Next-Generation Probiotics Bacteroides fragilis Alleviates Weaning-induced Stress Responses by Interacting Gut-Microbiome-Brain Axis = 차세대 프로바이오틱스 Bacteroides fragilis 의 장-마이크로바이옴-뇌 축 조절을 통한 이유 스트레스 완화 기능성 평가

다국어 초록 (Multilingual Abstract)

The post-weaning period in mammals brings nutritional, microbial, and psychological stresses that can impair growth and development. In this study, we investigated the potential of next-generation probiotics (NGPs) as a strategy to alleviate post-weaning stress. NGPs are gut commensals with targeted functional properties positively affecting gut integrity, systemic inflammation, and gut-brain axis interactions. However, the application of strict anaerobes such as Bacteroides fragilis is often limited due to cultivation challenges. We applied a mild heat-inactivation method to the porcine-derived B. fragilis SLAM_BAF01 to examine how the heat-killed strain affects the gut–microbiome–brain axis under stress conditions related to weaning. Heat-killed B. fragilis SLAM_BAF01 preserved structural integrity and lacked enterotoxicity. In Caenorhabditis elegans, both the live and heat-killed B. fragilis SLAM_BAF01 extended lifespan by 12% and 8%, respectively. In a fecal microbiota transplantation (FMT) mouse model designed to mimic weaning stress conditions, heat-killed B. fragilis SLAM_BAF01 positively modulated gut microbiota, gut integrity, and stress-related markers. Specifically, brain gamma-aminobutyric acid levels increased by 150% and serum corticosterone levels decreased by 17% compared with the control. In a pig model under stress conditions after weaning, heat-killed B. fragilis SLAM_BAF01 significantly improved growth by 7%, with reduced systemic stress and inflammation. Collectively, these findings demonstrate that heat-killed B. fragilis SLAM_BAF01 is a potent next-generation probiotic candidate capable of enhancing gut–brain health and mitigating weaning-associated stress across mammalian hosts.

국문 초록 (Abstract)

포유류에서 이유 과정은 개체의 영양 흡수, 장내미생물 균형, 나아가 심리적인 부분에서의 스트레스를 수반한다. 이로 인해 정상적인 성장과 발달이 저해될 수 있다. 해당 연구에서는, 이유 스트레스의 완화 방안으로 ‘차세대 프로바이오틱스 (Next-generation probiotics, NGPs)’를 이용하는 것의 가능성을 확인하였다. NGP는 장 장벽, 전신 염증, 그리고 장-뇌 축 상호작용에 대해 긍정적인 기능적 특성을 지닌 장내 공생균으로 정의된다. 하지만 Bacteroides fragilis와 같은 절대혐기성 균주는 배양의 어려움으로 산업적 적용이 제한된다. 이에 본 연구는 돼지 유래 B. fragilis SLAM_BAF01의 구조를 유지하는 저온 열사멸 방안을 채택하여 기능성과 안정성을 동시에 구현하였다. 더불어 생균과의 비교를 통해 사균체가 이유기 스트레스 조건에서 장-마이크로바이옴-뇌 축에 미치는 영향을 평가하였다. B. fragilis SLAM_BAF01은 위산 및 담즙산에 대한 내성을 보였으며, 전장유전체 분석에서 장내독소 관련 유전자가 확인되지 않았다. 열사멸 B. fragilis SLAM_BAF01은 열처리 이후에도 간균 형태를 유지하였다. 예쁜꼬마선충을 이용한 기능성 평가 실험에서 생균과 사균은 각각 12%, 8%의 수명 연장 효과를 나타냈다. 또한, 이유기 스트레스 모사 모델인 ‘분변 미생물 이식 마우스 모델’에서는 열사멸 B. fragilis SLAM_BAF01 투여 시 장내 미생물총 조성, 장 점막 무결성, 스트레스 관련 지표가 긍정적으로 조절 되었다. 특히 뇌 내 GABA 수치는 대조군 대비 150% 증가하였고, 혈중 코르티코스테론은 17% 감소하였다. 실제 이유 자돈 모델에서도 열사멸 B. fragilis SLAM_BAF01 급여는 성장률을 7% 향상시켰으며, 전신 스트레스와 염증을 완화하였다. 종합적으로, 본 연구는 열사멸 B. fragilis SLAM_BAF01이 장-뇌 건강을 증진하고 이유 유래 스트레스를 완화할 수 있는 차세대 프로바이오틱스 후보임을 입증하였다.

