Protective and Anti-Inflammatory Effects of Lactobacillus johnsonii JNU3402 in Gastric Injury and Colitis Models = 위점막 손상 및 대장염 모델에서 Lactobacillus johnsonii JNU3402의 점막 보호 활성과 염증 반응 조절 효과

다국어 초록 (Multilingual Abstract)

This study aimed to evaluate the anti-inflammatory activity and industrial applicability of Lactobacillus johnsonii JNU3402 (JNU3402), a strain previously reported to exert body weight–regulating effects, by establishing its functional efficacy and large-scale production conditions. To this end, a comprehensive safety assessment based on whole-genome sequencing was conducted, including analyses of antibiotic resistance and hemolytic activity, followed by in vivo evaluation of its anti-inflammatory effects. Gastric and intestinal protective activities were assessed using an HCl/ethanol-induced gastritis model and a dextran sulfate sodium (DSS)–induced colitis model, respectively. In addition, freeze-drying protectant optimization using statistical mixture design was performed to support industrial production. JNU3402 exhibited strong acid tolerance and high adhesion capacity to HT-29 intestinal epithelial cells compared with commercially available probiotic strains. Whole-genome analysis revealed that JNU3402 harbors key functional genes associated with stress tolerance (trxA/B, tpx, groES, dnaK/J), immunomodulation (dltA–D, lspA), and adhesion (comGA/GC/GF). Although the tetracycline resistance gene tet(W) was detected, no transferable resistance elements were identified, and no hemolytic activity was observed, supporting the strain’s safety. In the gastritis model, JNU3402 alleviated gastric epithelial damage by enhancing MUC5AC expression and suppressing pro-inflammatory cytokine production. In the DSS-induced colitis model, JNU3402 significantly reduced disease activity, prevented DSS-induced colon shortening, and attenuated epithelial barrier disruption and mucus layer deterioration. These protective effects were accompanied by increased expression of the tight junction proteins ZO-1 and Claudin-1, indicating reinforcement of intestinal barrier integrity. Furthermore, tissue and serum analyses demonstrated increased IL-10 levels alongside reduced TNF-α and IL-6 levels, suggesting effective attenuation of inflammatory responses and restoration of intestinal mucosal homeostasis. JNU3402 possesses a thioredoxin-based antioxidant system (trxA–trxB–tpx), which may contribute to reactive oxygen species detoxification and modulation of NF-κB signaling, thereby providing a mechanistic basis for the observed mitigation of oxidative epithelial damage under inflammatory and oxidative stress conditions. These molecular characteristics partially explain the strain’s anti-inflammatory and cytoprotective effects. Optimization of freeze-drying protectants was conducted using a statistical mixture design in which the proportions of three components were varied while maintaining a constant total composition, with viable cell count as the response variable. The optimal formulation (MSG 0.01–trehalose 0.01–whey powder 0.98) maximized cell viability and resulted in high post-lyophilization survival. Storage stability evaluation revealed distinct temperature-dependent viability trends: viability decreased most markedly under room-temperature storage, showed a more gradual decline under refrigerated conditions, and remained highly stable under frozen storage. Consistent long-term stability at low temperatures, together with the achievement of high viable counts at pilot-scale fermentation, supports the feasibility of large-scale production of JNU3402. In conclusion, JNU3402 exhibits robust gastrointestinal survivability, mucosal protective activity, immunomodulatory and antioxidant properties, and industrial stability, highlighting its potential as a next-generation probiotic candidate for the prevention and alleviation of mucosal inflammatory disorders, including gastritis and colitis, as well as for industrial application.

