Molecular and physiological studies on the functionality of probiotic lactobacilli:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Buch |
| Sprache: | English |
| Veröffentlicht: |
München
Verl. Dr. Hut
2007
|
| Ausgabe: | 1. Aufl. |
| Schriftenreihe: | Biologie
|
| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | Zugl.: Karlsruhe, Univ., Diss., 2006 |
| Beschreibung: | VII, 160 S. Ill., graph. Darst. |
| ISBN: | 9783899634914 |
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| 245 | 1 | 0 | |a Molecular and physiological studies on the functionality of probiotic lactobacilli |c von Maria Guadalupe Vizoso Pinto |
| 250 | |a 1. Aufl. | ||
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Datensatz im Suchindex
| _version_ | 1804136455574913024 |
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| adam_text | CONTENTS
Dedication...................................................................................................................................i
Zusammenfassung......................................................................................................................ii
Abstract.....................................................................................................................................iii
Acknowledgements...................................................................................................................iv
Contents......................................................................................................................................v
1 INTRODUCTION_______________________________________________________1
1.1 Development of the human gut microbiota....................................................................1
1.2 Microbial balance...........................................................................................................2
1.3 Probiotics........................................................................................................................3
1.3.1 Definition ..................................................................................................................3
1.3.2 Postulated mechanisms of action........................................................................................3
1.3.3 New probiotic strains and sources of isolation...................................................................5
1.3.4 Safety considerations..........................................................................................................6
1.3.5 Site of action of probiotics: the small intestine..................................................................7
1.4 Tolerance and immunity in the gut.................................................................................7
1.4.1 Bridging innate immunity and adaptive immunity..............................................9
1.4.2 Toll-like receptors..................................................................................11
1.4.3 The enterocyte as sentinel of the gut.............................................................13
1.5 Probiotics and their effect in the immune system........................................................16
1.6 Probiotics and acute gastroenteritis..............................................................................17
1.7 Study objective.............................................................................................................18
2 MATERIALS AND METHODS__________________________________________19
2.1 Culture media...............................................................................................................19
2.2 Buffers and solutions....................................................................................................20
2.2.1 Buffers and solutions used in molecular biology.................................................20
2.2.2 Buffers and solutions required for sandwich ELISA...........................................22
2.2.3 Buffers and solutions required for FACS analysis...............................................22
2.2.4 Other buffers and solutions..................................................................................23
2.3 Isolation, selection and identification of Lactobadllus strains....................................24
2.3.1 Isolation of bacterial strains.................................................................................24
2.3.2 Strain selection and identification........................................................................24
2.3.3 Phenotypic characterisation......................................................................................
2.3.3.1 Growth at différent temperatures................................................................................................................24
2.3.3.2 Production of gas from glucose..................................................................................................................24
2.3.3.3 Hydrolysis of argmine..........................................._...................................................................................26
2.3.3.4 Presence of muo-diaminopinielic actd(mDAP) in the cell walls.............-...............................................26
2.3.3.5 Determination of lactic acid enantiomers produced....................................................................................26
2.3.3.6 Sugar fermentation profiles............._.........................................................................................................27
2.3.4 Genotypic characterisation...................................................................................27
2.3.4.1 Isolation of genomic DNA ttom bacteria............................._...................................-...............................27
2.3.4.2 Determination of DNA and RNA concentration and purity......................_...............................................27
2.3.4.3 Rep-PCR...................................................._..............................._.............................................................28
2.3.4.4 16 S rDNA sequence................................................................................................................................28
2.3.4.5 Analytical scale purification of PCR products............................................................................................29
2.4 Testing Lactobaciuus strains for in vitro probiotic properties.....................................29
2.4.1 Studies on resistance to gastrointestinal conditions.............................................29
2.4.1.1 Evaluation of parameters which affect survival of probiotic strains during
simulated stomach passage........................................................................................................................29
2.4.1.2 Survival aSLactobàcïïhu strains after e successive passages ätrougii artificial saliva,
viittliTf j^rirf* awl fnfnfiiwl jró* .........................................................................................«..........3t
2.4.0 Resistance to 0.4 % phenol........_____...........-.........................................................................................32
2.4.2 Determination of antimicrobial potential of probiotic strains..............................32
2.4.2.1 Production of H2O,.....................................................................................................................................32
2.4.2.2 Screening for antagonistic activity.............................................................................................................32
2.4.3 Bile salt hydrolase activity...................................................................................33
2.4.4 Production of ß-galactosidase..............................................................................33
2.4.5 Growth on prebiotics............................................................................................34
2.5 Safety considerations: antibiotic resistance profiles of Lactobacillus strains..............34
2.6 Adhesion properties of selected Lactobacillus strains.................................................34
2.4.6.1 Microbial adhesion to solvents...................................................................................................................34
2.4.6.2 Aggregation................................................................................................................................................35
2.4.6.3 Coaggregation............................................................................................................................................35
2.4.6.4 Lactobacilli adhesion to HT29 cells...........................................................................................................35
