Anti-Adipogenic and Intestinal Health- Promoting Effects of Lactiplantibacillus plantarum strains Isolated from Kimchi = 김치 분리 프로바이오틱 유산균의 항비만 및 장 건강 개선 효과

다국어 초록 (Multilingual Abstract)

Probiotics, beneficial microorganisms derived from fermented foods, have attracted increasing attention for their multifaceted health-promoting effects, including anti- obesity, anti-inflammatory, antimicrobial, and barrier-protective activities. The purpose of this study was to evaluate Lactiplantibacillus plantarum WB4201, WB4202, and WB4203, isolated from kimchi, for their potential as probiotic and paraprobiotic candidates. Firstly, L. plantarum WB4201, WB4202, and WB4203 were characterized for fundamental probiotic properties. All strains exhibited tolerance to simulated gastrointestinal conditions, including acidic and bile environments, and demonstrated strong adhesion ability to intestinal epithelial cells. Safety was confirmed through hemolytic activity on blood agar, bile salt hydrolase activity assays, and antibiotic susceptibility profiling, which indicated no harmful traits. Functionally, their heat-killed forms were assessed in 3T3-L1 adipocytes and found to suppress lipid accumulation. Mechanistically, heat-killed strains downregulated adipogenesis-related transcription factors (PPAR-γ, C/EBP-α, and AKT2) and lipogenic regulators (SREBP-1c, FAS, ACC1, and GPAT), while modulating fatty acid metabolism by reducing CD36 and enhancing CPT1 expression. These findings suggest that the heat-killed forms exert anti- obesity effects through the coordinated regulation of adipogenesis, lipogenesis, and fatty acid β-oxidation. Secondly, L. plantarum WB4201, WB4202, and WB4203 demonstrated intestinal health-promoting functions. The strains exhibited antimicrobial and anti-adhesion activities against pathogenic bacteria, limiting colonization on the epithelial surface. In LPS-induced HT-29 cells, treatment with the strains downregulated pro-inflammatory cytokines (IL-8, IL-1β, TNF-α, COX-2, and iNOS) while enhancing the expression of tight junction proteins (Occludin, Claudin-1, and ZO-1) and MUC2, thereby reinforcing epithelial barrier integrity. Western blot analysis further confirmed that these effects involved the inhibition of phosphorylation of NF-κB (p65 and IκBα) and MAPK (p38, ERK1/2, and JNK). The findings of this study revealed that heat-killed forms of L. plantarum WB4201, WB4202, and WB4203 showed paraprobiotic potential by exerting anti-obesity effects in adipocyte models. In addition, live forms possess probiotic characteristics, including antimicrobial, anti-inflammatory, and barrier-protective activities in intestinal models. Collectively, these results highlight the dual functionality of these kimchi-derived strains for improving intestinal health and metabolic regulation.

국문 초록 (Abstract)

프로바이오틱스는 발효 식품에서 유래한 유익한 미생물로, 항비만, 항염증, 항균 및 장 장벽 보호 등 다양한 건강 증진 효과가 보고되며 큰 주목을 받고 있다. 본 연구의 목적은 김치에서 분리한 Lactiplantibacillus plantarum WB4201, WB4202, WB4203의 기능성을 평가하고, 이들이 프로바이오틱스와 파라프로바이오틱스로서의 가능성을 확인하는 것이었다. 첫째, L. plantarum WB4201, WB4202, WB4203의 기본적인 프로바이오틱 특성을 평가하였다. 모든 균주는 위산과 담즙을 포함한 모의 위장관 조건에서 우수한 내성을 보였으며, 장 상피세포에 대한 강한 부착능을 나타냈다. 안전성은 혈액한천 배지를 이용한 용혈 활성, 담즙염 가수분해 활성, 항생제 감수성 평가를 통해 확인되었으며, 유해한 특성은 나타나지 않았다. 기능적으로, 열처리 사균체를 3T3-L1 지방세포에 처리했을 때 지방 축적이 억제되었다. 기전적으로 열처리 균주는 지방세포 분화 관련 전사인자(PPAR-γ, C/EBP-α, AKT2)와 지질합성 관련 인자(SREBP-1c, FAS, ACC1, GPAT)의 발현을 억제하는 동시에, 지방산 수송 단백질 CD36을 감소시키고 β-산화 효소 CPT1의 발현을 증가시켜, 지방합성 억제와 지방산 산화 촉진을 통한 항비만 효과를 나타냈다. 둘째, L. plantarum WB4201, WB4202, WB4203은 장 건강 증진 효과를 나타내었다. 모든 균주는 병원성 세균에 대해 항균 및 부착 저해 활성을 나타내어 장 상피 표면에서의 병원균 정착을 억제하였다. 또한 LPS로 자극된 HT-29 세포에서 염증성 사이토카인(IL-8, IL-1β, IL-6, TNF-α, COX-2)의 발현을 억제하고, 밀착연접 단백질(Occludin, Claudin-1, ZO-1)과 MUC2의 발현을 증가시켜 장 장벽의 무결성을 강화하였다. 웨스턴 블롯 분석 결과, 이러한 효과는 NF-κB 경로(p65, IκBα)와 MAPK 경로(p38, ERK1/2, JNK)의 인산화 억제를 통해 매개됨이 확인되었다. 본 연구 결과, L. plantarum WB4201, WB4202, WB4203의 사균체는 지방세포 모델에서 항비만 효과를 나타내어 파라프로바이오틱으로서의 잠재력을 입증하였다. 또한 생균체는 장 모델에서 항균, 항염증, 장 장벽 보호 효과를 포함한 프로바이오틱 특성을 보유하고 있음을 확인하였다. 종합적으로, 이들 김치 유래 균주는 장 건강 개선과 대사 조절을 동시에 기대할 수 있는 이중 기능성을 지닌 유망한 후보임을 시사한다.

