Title Page
Contents
ABSTRACT 20
I. INTRODUCTION 23
II. LITERATURE REVIEW 26
1. Periodontitis and systemic disease 26
1.1. Definition of periodontitis 26
1.2. Correlation of periodontitis and systemic disease 27
1.3. Treatment of periodontitis 28
2. Probiotics-mediated bioconversion 29
2.1. Probiotics 29
2.2. Microbial bioconversion 30
2.3. Probiotics-mediated bioconversion for periodontitis treatment 31
3. Artemisia herba-alba 32
4. Gut microbiome and periodontitis 33
III. MATERIALS AND METHODS 35
1. Selection of lactic acid bacteria 35
1.1. Preparation of probiotics 35
1.2. Hemolytic analysis 36
1.3. β-glucosidase and β-glucuronidase activities 36
1.4. Acid and bile salt tolerance 37
1.5. Antioxidant activity 38
2. Preparation of Artemisia herba-alba extracts 40
2.1. Extracts of Artemisia herba-alba 40
2.2. Minimum bactericidal concentration (MBC) of Artemisia herba-alba against periodontal pathogens 41
2.3. Growth of the lactic acid bacteria in the presence of Artemisia herba-alba extract 42
2.4. Antimicrobial effects of bioconversion broths against periodontal pathogens 43
2.5. Anti-biofilm activity of bioconversion broth against Porphyromonas gingivalis 44
2.6. Lipase inhibitory activity of bioconversion broth 45
2.7. α-glucosidase inhibitory activity of bioconversion broth 46
3. Preparation of bioconversion of milk 47
3.1. Production of bioconversion of milk containing Artemisia herba-alba extract by probiotic candidate strain 47
3.2. Antimicrobial effects of bioconversion of milk against periodontal pathogens 48
3.3. Lipase inhibitory activity of bioconversion of milk 49
3.4. α-glucosidase inhibitory activity of bioconversion of milk 50
3.5. Determination of total phenol and flavonoid contents in bioconversion of milk 50
3.6. Cytotoxicity of bioconversion of milk 51
4. Whole-genome and morphological characteristic analysis of novel probiotics 53
4.1. Whole-genome sequencing 53
4.2. Scanning electron microscope (SEM) imaging 55
5. Efficacy analysis of bioconversion of milk on periodontitis in rat model 57
5.1. Development of periodontitis-induced rat model 57
5.2. Treatment of bioconversion of milk 59
5.3. Micro-CT analysis for periodontal tissue 59
5.4. Histopathology of periodontal tissue 61
5.5. Serum biochemistry analysis 61
5.6. Transcriptomic analysis 63
5.7. Quantification of protein expression 68
5.8. Gut microbiome analysis 71
6. Statistical analysis 72
IV. RESULTS AND DISCUSSION 73
1. Selection of lactic acid bacteria 73
1.1. Hemolytic analysis 73
1.2. β-glucosidase and β-glucuronidase activities 73
1.3. Acid and bile salt tolerance 74
1.4. Antioxidant activity 77
2. Preparation of Artemisia herba-alba extracts 82
2.1. Minimum bactericidal concentration (MBC) of Artemisia herba-alba against periodontal pathogens 82
2.2. Growth of the lactic acid bacteria in the presence of Artemisia herba-alba extract 86
2.3. Antimicrobial effects of bioconversion broths against periodontal pathogens 89
2.4. Anti-biofilm acitivity of bioconversion broths against Porphyromonas gingivalis 93
2.5. Lipase inhibitory activity of bioconversion broth 95
2.6. α-glucosidase inhibitory activity of bioconversion broth 97
3. Preparation and in vitro evaluation of bioconversion of milk 100
3.1. Preparation of bioconversion of milk 100
3.2. Antimicrobial effects of bioconversion of milk against periodontal pathogens 102
3.3. Lipase inhibitory activity of bioconversion of milk 104
3.4. α-glucosidase inhibitory activity of bioconversion of milk 106
3.5. Total phenol and flavonoid contents of bioconversion of milk 108
3.6. Cytotoxicity of bioconversion of milk 111
4. Whole-genome and morphological characteristics of probiotics 113
4.1. Whole-genome of probiotics 113
4.2. Cell morphology of probiotics 124
5. Efficacy of bioconversion of milk on periodontitis in rat model 130
5.1. Weight change of rats during bioconversion of milk treatment 130
5.2. Micro-CT results for periodontal tissue 132
5.3. Histopathology results of periodontal tissue 136
5.4. Measurement of biochemical parameters in serum 139
5.5. Gene expression levels in oral cavity 147
5.6. Gene expression levels in gut 154
5.7. Gene expression levels in liver 159
5.8. Protein expression levels in oral cavity 164
5.9. Protein expression levels in gut 168
5.10. Protein expression levels in liver 172
5.11. Changes in gut microbiome 175
V. SUMMARY AND CONCLUSION 184
VI. REFERENCES 190
ABSTRACT IN KOREAN 214
Table 1. Grouping of experimental animals for bioconversion of... 58
Table 2. Histomorphology scoring for jawbone in rats 62
Table 3. Primers for quantitative real-time transcription polymerase chain reaction (qRT-PCR) 65
Table 4. Primary antibody information used in this study 70
Table 5. Minimum bactericidal concentration of Artemisia... 84
Table 6. Specific growth rate of lactic acid bacteria in MRS broth... 87
Table 7. Generation time of lactic acid bacteria in MRS broth... 88
Table 8. Antimicrobial effect of bioconversion broths against... 90
Table 9. Antimicrobial effect of bioconversion broths against... 91
Table 10. Anti-biofilm effect of bioconversion broths against... 94
Table 11. Comparison of lipase inhibitory activity on treatment with... 96