2.4.6.5 Competitive exclusion: adhesion inhibition assays.....................................................................................36
2.7 Interaction oí Lactobacillus strains with intestinal epithelial cells:
activation of innate responses......................................................................................37
2.7.1 Co-culture of HT29 cells with bacteria for induction of cytokines......................37
2.7.2 Co-culture of HT29 cells with bacteria, LPS, LTA and TNF- o for
induction of cytokines.........................................................................................37
2.7.3 Co-culture of HT29 cells with bacterial cell components, supernatant
and bacteria pre-treated with proteinase K and KIO4.........................................38
2.7.3.1 Isolation of crude cell walls from selected Lactobacillus strains................................................................38
2.7.3.2 Isolation of cell walls with SDS.................................................................................................................38
2.7.3.3 Isolation of LTA.........................................................................................................................................39
2.7.3.4 Isolation of DNA........................................................................................................................................39
2.6.4 Flow cytometric detection of TLRs in non-stimulated HT29 cells
and HT29 cells stimulated with lactobacilli.........................................................40
2.6.5 Treatment of HT29 cells with lactobacilli before challenge
withiS. Typhimurium............................................................................................41
2.6.6 Treatment of HT29 cells with Ljohnsonii BFE 6128 before
challenge with S. Typhimurium components.......................................................42
2.8 Cell culture conditions and isolation of peripheral blood mononuclear cells..............42
2.8.1 Culturing of HT29 cells........................................................................................42
2.8.2 Isolation of peripheral blood mononuclear cells (PBMQ...................................43
2.8.3 Cell vitality assays................................................................................................43
2.8.3.1 LDH cytotoxiciry test.................................................................................................................................43
2.8.3.2 MTTTest............................................................... . ...........................44
2.9 Cytokme ELISA.....................................................Z......Z..Z........ ... ......................44
2.10 Transcriptomics...........................................................................................................45
2.10.1 Working with RNA..............................................................................................45
2.10.2 Isolation of RNA from HT-29 cells by a modified Single-step method...........45
2.10.3 RNA gel electrophoresis under denaturing conditions........................................46
2.10.4 One step protocol for RT-PCR.............................................................................47
2.10.5 Real-time RT-PCR...............................................................................................48
2.10.5.1 Synthesis of cDNA....................................................................................................................................«
2.10.5.2 Real tnne-FCR general protocol.................................................................................................................49
2.10.5.3 Calibration cunrcs for ral time PCR..........................................................................................................50
2.10.6 Kl2Profiler* PCR Array...................................... ...............................................52
2.10.7 Data analysis of gene expression modulation detected
with the RT2Profiler* PCR Array: the AACt method..........................................55
2.11 Statistical analysis......................................................................................................56
3 RESULTS______________________________________________________________57
3.1 Survival of lactobacilli to gastrointestinal conditions.........____.................................57
3.1.1 Effect of parameters which affect survival of probioric strains
during a simulated stomach passage....................................................................57
3.1.2 Effect of simulated gastric and intestinal passage on
the viability of lactobacilli....................................................................................59
3.1.3 Phenol resistance of Lactobacillus strains to 0.4% phenol..................................61
3.2 Selection of strains and identification..........................................................................62
3.3 Antagonism against pathogens.....................................................................................65
3.4 Bile salt hydrolase and ß-galactosidase activities........................................................67
3.5 Antibiotic Resistance....................................................................................................68
3.6 Growth of selected probiotic candidates on prebiotics.................................................70
3.6.1 Growth of L.johnsonii probiotic candidate strains on prebiotics........................70
3.6.2 Growth of L. plantarum probiotic candidate strains on prebiotics......................71
3.7 Adhesive properties......................................................................................................75
3.7.1 Microbial adhesion to solvents.............................................................................75
3.7.2 Adhesion of Lactobacillus strains to HT29 cells.................................................77
3.7.3 Competitive exclusion: effect of adherent Lactobacillus strains
on the adhesion of pathogens to HT29 cells........................................................78
3.7.4 Auto-aggregation of Lactobacillus strains...........................................................79
3.7.5 Coaggregation of Lactobacillus strains with foodborne pathogens....................80
3.8 Interaction of selected probiotic candidate strains with HT29 cells.............................83
3.8.1 HT29 cell viability in coculture experiments.......................................................83
3.8.2 Modulation of cytokine secretion in HT29 cells..................................................86
3.8.3 Identification of probiotic cellular components responsible
for IL-8 induction on HT29 cells after 24 h.........................................................87
3.8.4 TNF-a sensitises HT29 cells to probiotic lactobacilli..........................................90
3.8.5 Effect of lactobacilli on gene expression in HT29 cells.......................................91
3.8.5.1 RT-PCR results of modulation of TLR2-,TLR4-, TLR9- and HBD-2-gene expression
in HT29 cells by lactobacilli.......................................................................................................................91
3.8.5.2 Real-time RT-PCR results of modulation of TLR2-,TLR4-, TLR9- and
HBD-2-gene expression on HT29 cells by lactobacilli...............................................................................93
3.8.6 Effect of lactobacilli on the expression TLR2, TLR5 and TLR9
at protein level inHT29 cells................................................................................95
3.8.6.1 HT29 cells express TLR2, TLR5 and TLR9...............................................................................................95
3.8.6.2 Lactobacilli modulate TLR2, TLR5 and TLR9 protein expression in HT29 cells......................................97
3.9 Modulation of HT29 cell responses to S. Typhimurium trough probiotics..................99
3.9.1 Lactobacilli increased the IL-8 response of HT29 cells
to S. Typhimurium infection...............................................................................99
3.9.2 L. johnsonii BFE 6128 increased the IL-8 response
of HT29 cells to cellular components of S. Typhimurium.................................102
3.9.3 Transcriptomics: modulation of genes involved in TLR pathways...................103
4 DISCUSSION_________________________________________________________113
5 CONCLUSIONS_______________________________________________________137
5.1 Outlook.........................................................................................................................139
6 REFERENCES________________________________________________________140
ABBREVIATIONS.........................................................................................................159
