Immune Enhancement and Vaginal Health Promotion through Anti-Biofilm and Anti-Inflammatory Activities of Kimchi-Derived Lactic Acid Bacteria = 김치 유래 유산균의 면역증진 효과 및 항바이오필름과 항염증 활성을 통한 질 건강 개선 효과

다국어 초록 (Multilingual Abstract)

Probiotics are live microorganisms that confer health benefits to the host when administered in adequate amounts. Among them, lactic acid bacteria (LAB) constitute a major group with diverse biological activities, including anti-inflammatory, antioxidant, anticancer, anti-obesity, and neuroprotective effects. This study aimed to isolate novel LAB strains from the traditional Korean fermented food, kimchi and to evaluate their potential in immune enhancement and vaginal health promotion.
Firstly, the probiotic potential of Lactiplantibacillus plantarum WB4304, WB4305, and Levilactobacillus brevis WB4306, was assessed based on their resistance to simulated gastrointestinal conditions, adhesion to intestinal epithelial cells, cell surface hydrophobicity, and autoaggregation. In addition, their safety was confirmed through evaluations of hemolytic activity, bile salt hydrolase activity, and antibiotic susceptibility. Heat-treated LAB strains were subsequently analyzed for antioxidant and immunomodulatory activities in RAW 264.7 macrophages. These paraprobiotics exhibited strong free radical-scavenging and phagocytic activities. They also markedly enhanced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines—including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)—via activation of the nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. Among them, L. plantarum WB4304 and WB4305 induced the most pronounced immunostimulatory effects.
Secondly, regarding vaginal health, the dual functionality of L. plantarum WB4305 in inhibiting biofilm formation by vaginal pathogens and modulating inflammation in HeLa cells was investigated. Six Lactobacillus strains isolated from kimchi were initially screened for anti-biofilm activity against Escherichia coli O157, Staphylococcus aureus, Streptococcus agalactiae, E. coli, and Candida albicans using crystal violet assays and nitric oxide inhibition assays. Among them, L. plantarum WB4305 exhibited the highest efficacy. Its cell-free supernatants reduced insoluble extracellular polysaccharide production in all tested pathogens, disrupted biofilm structure, and prevented pathogen adhesion, as observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). In a lipopolysaccharide (LPS)-stimulated HeLa cell model, L. plantarum WB4305 suppressed NF-κB and MAPK activation, resulting in lower expression of pro-inflammatory cytokines.
These results demonstrate that L. plantarum WB4304, WB4305, and L. brevis WB4306 exhibited notable probiotic potential, along with antioxidant and immune-enhancing activities. In particular, L. plantarum WB4305 showed marked antibiofilm and anti-inflammatory effects, supporting its potential as a multifunctional probiotic candidate for both immune support and vaginal health.

국문 초록 (Abstract)

프로바이오틱스는 적절한 양을 섭취하였을 때 숙주에게 건강상의 이로움을 주는 살아있는 미생물로, 유산균은 그 대표적인 집단으로 다양한 생리활성을 지닌다. 항염증, 항산화, 항암, 항비만 및 신경보호 효과 등이 보고된 바 있으며, 본 연구에서는 전통 발효식품인 김치로부터 신규 유산균 균주를 분리하여 면역 증진 및 질 건강 개선에 대한 기능성을 평가하고자 하였다. 첫째, Lactiplantibacillus plantarum WB4304, WB4305 및 Levilactobacillus brevis WB4306 균주의 위장관 모의 조건 내 생존력, 장세포 부착 능력, 세포 표면 소수성 및 자가응집성을 평가하여 프로바이오틱 특성을 확인하였다. 또한, 용혈 활성, 담즙산염 가수분해효소 활성 및 항생제 감수성 시험을 통해 안전성을 검증하였다. 이어 열처리한 유산균을 RAW 264.7 대식세포에 처리하여 항산화 및 면역조절 활성을 분석하였다. 파라프로바이오틱스는 높은 자유라디칼 소거능과 식세포 활성화과 더불어 inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α) 등 염증 관련 인자의 발현을 유의적으로 증가시켰다. 이는 nuclear factor-kappa B (NF-κB) 및 mitogen-activated protein kinase (MAPK) 경로의 활성화를 통해 매개됨을 확인하였다. 특히 L. plantarum WB4304 와 WB4305 에서 가장 뚜렷한 면역 증진 효과가 관찰되었다. 둘째, 질 건강 관련 기능성을 검증하기 위해 L. plantarum WB4305 의 항바이오필름 및 항염증 활성을 분석하였다. 김치 유래 6 종의 유산균을 대상으로 Escherichia coli O157, Staphylococcus aureus, Streptococcus agalactiae, E. coli, Candida albicans 에 대한 항바이오필름 활성을 선별한 결과, L. plantarum WB4305 가 가장 우수한 활성을 나타냈다. 이 균주의 세포상등액은 모든 병원체에서 불용성 세포외다당류 생성을 억제하였고, 주사전자현미경(SEM)과 공초점레이저주사현미경(CLSM) 분석을 통해 병원체의 부착과 생물막 구조 형성을 저해하는 것으로 확인하였다. 또한, lipopolysaccharide (LPS)로 염증을 유도한 HeLa 세포 모델에서 NF-κB 와 MAPK 경로의 활성이 억제되면서 염증성 사이토카인의 발현이 감소하였다. 종합적으로, L. plantarum WB4304, WB4305 및 L. brevis WB4306 은 프로바이오틱 특성과 더불어 항산화 및 면역 증진 활성을 보였다. 특히 L. plantarum WB4305 는 항바이오필름 및 항염증 효과를 동시에 나타내어 면역 증진과 질 건강 개선을 위한 다기능성 프로바이오틱 후보로서의 가능성을 제시하였다.

