 13 1.5 Probiotic effects of L. casei group strains .......................... 15 1.6 Stress response in L. casei group strains ............................ 16 Heat stress ............................................................................................... 17 Cold stress .............................................................................................. 18 Acid stress .............................................................................................. 19 Bile salt tolerance ................................................................................... 20 Osmotic stress ......................................................................................... 21 Oxidative stress ...................................................................................... 22 1.7 Potential application of the stress response in food and probiotic industry ...................................................................... 22 References ................................................................................. 24 2. Collection and identification of strains belonging to the Lactobacillus casei group ............................................................ 41 2.1 Aim of the study ................................................................. 41 2.2 Materials and methods ........................................................ 41 2.2.1 Strains and culture conditions .......................................... 41 2.2.2 DNA extraction from pure cultures ................................. 44 2.2.3 L. casei group-specific PCR ............................................ 44 2.2.4 Species-specific PCRs ..................................................... 44 2.2.5 tuf multiplex PCR ............................................................ 46 1 2.2.6 Development and optimisation of High-Resolution Melting (HRM) analysis ......................................................................... 46 2.3 Results and discussion ........................................................ 47 2.3.1 Preliminary identification by L. casei group-specific PCR ................................................................................................... 47 2.3.2 Species identification by species-specific PCRs and tuf multiplex PCR ........................................................................... 48 2.3.3 High-resolution melting (HRM) analysis ........................ 51 2.4 Conclusions ......................................................................... 57 References ................................................................................. 58 3. Genetic characterization of Lactobacillus casei group strains . 63 3.1 Aim of the study .................................................................. 63 3.2 Materials and methods ........................................................ 63 3.2.1 Strains ............................................................................... 63 3.2.2 DNA extraction from pure colonies ................................. 63 3.2.3 REP-PCR analysis ........................................................... 63 3.2.4 RAPD-PCR analysis ........................................................ 64 3.2.5 Sau-PCR analysis ............................................................. 64 3.3 Results and discussion ........................................................ 65 3.3.1 Rep-PCR Analysis ........................................................... 65 Lactobacillus rhamnosus ....................................................................... 65 Lactobacillus casei and L. paracasei ..................................................... 67 3.3.2 RAPD-PCR Analysis ....................................................... 69 Lactobacillus rhamnosus ....................................................................... 69 Lactobacillus casei and L. paracasei ..................................................... 70 3.3.3 Sau-PCR Analysis ............................................................ 73 Lactobacillus rhamnosus ....................................................................... 73 Lactobacillus casei and L. paracasei ..................................................... 74 3.4 Conclusions ......................................................................... 77 2 References ................................................................................. 78 4. MLST and SAPD analysis to characterize the stress response in Lactobacillus casei group ............................................................ 81 4.1 Aim of the study ................................................................. 81 4.2 Materials and methods ........................................................ 81 4.2.1 Bacterial strains ................................................................ 81 4.2.2 DNA extraction ................................................................ 81 4.2.3 Selection of the genes for the MLST protocol ................ 83 4.2.4 Data analysis .................................................................... 85 4.2.5 Whole genome sequencing .............................................. 86 4.2.6 Specifically Amplified Polymorphic DNA (SAPD) PCR 86 4.2.7 Gel electrophoresis .......................................................... 87 4.2.8 Cluster analysis of SAPD-PCR patterns .......................... 87 4.3 Results ................................................................................. 87 4.3.1 UPGMA tree based on MLST data ................................. 91 4.3.2 Whole genome sequences comparison ............................ 95 4.3.3 UPGMA tree based on SAPD-PCR data ......................... 98 4.4 Discussion and Conclusions ............................................. 105 References ............................................................................... 108 5. Mucus binding properties of Lactobacillus casei group strains before and after oxidative stress ................................................. 113 5.1 Aim of the study ............................................................... 113 5.2 Material and methods........................................................ 113 5.2.1 Bacteria and growth conditions ..................................... 113 5.2.2 Mucin binding assay ...................................................... 115 5.2.3 Adhesion to Matrigel Matrix ......................................... 115 5.2.4 Mucin binding assay after in vitro digestion ................. 116 5.3 Results ............................................................................... 117 3 5.3.1 Bacterial adhesion to mucin ........................................... 117 5.3.2 Adhesion to Matrigel Matrix ......................................... 118 5.3.3 Mucin binding assay after in vitro digestion ................. 120 5.4 Discussion and Conclusions ............................................. 121 References ............................................................................... 123 6. Effect of different pH level at different EtOH concentration on the expression of stress response related gene of Lactobacillus paracasei B171 in wine .............................................................. 125 6.1 Aim of the study ................................................................ 125 6.2 Material and methods ........................................................ 125 6.2.1 Inoculation of must samples .......................................... 125 6.2.2 Microbial enumeration ................................................... 126 6.2.3 Determination of pH ...................................................... 127 6.2.4 Determination of lactic and malic acid by the use of enzymatic commercial kits ..................................................... 127 6.2.5 Determination of alcohol content .................................. 127 6.2.6 Determination of β-glucosidase activity with p-NPG ... 127 6.2.7 Determination of gene expression by RT-qPCR ........... 128 6.2.8 RNA extraction .............................................................. 128 6.2.9 Standardization