LIST OF PUBLICATIONS............................................................................................160
CURRICULUM VITAE.................................................................................................161
|
| adam_txt |
CONTENTS
Dedication.i
Zusammenfassung.ii
Abstract.iii
Acknowledgements.iv
Contents.v
1 INTRODUCTION_1
1.1 Development of the human gut microbiota.1
1.2 Microbial balance.2
1.3 Probiotics.3
1.3.1 Definition .3
1.3.2 Postulated mechanisms of action.3
1.3.3 New probiotic strains and sources of isolation.5
1.3.4 Safety considerations.6
1.3.5 Site of action of probiotics: the small intestine.7
1.4 Tolerance and immunity in the gut.7
1.4.1 Bridging innate immunity and adaptive immunity.9
1.4.2 Toll-like receptors.11
1.4.3 The enterocyte as sentinel of the gut.13
1.5 Probiotics and their effect in the immune system.16
1.6 Probiotics and acute gastroenteritis.17
1.7 Study objective.18
2 MATERIALS AND METHODS_19
2.1 Culture media.19
2.2 Buffers and solutions.20
2.2.1 Buffers and solutions used in molecular biology.20
2.2.2 Buffers and solutions required for sandwich ELISA.22
2.2.3 Buffers and solutions required for FACS analysis.22
2.2.4 Other buffers and solutions.23
2.3 Isolation, selection and identification of Lactobadllus strains.24
2.3.1 Isolation of bacterial strains.24
2.3.2 Strain selection and identification.24
2.3.3 Phenotypic characterisation.
2.3.3.1 Growth at différent temperatures.24
2.3.3.2 Production of gas from glucose.24
2.3.3.3 Hydrolysis of argmine._.26
2.3.3.4 Presence of muo-diaminopinielic actd(mDAP) in the cell walls.-.26
2.3.3.5 Determination of lactic acid enantiomers produced.26
2.3.3.6 Sugar fermentation profiles._.27
2.3.4 Genotypic characterisation.27
2.3.4.1 Isolation of genomic DNA ttom bacteria._.-.27
2.3.4.2 Determination of DNA and RNA concentration and purity._.27
2.3.4.3 Rep-PCR._._.28
2.3.4.4 16 S rDNA sequence.28
2.3.4.5 Analytical scale purification of PCR products.29
2.4 Testing Lactobaciuus strains for in vitro probiotic properties.29
2.4.1 Studies on resistance to gastrointestinal conditions.29
2.4.1.1 Evaluation of parameters which affect survival of probiotic strains during
simulated stomach passage.29
2.4.1.2 Survival aSLactobàcïïhu strains after e successive passages ätrougii artificial saliva,
viittliTf j^rirf* awl fnfnfiiwl jró*".«.3t
2.4.0 Resistance to 0.4 % phenol._.-.32
2.4.2 Determination of antimicrobial potential of probiotic strains.32
2.4.2.1 Production of H2O,.32
2.4.2.2 Screening for antagonistic activity.32
2.4.3 Bile salt hydrolase activity.33
2.4.4 Production of ß-galactosidase.33
2.4.5 Growth on prebiotics.34
2.5 Safety considerations: antibiotic resistance profiles of Lactobacillus strains.34
2.6 Adhesion properties of selected Lactobacillus strains.34
2.4.6.1 Microbial adhesion to solvents.34
2.4.6.2 Aggregation.35
2.4.6.3 Coaggregation.35
2.4.6.4 Lactobacilli adhesion to HT29 cells.35
2.4.6.5 Competitive exclusion: adhesion inhibition assays.36
2.7 Interaction oí Lactobacillus strains with intestinal epithelial cells:
activation of innate responses.37
2.7.1 Co-culture of HT29 cells with bacteria for induction of cytokines.37
2.7.2 Co-culture of HT29 cells with bacteria, LPS, LTA and TNF- o for
induction of cytokines.37
2.7.3 Co-culture of HT29 cells with bacterial cell components, supernatant
