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Screening and Evaluation of Lactobacillus paracasei M9-1 against Biofilm Formation by Streptococcus mutans : Streptococcus mutans의 biofilm 형성을 억제하는 Lactobacillus paracasei M9-1의 선발 및 평가

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dc.contributor.advisor허철성-
dc.contributor.author신희성-
dc.date.accessioned2019-05-07T03:26:22Z-
dc.date.available2021-04-13T05:51:39Z-
dc.date.issued2019-02-
dc.identifier.other000000155748-
dc.identifier.urihttps://hdl.handle.net/10371/150896-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 국제농업기술대학원 국제농업기술학과, 2019. 2. 허철성.-
dc.description.abstract충치는 치아 표면에 존재하는 enamel의 demineralization에 의하여 발생한다. 구강 내의 높은 산성도는 enamel 층의 주요 구성 성분인 수산화인회석의 분해를 일으키게 된다. 구강 미생물 중에서도 충치를 유발하는 미생물 들은 구강 내에서 산성도를 높이는 여러 요인 중 하나 이다. 충치 유발 미생물들은 구강 내 잔여 탄수화물을 에너지원으로 사용하고, glucosyltransferase 라는 효소를 이용하여 바이오필름을 형성하며, 유기산들을 생성한다. 건강한 구강 내에서는 일반적으로 인간의 침에 의해 낮은 pH 환경이 중화된다. 그러나 바이오필름이 형성 될 시, 바이오필름은 인간의 침이 바이오필름 내부로 들어오는 것을 방지하고, 내부의 유기산들이 밖으로 방출되는 것을 억제한다. 결과적으로 바이오필름 내부에서는 유기산들이 갇혀 높은 산성 환경이 유지되게 된다. Streptococcus mutans는 glucosyltransferase를 생성하지 못하는 타 미생물에게 glucosyltransferase를 전달하여 해당 미생물 또한 glucan 형성이 가능하게 되어 바이오필름 형성을 가속화시키기 때문에 충치 유발 주요 원인 균으로 잘 알려져 있다. 따라서 본 연구의 목적은 충치의 주요 원인 균인 S. mutans의 바이오필름 형성을 감소시켜 충치 발생을 억제 할 수 있는 유산균 주의 스크리닝 및 평가이다. 본 연구에서는 기존에 타 미생물에 대하여 항균활성 능력이 검증 된 유산균 21 종류를 사용하였다. 안전성 검사를 실시한 결과 EFSA가 제시하는 기준에 21가지 유산균이 모두 부합한 것을 확인하였고, 이는 구강으로 섭취하였을 때, 건강에 이상이 없음을 의미한다. Lactobacillus paracaei M9-1은 실험에 사용된 유산균 중에서 가장 높은 항균 활성 능력을 보였으며 (> 14mm), reference strain인 Lactobacillus rhamnosus GG보다 더 높은 co-aggregation 능력과 가장 높은 biofilm 형성을 억제 능력을 보였다. 따라서 L. paracasei M9-1이 S. mutans의 biofilm 형성을 억제할 수 있는 잠재적인 프로바이오틱스 후보로 선발 되었다. L. paracasei M9-1이 S. mutans의 충치 유발 관련 유전자들에 미치는 영향을 평가해 본 결과, 부착 기능과 관련된 유전자들의 발현을 감소시키는 것을 확인하였다. 그러므로 본 논문은 L. paracasei M9-1이 항균활성 능력, co-aggregation 능력, S. mutans의 부착 단백질들을 발현하는 유전자들의 발현을 억제하여 S. mutans의 바이오필름 생성을 억제할 수 있기 때문에 충치를 감소시킬 수 있는 프로바이오틱스임을 확인하였다.-
dc.description.abstractDental caries is a disease caused by demineralization of enamel layer in tooth. Acidic condition in oral cavity results in destruction of hydroxyapatite, which is a major component of enamel layer. Cariogenic microorganisms in oral microbiota are one of the reasons that causes acidic condition in oral cavity. They utilize carbohydrates as their energy source and produce organic acids and glucans by using glucosyltransferases to form biofilm. In normal oral cavity, acidic condition is usually neutralized by human saliva. However, when biofilm is formed, it blocks human saliva and keeps organic acids around the enamel layer. As a result, acidic condition is maintained within biofilm. Streptococcus mutans is well known for acceleration of biofilm formation by providing glucosyltransferases (Gtfs) to other microbiota which cant produce Gtfs, thereby accelerating biofilm formation. The aim of this study is to screen and evaluate lactic acid bacteria (LAB) which has inhibitory effects against biofilm formation of S. mutans. 21 LAB, which have been proven for their antimicrobial activity against pathogenic bacteria in previous study were used in this study, The results of safety assessment showed all of tested LAB satisfied criteria from EFSA, which suggests that all of tested LAB are safe when consumed by mouth. Lactobacillus paracasei M9-1 showed highest antimicrobial activity (> 14mm), higher co-aggregation ability than that of reference strain, Lactobacillus rhamnosus GG and highest inhibition ability in biofilm formation against S. mutans among tested LAB. Therefore, L. paracasei M9-1 was selected as a promising probiotic strain which has inhibitory effects against biofilm formation of S. mutans. The effects of L. paracasei M9-1 on the caries-inducing related gene expression in S. mutans caused the reduction of adhesion related gene expression. In conclusion, L. paracasei M9-1 is the promising probiotic strain which can reduce dental caries by their antimicrobial ability to reduce cell concentration of S. mutans, co-aggregation ability to prevent S. mutans from binding to tooth surface, and inhibition ability to reduce adhesion related gene expression of S. mutans, thereby reducing biofilm formation of S. mutans.-
dc.description.tableofcontentsAbstract i
Contents iv
List of Tables viii
List of Figures ix
List of Abbreviations xii