국문 초록 (Abstract)

본 연구에서는 체중조절 작용으로 알려져 있는 Lactobacillus johnsonii JNU3402(JNU3402)의 염증완화 작용을 규명하고 대량생산을 위한 조건을 정립함으로써 산업적 활용 가능성을 평가하고자 하였다. 이를 위하여 JNU3402의 전장유전체 분석을 기반으로 한 안전성 평가를 수행하고, 항생제 내성 및 용혈성을 검증하였으며, 동물실험을 통해 항염증 효과를 분석하였다. 위 점막 및 장 상피 보호 효과를 평가하기 위하여 HCl/ethanol로 유도한 위염 모델과 dextran sulfate sodium(DSS)으로 유도한 대장염 모델을 각각 적용하였다. 또한, JNU3402의 산업적 생산을 위하여 통계적 방법론을 적용한 동결보호제 최적화를 수행하였다. 내산성과 HT-29 장 상피세포에 대한 부착능을 평가한 결과, JNU3402는 상용화된 프로바이오틱스 균주들 과 비교하여 우수한 내산성과 부착 활성을 나타내었다. 전장유전체 분석 결과, JNU3402는 스트레스 저항성(trxA/B, tpx, groES, dnaK/J), 면역조절(dltA-D, lspA), 그리고 부착 능력(comGA/GC/GF)과 관련된 주요 기능 유전자들을 보유하고 있었다. 그러나, tet(W) 유전자를 보유하고 있는 것으로 나타났지만, 전이 관련 유전자는 검출되지 않았고, 용혈 반응은 관찰되지 않았다. 위염 모델에서 JNU3402는 MUC5AC 발현 증진시키고 염증성 사이토카인을 억제함으로써 위상피 손상을 완화하였다. DSS 유도 대장염 모델에서 JNU3402는 질병활성도 지수를 유의적으로 감소시키고, DSS에 의해 유도된 결장 단축을 억제하였으며, 상피 장벽 손상과 점액층 붕괴를 완화시켰다. 또한 ZO-1과 Claudin-1의 발현을 증가시켜 장 장벽 기능을 강화하였다. 또한 조직·혈청 분석에서 IL-10 증가와 TNF-α 및 IL-6 감소를 통하여 염증 반응을 완화시켜 장 점막의 항상성 유지에 기여하였다. JNU3402는 thioredoxin 기반 항산화 시스템(trxA–trxB–tpx)을 보유하고 있는데, 이는 ROS 제거 및 NF-κB 신호 조절에 관여할 가능성이 있어, 관찰된 산화적 상피 손상 완화 효과와 연관되었을 것으로 추정된다. 이러한 분자적 특성은 염증 및 산화 스트레스 환경에서 나타난 항염증 효과를 부분적으로 설명할 수 있는 기전적 배경으로 해석될 수 있다. JNU3402의 동결보호제 조건 정립은 3종류의 보호제를 대상으로 각 성분의 비율로 구성하고 모든 성분의 합이 1(또는 100%)로 고정되는 조건에서 생균수를 반응값(response)로 하여 수행하였다. 통계적 모델을 적용한 결과, MSG 0.01–trehalose 0.01–whey powder 0.98 조성이 생균수를 극대화하는 최적 보호제 조건으로 도출되었으며, 해당 조건에서 동결건조 후 생존율은 88%를 나타냈다. JNU3402 균주를 최적 보호제 조건에서 저장 기간에 따라 평가한 결과, 저장 온도에 따라 생균수 감소 양상에 뚜렷한 차이가 관찰되었다. 25 ℃ 저장 조건 에서는 생균수 감소가 가장 크게 나타났으며, 5 ℃ 저장 조건에서는 상대적으로 완만한 감소 경향을 보였다. 반면 –20 ℃ 저장 조건에서는 생균수가 유지되어 가장 우수한 저장 안정성을 나타냈다. 이러한 결과는 5 ℃ 및 –20 ℃ 저장 조건에서 우수한 장기 안정성이 유지됨을 보여주며, 파일럿 규모 배양에서도 1010 CFU/g 이상의 높은 생균수를 확보함으로써 본 균주의 대량 생산 가능성을 뒷받침한다. 종합하면, JNU3402는 장내 생존성, 점막 보호, 면역 조절, 항산화 능력, 산업적 안정성을 동시에 갖춘 차세대 프로바이오틱스 후보로서, 위염 및 장염 등 점막 손상 질환의 예방·완화와 산업적 활용 가능성이 높음을 보여준다.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. Screening and safety evaluation of Lactobacillus johnsonii JNU3402 as a probiotic candidate 8
• 2.1 Abstract 8
• 2.2 Introduction 10
• 2.3 Materials and Methods 13
• 2.3.1 LAB Used for Screening 13
• 2.3.2 Assessment of acid tolerance in bacterial strains 13
• 2.3.3 Adhesion assay 14
• 2.3.4 Antibiotic susceptibility and hemolytic activity assays 14
• 2.3.5 Whole-Genome Sequencing 15
• 2.4 Results 17
• 2.4.1 Acid tolerance screening of candidate strains 17
• 2.4.2 Secondary screening based on adhesion to HT-29 cells 19
• 2.4.3 Antibiotic Susceptibility and Hemolytic Activity of JNU3402 21
• 2.4.4 Genome features and functional annotation 25
• 2.5 Discussion 31
• 3. Gastroprotective effects of JNU3402 in ethanol/HCl-induced gastric injury 35
• 3.1 Abstract 35