목차 (Table of Contents)

• List of Tables ⅲ
• List of Figures ⅳ
• Abstract ⅴ
• Chapter 1. Immunoenhancing effects of Lactobacillus strains through intracellular
• signaling modulation in RAW 264.7 cells 1
• 1.1. Introduction 1
• 1.2. Materials and methods 4
• 1.2.1. Bacterial strains and sample preparation 4
• 1.2.2. Cell culture 4
• 1.2.3. Evaluation of probiotics properties 5
• 1.2.3.1. Tolerance to artificial gastric juice and bile salts 5
• 1.2.3.2. Adhesion ability to HT-29 cells 5
• 1.2.3.3 Cell surface hydrophobicity and auto-aggregation 6
• 1.2.4. Safety assessment 7
• 1.2.4.1. Hemolytic and bile salt hydrolase activity 7
• 1.2.4.2. Antibiotic resistance 7
• 1.2.5. Antioxidant assay 8
• 1.2.6. Cell cytotoxicity assay 8
• 1.2.7. Quantification of NO production 9
• 1.2.8. Phagocytic uptake assay 9
• 1.2.9. Quantitative real-time polymerase chain reaction (qRT-PCR) 10
• 1.2.10. Western blot analysis 12
• 1.2.11. Statistical analysis 12
• 1.3. Results and discussion 13
• 1.3.1. Probiotic properties of LAB strains 13
• 1.3.2. Safety evaluation of LAB strains 16
• 1.3.3. Antioxidant activity of LAB strains 18
• 1.3.4. Cell cytotoxicity and NO production 20
• 1.3.5. Phagocytic activity of LAB strains 22
• 1.3.6. Effect of LAB strains on pro-inflammatory cytokines 24
• 1.3.7. Activation of NF-κB signaling pathway 26
• 1.3.8. Activation of MAPK signaling pathway 28
• 1.3.9. Activation of iNOS and COX-2 expression 30
• 1.4. Conclusion 32
• Chapter 2. Anti-biofilm and anti-inflammatory activities of Lactiplantibacillus
• plantarum WB4305 in vaginal health models 33
• 2.1. Introduction 33
• 2.2. Materials and methods 36
• 2.2.1. Bacterial strains and probiotic sample preparation 36
• 2.2.2. Antimicrobial assay 36
• 2.2.3. Biofilm formation assay 37
• 2.2.4. EPS quantification 38
• 2.2.5. Scanning electron microscopy (SEM) 38
• 2.2.6. Confocal laser scanning microscopy (CLSM) 39
• 2.2.7. Evaluation of anti-inflammatory effects 39
• 2.2.7.1. Bacterial strains and sample preparation 39
• 2.2.7.2. Cell culture condition 39
• 2.2.7.3 Cell cytotoxicity assay 40
• 2.2.7.4. Quantification of NO production 40
• 2.2.7.5. Quantitative real-time polymerase chain reaction (qRT-PCR) 40
• 2.2.7.6 Western blot analysis 43
• 2.2.8. Statistical analysis 43
• 2.3. Results and discussion 44
• 2.3.1. Screening of probiotic strains for vaginal health 44
• 2.3.2. Minimum inhibitory concentration (MIC) 44
• 2.3.3. Biofilm inhibition and degradation effects of CFS 46
• 2.3.4. Anti-inflammatory effect of LAB strains based on NO inhibition 49
• 2.3.5 Effects of CFS on EPS production 51
• 2.3.6 Visualization of biofilm disruption by CFS 53
• 2.3.6.1. Scanning electron microscopy (SEM) 53
• 2.3.6.2. Confocal laser scanning microscopy (CLSM) 55
• 2.3.7. Suppression of pro-inflammatory cytokines 58
• 2.3.8. Inhibition of NF-κB and MAPK signaling pathways 60
• 2.4. Conclusion 63
• References 64
• Abstract (in Korean) 77