Table 12. Comparison of α-glucosidase inhibitory activity on... 98
Table 13. pH and lactic acid bacteria cell count of the bioconversion of milk by fermentation time 101
Table 14. Antimicrobial effect of bioconversion of milk against... 103
Table 15. De novo assembly for Lactobacillus plantarum... 114
Table 16. Description of the best hit results matches for Lactobacillus plantarum SMFM2016-RK contig 1 using... 115
Table 17. Description of the best hit results matches for Lactobacillus plantarum SMFM2016-RK contig 2 using... 116
Table 18. Description of the best hit results matches for Lactobacillus plantarum SMFM2016-RK contig 3 using... 117
Table 19. Gene ontology by gene prediction of Lactobacillus... 121
Table 20. Distribution of predicted open reading frames (ORFs) over... 123
Table 21. Chromosomal comparison among Lactobacillus plantarum strains registered at NCBI database 125
Table 22. Trabecular resorption volume and cementum-enamel... 133
Table 23. Histopathology results after a 6-week animal experiment for the bioconversion of milk effects on periodontitis 137
Table 24. Taxonomy abundance ratio of phylum level for gut microbiome after bioconversion of milk... 176
Table 25. Taxonomy abundance ratio of species level of Actinobacteria for gut microbiome after bioconversion... 179
Figure 1. Experimental design of animal experiment for the effect of bioconversion of milk on periodontitis. 60
Figure 2. Acid tolerance of the lactic acid bacteria isolates. 75
Figure 3. Bile salt tolerance of the lactic acid bacteria isolates. 76
Figure 4. 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS)... 78
Figure 5. Superoxide dismutase (SOD)-like activity of the lactic... 80
Figure 6. Bacterial complexes as described by Socransky et al. (1998). 83
Figure 7. Comparison of lipase inhibitory activity on treatment... 105
Figure 8. Comparison of α-glucosidase inhibitory activity on... 107
Figure 9. Total phenolic contents of bioconversion of milk. 109
Figure 10. Total flavonoid contents of bioconversion of milk. 110
Figure 11. Cell viability of HT-29 cells influenced by... 112
Figure 12. Chromosomal genome properties of Lactobacillus... 119
Figure 13. Gene ontology classification of transcriptome for... 120
Figure 14. The average nucleotide identity (ANI) of Lactobacillus plantarum SMFM2016-RK. 126
Figure 15. Unweighted pair group method with arithmetic mean (UPGMA) tree by average nucleotide identity... 127
Figure 16. Comparison of genome annoation between Lactobacillus plantarum SMFM2016-RK and L. plantarum E1... 128
Figure 17. Morphological characteristic of Lactobacillus plantarum... 129
Figure 18. Weight change of rats during animal experiment for the... 131
Figure 19. Trabecular resorption volumes for the mandible first... 134
Figure 20. Histopathological analysis for the mandible first molar... 138
Figure 21. Serum level of alanine amino-transferase (A) and... 140
Figure 22. Serum glucose level (A) and insulin level (B) in the... 141
Figure 23. Serum total cholesterol level (A), HDL-cholesterol level (B), and LDL-cholesterol level (C) in the rats... 144
Figure 24. Serum triglyceride level (A) and adiponectin level (B) in the rats after treatment of bioconversion of... 145
Figure 25. Serum high sensitivity C-reative proteion (hsCRP) level... 146
Figure 26. Relative gene expression level of inflammatory cytokines (A, tumor necrosis-α; B, intreleukin-1β) in... 148
Figure 27. Relative gene expression level of inflammatory cytokines (A, interluekin-6; B, intreleukin-10) in gingiva... 149
Figure 28. Ratio of inflammatory cytokines in gingiva after treatment of bioconversion of milk for 6 weeks. 150
Figure 29. Relative gene expression level of extracellular matrix destruction related molecules (A, matrix... 151
Figure 30. Relative gene expression level of inflammatory cytokines (A, tumor necrosis-α; B, intreleukin-1β) in colon after... 155
Figure 31. Relative gene expression level of inflammatory cytokines (A, interluekin-6; B, intreleukin-10) in gingiva... 156
Figure 32. Relative gene expression level of tight junction related molecules (A, occludin; B, zonula occludens-1)... 158
Figure 33. Relative gene expression level of inflammatory cytokines (A, tumor necrosis-α; B, intreleukin-1β; C,... 160
Figure 34. Relative gene expression level of macrophage attraction chemokines (A, monocyte chemoattractant... 161
Figure 35. Relative gene expression level of glucose-6-phosphatase... 163
Figure 36. The immunoreactive intensity of tumor necrosis factor-α (A), interleukin-6 (B), and interleukin-10 (C)... 166
Figure 37. The immunoreactive intensity of superoxide dismutase 1 (A), glutathione peroxidase 1 (B), and catalase... 167
Figure 38. The immunoreactive intensity of tumor necrosis factor-α (A), interleukin-6 (B), and interleukin-10 (C)... 170
Figure 39. The immunoreactive intensity of occludin of colon in... 171
Figure 40. The immunoreactive intensity of tumor necrosis factor-α (A), interleukin-6 (B), and interleukin-10 (C)... 173
Figure 41. The immunoreactive intensity of glucose-6-phophatase... 174
Figure 42. α-diversity of gut microbiome by inverse Simpson... 181
Figure 43. PCoA analysis of gut microbiome after bioconversion of... 182