Chapter 1. Introduction 1

Chapter 2. Review of Literature 3
2.1. Dental caries in oral cavity 3
2.1.1. Dental caries 3
2.1.2. Demineralization of hydroxyapatite 7
2.1.3. Acidic condition is maintained by cariogenic oral
microbiota 10
2.1.4. Streptococcus mutans 14
2.2. The caries-inducing factors of S. mutans 15
2.2.1. Biofilm formation 15
2.2.2. Acid tolerance response 22
2.2.3. Adhesion factors 24
2.3. Probiotics and their anti-cariogenic role 26
2.3.1. Probiotics 26
2.3.2. Benefical effects of probiotics in oral cavity 27
2.3.3. Potential cariogenicity of probiotics 30

Chapter 3. Materials and Methods 31
3.1. Screening of potential probiotics with inhibition ability
against biofilm formation by S. mutans 31
3.1.1. Bacterial strains and growth condition 31
3.1.2. Preparation of culture supernatant 33
3.1.3. Safety assessment 33
3.1.4. Antimicrobial activity test 37
3.1.5. Co-aggregation assay 37
3.1.6. Biofilm inhibition assay by LAB against S. mutans
38
3.1.7. Carbohydrate fermentative pattern and enzymatic
profiling 41
3.2. Potential cariogenic ability of selected probiotic
strains 41
3.2.1. Biofilm formation of the selected probitoic strains
41
3.2.2. The hydroxyapatite degradation 42
3.3. Inhibitory effects of selected probiotic strains on the
gene expression in S. mutans 42
3.3.1. Extraction of total RNA 42
3.3.2. Quantitative real-time polyermerase chain reaction
(qRT-PCR) and data analysis 44

Chapter 4. Results 48
4.1. Screening of potential probiotics with inhibition
ability against biofilm formation by S. mutans 48
4.1.1. Safety assessment 48
4.1.2. Antimicrobial activitiy 52
4.1.3. Co-aggregation ability with S. mutans 54
4.1.4. Biofilm inhibition ability against S. mutans 56
4.1.5. Selection of potential probiotics 60
4.1.6. Carbohydrate fermentative pattern and enzymatic
profiling 60
4.2. Potential cariogenic ability of selected probiotic
strains 62
4.2.1. Biofilm formation of selected probiotic strains 62
4.2.2. Degradation of hydroxyapatite 64
4.3. Inhibitory effects of probiotic strains on the gene
expressions in S. mutans 67
4.3.1. Alteration in biofilm formation associated
gene expression 67
4.3.2. Alteration in acid tolerance associated gene
expression 74
4.3.3. Alteration in adhesion associated gene
expression 78