and bacteria pre-treated with proteinase K and KIO4.38
2.7.3.1 Isolation of crude cell walls from selected Lactobacillus strains.38
2.7.3.2 Isolation of cell walls with SDS.38
2.7.3.3 Isolation of LTA.39
2.7.3.4 Isolation of DNA.39
2.6.4 Flow cytometric detection of TLRs in non-stimulated HT29 cells
and HT29 cells stimulated with lactobacilli.40
2.6.5 Treatment of HT29 cells with lactobacilli before challenge
withiS. Typhimurium.41
2.6.6 Treatment of HT29 cells with Ljohnsonii BFE 6128 before
challenge with S. Typhimurium components.42
2.8 Cell culture conditions and isolation of peripheral blood mononuclear cells.42
2.8.1 Culturing of HT29 cells.42
2.8.2 Isolation of peripheral blood mononuclear cells (PBMQ.43
2.8.3 Cell vitality assays.43
2.8.3.1 LDH cytotoxiciry test.43
2.8.3.2 MTTTest. . .44
2.9 Cytokme ELISA.Z.Z.Z.".'.44
2.10 Transcriptomics.45
2.10.1 Working with RNA.45
2.10.2 Isolation of RNA from HT-29 cells by a modified "Single-step" method.45
2.10.3 RNA gel electrophoresis under denaturing conditions.46
2.10.4 One step protocol for RT-PCR.47
2.10.5 Real-time RT-PCR.48
2.10.5.1 Synthesis of cDNA.«
2.10.5.2 Real tnne-FCR general protocol.49
2.10.5.3 Calibration cunrcs for ral time PCR.50
2.10.6 Kl2Profiler* PCR Array.\.52
2.10.7 Data analysis of gene expression modulation detected
with the RT2Profiler* PCR Array: the AACt method.55
2.11 Statistical analysis.56
3 RESULTS_57
3.1 Survival of lactobacilli to gastrointestinal conditions._.57
3.1.1 Effect of parameters which affect survival of probioric strains
during a simulated stomach passage.57
3.1.2 Effect of simulated gastric and intestinal passage on
the viability of lactobacilli.59
3.1.3 Phenol resistance of Lactobacillus strains to 0.4% phenol.61
3.2 Selection of strains and identification.62
3.3 Antagonism against pathogens.65
3.4 Bile salt hydrolase and ß-galactosidase activities.67
3.5 Antibiotic Resistance.68
3.6 Growth of selected probiotic candidates on prebiotics.70
3.6.1 Growth of L.johnsonii probiotic candidate strains on prebiotics.70
3.6.2 Growth of L. plantarum probiotic candidate strains on prebiotics.71
3.7 Adhesive properties.75
3.7.1 Microbial adhesion to solvents.75
3.7.2 Adhesion of Lactobacillus strains to HT29 cells.77
3.7.3 Competitive exclusion: effect of adherent Lactobacillus strains
on the adhesion of pathogens to HT29 cells.78
3.7.4 Auto-aggregation of Lactobacillus strains.79
3.7.5 Coaggregation of Lactobacillus strains with foodborne pathogens.80
3.8 Interaction of selected probiotic candidate strains with HT29 cells.83
3.8.1 HT29 cell viability in coculture experiments.83
3.8.2 Modulation of cytokine secretion in HT29 cells.86
3.8.3 Identification of probiotic cellular components responsible
for IL-8 induction on HT29 cells after 24 h.87
3.8.4 TNF-a sensitises HT29 cells to probiotic lactobacilli.90
3.8.5 Effect of lactobacilli on gene expression in HT29 cells.91
3.8.5.1 RT-PCR results of modulation of TLR2-,TLR4-, TLR9- and HBD-2-gene expression
in HT29 cells by lactobacilli.91
3.8.5.2 Real-time RT-PCR results of modulation of TLR2-,TLR4-, TLR9- and
HBD-2-gene expression on HT29 cells by lactobacilli.93
3.8.6 Effect of lactobacilli on the expression TLR2, TLR5 and TLR9
at protein level inHT29 cells.95
3.8.6.1 HT29 cells express TLR2, TLR5 and TLR9.95
3.8.6.2 Lactobacilli modulate TLR2, TLR5 and TLR9 protein expression in HT29 cells.97