• 3.2 Introduction 37
• 3.3 Materials and Methods 39
• 3.3.1 Preparation of JNU3402 39
• 3.3.2 Cell-Based Experiments (MTT, RT-qPCR) 40
• 3.3.3 Animal Experiments (Rat Gastritis Model) 43
• 3.3.3.1 Induction of Gastritis and Lesion Evaluation 43
• 3.3.3.2 Histological Analysis (H&E) 44
• 3.3.3.3 Gene Expression (RT-qPCR) 44
• 3.3.3.4 Western Blot Analysis 45
• 3.3.4 Statistical Analysis 45
• 3.4 Results 47
• 3.4.1 Viability and Cytotoxicity in Gastric Cells 47
• 3.4.2 Morphological and Histological Changes and Proposed Mechanism of Gastroprotection 51
• 3.4.3 Regulation of Inflammatory Gene Expression (Il6, Tnf-α, Il10) 57
• 3.4.4 Western Blot Analysis of MUC5AC, TNF-α, and IL-10 in EtOH/HCl-Induced Gastric Injury 61
• 3.5 Discussion 64
• 4. Anti-inflammatory effects of JNU3402 in DSS-induced intestinal colitis 67
• 4.1 Abstract 67
• 4.2 Introduction 69
• 4.3 Materials and Methods 71
• 4.3.1 Preparation of JNU3402 71
• 4.3.2 Cell-Based Experiments (HT-29 Viability and Cytokine Profiling) 71
• 4.3.3 Animal Experiments (DSS-Induced Colitis in Mice) 73
• 4.3.3.1 Experimental Design and DAI Assessment 73
• 4.3.3.2 Serum and Colon Cytokine Analysis (RT-qPCR, ELISA) 75
• 4.3.3.3 Histopathological Examination 76
• 4.3.3.4 Western Blot Analysis 76
• 4.3.3.5 Malondialdehyde (MDA), and Superoxide Dismutase (SOD) Assays 77
• 4.3.4 Statistical Analysis 77
• 4.4 Results 78
• 4.4.1 Cytotoxicity and Cell Viability of JNU3402 78
• 4.4.2 Modulation of Inflammatory Cytokines in HT-29 Cells 80
• 4.4.3 Improvement of DAI and Colon Length in DSS-Induced Colitis 82
• 4.4.4 Suppression of Pro-Inflammatory Mediators (Tnf-α, Il6) 85
• 4.4.5 Histopathological Recovery, Mucosal Integrity 89
• 4.4.6 Antioxidant Enzyme Activity and Lipid Peroxidation (MDA, SOD) 91
• 4.4.7 Tight Junction Proteins and Cytokine Expression (Western blot) 93
• 4.5 Discussion 96
• 5. Optimization of freeze-drying protectants for JNU3402 survival and stability 99
• 5.1 Abstract 99
• 5.2 Introduction 100
• 5.3 Materials and Methods 102
• 5.3.1 Mixture Design and Statistical Optimization 102
• 5.3.2 Preparation of Protectant Combinations and Freeze-Drying Procedure 106
• 5.3.3 Scanning Electron Microscopy (SEM) 106
• 5.3.4 Pilot-Scale Fermentation and Lyophilization 107
• 5.3.5 Storage Stability Evaluation 109
• 5.4 Results 110
• 5.4.1 Optimization by Mixture Design and Response Surface Analysis 110
• 5.4.2 SEM Observation of Microstructural Changes 116
• 5.4.3 Pilot-Scale Cultivation and Viability Assessment of JNU3402 118
• 5.4.4 Storage Stability of Freeze-Dried JNU3402 under Different Temperature Conditions 120
• 5.5 Discussion 122
• 6. CONCLUSIONS 125
• 7. REFERENCES 128
• 국문초록 145