목차 (Table of Contents)

• Abstract I
• Table of Contents III
• List of Tables VIII
• List of Figures IX
• List of Abbreviations XIII
• Chapter 1. Literature review
• 1.1. Probiotics, gut microbiome, and gut-brain axis 1
• 1.1.1. Definition and importance of probiotics 1
• 1.1.2. Probiotics in gut and systemic regulation 2
• 1.1.3. Gut-brain axis 7
• 1.1.3.1. SCFAs, GABA, and neuroactive metabolites 10
• 1.1.3.2. Gut dysbiosis and stress-related disorders 11
• 1.1.4. Next-generation probiotics 13
• 1.1.4.1. Definition and examples 13
• 1.1.4.2. Challenges and prospects 14
• 1.1.5. Bacteroides fragilis as a NGP 15
• 1.1.5.1. Structure and metabolism 15
• 1.1.5.2. Roles in immunity, cognition and gut-brain axis 16
• 1.2. Weaning stress and gut microbiome 17
• 1.2.1. Physiological and microbial impact of weaning 17
• 1.2.2. Gut microbiota shift and dysbiosis 18
• 1.2.3. Probiotics and weaning stress management 19
• Chapter 2. Comprehensive characterization and probiotic functional assessment of porcine-derived B. fragilis in vitro.
• 2.1. Introduction 21
• 2.2. Materials and methods 24
• 2.2.1. Functional characterization of B. fragilis SLAM_BAF01 24
• 2.2.1.1. Bacterial strains culture 24
• 2.2.1.2. Total DNA extraction 24
• 2.2.1.3. Bacterial whole-genome sequencing 25
• 2.2.1.4. Bile and acid tolerance test 26
• 2.2.1.5. Intestinal epithelial cell culture and adhesion assay 27
• 2.2.1.6. Scanning Electron Microscopy 28
• 2.2.2. C. elegans lifespan and GFP fluorescence analysis 28
• 2.2.3. Statistical analysis 29
• 2.3. Results 31
• 2.3.1. Functional characterization of B. fragilis SLAM_BAF01 31
• 2.3.1.1. Bacterial whole-genome analysis 31
• 2.3.1.2. In vitro evaluation of the probiotic potential of B. fragilis SLAM_BAF01 39
• 2.3.1.3. SEM analysis of live and heat-killed SLAM_BAF01 43
• 2.3.2. C. elegans lifespan and GFP fluorescence analysis 45
• 2.4. Discussion 48
• Chapter 3. Administration of porcine-derived B. fragilis for alleviating weaning-induced stress in various in vivo models.
• 3.1. Introduction 51
• 3.2. Materials and methods 54
• 3.2.1. Bacterial strains culture 54
• 3.2.2. Mouse FMT experiment 54
• 3.2.2.1. Animals and experimental design 55
• 3.2.2.2. Molecular analysis 58
• 3.2.2.2.1. Real-time qPCR 58
• 3.2.2.2.2. Enzyme-Linked Immunosorbent Assay (ELISA) 59
• 3.2.2.3. Multi-omics analysis 59
• 3.2.2.3.1. 16S rRNA amplicon sequencing 60
• 3.2.2.3.2. Microbiome abundance and diversity analysis 60
• 3.2.2.3.3. GC/MS analysis of mouse fecal metabolites 62
• 3.2.2.4. Behavioral tests 64
• 3.2.3. Weaning-pig experiment 65
• 3.2.3.1. Animals and experimental design 65
• 3.2.3.2. Blood profiles analysis 70
• 3.2.3.3. Multi-omics analysis 70
• 3.2.3.3.1. 16S rRNA amplicon sequencing 70
• 3.2.3.3.2. Microbiome abundance and diversity analysis 71
• 3.2.3.3.3. GC/MS analysis of pig fecal metabolites 73
• 3.2.4. Statistical analysis 74
• 3.3. Results 76
• 3.3.1. Mouse FMT experiment 76
• 3.3.1.1. FMT model validation (Pre-FMT vs Post-FMT) 76
• 3.3.1.2. Weaning stress alleviating function of B. fragilis SLAM_BAF01 80
• 3.3.1.2.1. Growth performance and colon length restoration 80
• 3.3.1.2.2. PSA-mediated colon inflammation alleviation 83
• 3.3.1.3. Brain function restoration 86
• 3.3.1.4. Fecal metagenome analysis 92
• 3.3.1.4.1. Pre- and post-weaning pig 92
• 3.3.1.4.2. Fecal metagenomic dynamics during Mouse FMT 96
• 3.3.1.4.3. Live or heat-killed B. fragilis SLAM_BAF01-treated groups 100
• 3.3.1.4.4. PICRUSt2 analysis 104
• 3.3.1.5. Fecal metabolite analysis 107
• 3.3.2. Weaning pig experiment 111
• 3.3.2.1. Growth and feed indices 111
• 3.3.2.2. Blood profile analysis 114
• 3.3.2.3. Fecal metagenome and metabolite analysis 117
• 3.4. Discussion 122
• References 132
• Summary in Korean 159