Chapter 5. Discussion 82

References 99

Abstract in Korean 121

List of Tables

Table 1. The summary of probiotics and their actions in oral
cavity 29
Table 2. List of bacterial strains used in this study 32
Table 3. Composition (%) of decarboxylase medium 34
Table 4. List of primers used in this study 46
Table 5. Minimum inhibitory concentration (ug/ml) of
antibiotics to LAB 49
Table 6. The antimicrobial activity of LAB against S. mutans
ATCC25175 53


List of Figures

Figure 1. The global prevalence of dental caries in 12 years- old children 4
Figure 2. Structure of normal tooth 6
Figure 3. The demineralization of tooth under low pH condition 9
Figure 4. The biofilm formation by cariogenic oral microbiota 13
Figure 5. Synthesis of water-insoluble glucan by
glucosyltransferase B in S. mutans 16
Figure 6. Model of glucan-mediated bacterial adherence 18
Figure 7. Schematic representation of two component signal
transduction system VicKR 21
Figure 8. Competence stimulating peptide and role of comCDE
system 23
Figure 9. Proposed role of acid tolerance system in S. mutans
23
Figure 10. The role of srtA in Gram-positive bacteria 25
Figure 11. The schematic diagram of safety assessment 36
Figure 12. Time schedule for the (a) prevention and (b)
inhibition assays of biofilm formation by S. mutans
40
Figure 13. Co-aggregation ability of LAB with S. mutans (a)
ATCC 25175, and (b) ATCC700610 55
Figure 14. Inhibitory effects of LAB on biofilm-forming
S. mutans (a) ATCC25175, and (b) ATCC700610
58
Figure 15. Inhibitory effects of LAB on preformed biofilm of
S. mutans (a) ATCC25175, and (b) ATCC700610
59
Figure 16. Heatmap of carbohydrates fermentative pattern and
enzymatic profiling of selected probiotic strains and
S. mutans strains 61
Figure 17. Biofilm formation ability of selected probiotic strains
63
Figure 18. The calcium releasing ability of S. mutans and
selected probiotic strains 66
Figure 19. Effects of probiotics on expression of biofilm-
associated genes in biofilm-forming S. mutans 69

Figure 20. Effects of probiotics on expression of sacB, vicK,
and vicR genes in biofilm-forming S. mutans (a)
ATCC25175, and (b) ATCC700610 70
Figure 21. Effects of probiotics on expression of biofilm-
associated genes in planktonic S. mutans 72
Figure 22. Effects of probiotics on expression of sacB, vicK,
and vicR genes in planktonic S. mutans (a) ATCC
25175, and (b) ATCC700610 73
Figure 23. Effects of probiotics on expression of acid tolerance
genes in biofilm-forming S. mutans (a) ATCC
25175, and (b) ATCC700610 75
Figure 24. Effects of probiotics on expression of acid tolerance
genes in planktonic S. mutans (a) ATCC 25175, and
(b) ATCC700610 77
Figure 25. Effects of probiotics on expression of adhesion
genes in biofilm-forming S. mutans (a) ATCC
25175, and (b) ATCC700610 79
Figure 26. Effects of probiotics on expression of adhesion
genes in planktonic S. mutans (a) ATCC 25175, and
(b) ATCC700610 81		-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc631-
dc.titleScreening and Evaluation of Lactobacillus paracasei M9-1 against Biofilm Formation by Streptococcus mutans-
dc.title.alternativeStreptococcus mutans의 biofilm 형성을 억제하는 Lactobacillus paracasei M9-1의 선발 및 평가-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorHeesung Shin-
dc.description.degreeMaster-
dc.contributor.affiliation국제농업기술대학원 국제농업기술학과-
dc.date.awarded2019-02-
dc.identifier.uciI804:11032-000000155748-
dc.identifier.holdings000000000026▲000000000039▲000000155748▲-
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