3.9 Modulation of HT29 cell responses to S. Typhimurium trough probiotics.99
3.9.1 Lactobacilli increased the IL-8 response of HT29 cells
to S. Typhimurium infection.99
3.9.2 L. johnsonii BFE 6128 increased the IL-8 response
of HT29 cells to cellular components of S. Typhimurium.102
3.9.3 Transcriptomics: modulation of genes involved in TLR pathways.103
4 DISCUSSION_113
5 CONCLUSIONS_137
5.1 Outlook.139
6 REFERENCES_140
ABBREVIATIONS.159
LIST OF PUBLICATIONS.160
CURRICULUM VITAE.161 |
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| illustrated | Illustrated |
| index_date | 2024-07-02T17:16:27Z |
| indexdate | 2024-07-09T20:56:40Z |
| institution | BVB |
| isbn | 9783899634914 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-015604467 |
| oclc_num | 160074608 |
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| owner_facet | DE-19 DE-BY-UBM DE-12 |
| physical | VII, 160 S. Ill., graph. Darst. |
| publishDate | 2007 |
| publishDateSearch | 2007 |
| publishDateSort | 2007 |
| publisher | Verl. Dr. Hut |
| record_format | marc |
| series2 | Biologie |
| spelling | Vizoso Pinto, Maria Guadalupe Verfasser aut Molecular and physiological studies on the functionality of probiotic lactobacilli von Maria Guadalupe Vizoso Pinto 1. Aufl. München Verl. Dr. Hut 2007 VII, 160 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Biologie Zugl.: Karlsruhe, Univ., Diss., 2006 Probiotikum (DE-588)4338112-1 gnd rswk-swf Lactobacillus (DE-588)4166370-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Probiotikum (DE-588)4338112-1 s Lactobacillus (DE-588)4166370-6 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015604467&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Vizoso Pinto, Maria Guadalupe Molecular and physiological studies on the functionality of probiotic lactobacilli Probiotikum (DE-588)4338112-1 gnd Lactobacillus (DE-588)4166370-6 gnd |
| subject_GND | (DE-588)4338112-1 (DE-588)4166370-6 (DE-588)4113937-9 |
| title | Molecular and physiological studies on the functionality of probiotic lactobacilli |
| title_auth | Molecular and physiological studies on the functionality of probiotic lactobacilli |
| title_exact_search | Molecular and physiological studies on the functionality of probiotic lactobacilli |
| title_exact_search_txtP | Molecular and physiological studies on the functionality of probiotic lactobacilli |
| title_full | Molecular and physiological studies on the functionality of probiotic lactobacilli von Maria Guadalupe Vizoso Pinto |
| title_fullStr | Molecular and physiological studies on the functionality of probiotic lactobacilli von Maria Guadalupe Vizoso Pinto |
| title_full_unstemmed | Molecular and physiological studies on the functionality of probiotic lactobacilli von Maria Guadalupe Vizoso Pinto |
| title_short | Molecular and physiological studies on the functionality of probiotic lactobacilli |
| title_sort | molecular and physiological studies on the functionality of probiotic lactobacilli |
| topic | Probiotikum (DE-588)4338112-1 gnd Lactobacillus (DE-588)4166370-6 gnd |
| topic_facet | Probiotikum Lactobacillus Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=015604467&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT vizosopintomariaguadalupe molecularandphysiologicalstudiesonthefunctionalityofprobioticlactobacilli |