목차 (Table of Contents)

• List of Tables ⅲ
• List of Figures ⅳ
• Abstract ⅴ
• Chapter 1. Anti-adipogenic effects of heat-killed Lactiplantibacillus plantarum
• WB4201, WB4202, and WB4203 on 3T3-L1 adipocytes 1
• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 4
• 1.2.1. Bacterial strains and sample preparation 4
• 1.2.2. Evaluation of probiotic characteristics 4
• 1.2.2.1. Tolerance to gastrointestinal tract condition 4
• 1.2.2.2. Adhesion to HT-29 cells 5
• 1.2.3. Safety evaluation 6
• 1.2.3.1. Hemolytic and bile salt hydrolase activity 6
• 1.2.3.2. Antibiotic susceptibility 6
• 1.2.4. Cell culture and differentiation of 3T3-L1 adipocytes 7
• 1.2.5. Cell viability 7
• 1.2.6. Oil red O staining and triglyceride quantification 8
• 1.2.7. Quantitative real-time PCR (qRT-PCR) 8
• 1.2.8. Western blot analysis 11
• 1.2.9. Statistical analysis 11
• Ⅲ. Results and discussion 12
• 1.3.1. Probiotic characteristics of isolated LAB strains 12
• 1.3.2. Safety evaluation of isolated LAB strains 15
• 1.3.3. Cell viability of heat-killed LAB strains 17
• 1.3.4. Anti-adipogenic effects of heat-killed LAB strains 19
• 1.3.5. Suppression of lipid metabolism-related gene expression 21
• 1.3.6. Protein expression related to adipogenesis using Western blot analysis 26
• Ⅳ. Conclusion 28
• Chapter 2. Intestinal protective effects of Lactiplantibacillus plantarum strains on
• inflammation and tight junction regulation in HT-29 cells 29
• Ⅰ. Introduction 29
• Ⅱ. Materials and methods 32
• 2.2.1. Bacterial strains and sample preparation 32
• 2.2.2. Antimicrobial activity 32
• 2.2.3. Anti-adhesion of pathogenic strains to intestinal cells 33
• 2.2.4. Cell culture 34
• 2.2.5. Cell viability and quantification of NO production 34
• 2.2.6. Quantitative real-time PCR (qRT-PCR) 35
• 2.2.7. Western blot analysis 37
• 2.2.8. Statistical analysis 37
• Ⅲ. Results and discussion 39
• 2.3.1. Antimicrobial and anti-adhesion activity of LAB strains 39
• 2.3.2. Cell viability and NO production in SNP-stimulated HT-29 cells 43
• 2.3.3. Downregulation of pro-inflammatory cytokines gene expression 45
• 2.3.4. Upregulation of tight junction and mucin-related gene expression 47
• 2.3.5. Regulation effect on the NF-κB signaling pathway 50
• 2.3.6. Regulation effect on the MAPK signaling pathway 52
• Ⅳ. Conclusion 54
• References 55
• Abstract (in Korean) 66