Bacillus subtilis BSC35 포자의 혐기성 발아 중 박테리오신 생산을 통한 Clostridioides difficile 및 Clostridium perfringens 증식 억제와 포자 생산 배지 최적화 연구 = Growth Inhibition of Clostridioides difficile and Clostridium perfringens via Bacteriocin Production during Anaerobic Germination of Bacillus subtilis BSC35 Spores, and Optimization of Sporulation Medium

다국어 초록 (Multilingual Abstract)

Bacillus subtilis BSC35 spores are investigated for its ability, during germination, to produce a bacteriocin-like inhibitory substance (BLIS) against Clostridioides difficile and Clostridium perfringens, and its sporulation medium is optimized. In Chapter Ⅰ, the cell-free supernatant (CFS) of B. subtilis BSC35 is shown to exhibit quantifiable and reproducible antibacterial activity against both Clostridioides difficile and Clostridium perfringens. Despite inherent variability in inhibition zone diameters among C. perfringens strains reported in previous studies, BLIS BSC35 consistently inhibited two phylogenetically distinct pathogens, suggesting cross-genus activity uncommon among Bacillus-derived bacteriocins. Transmission electron microscopy of CFS-treated C. difficile revealed disrupted cell walls, compromised membranes, and cytoplasmic leakage, indicating a bactericidal mechanism. Medium composition significantly influenced sizes of inhibition zone by BLIS; CFS derived from DSM medium exhibited reduced inhibition compared with TSB-derived CFS, implying medium composition-dependent BLIS production or stability. Notably, BSC35 spores successfully germinated and produced BLIS under aerobic and anaerobic conditions, highlighting their potential for gastrointestinal applications where oxygen availability is limited. The B. subtilis BSC35 CFS concentration-dependent antibacterial activity observed in liquid culture further confirmed that BLIS production is intrinsic to BSC35 and not due to medium carryover. Although the chemical identity of BLIS remains to be elucidated, its dual activity against C. difficile and C. perfringens underscores its potential as a candidate for gut-targeted biocontrol strategies.
In Chapter Ⅱ, a stepwise optimization strategy incorporating One Variable At a Time (OVAT), Plackett-Burman Design (PBD), and Central Composite Design (CCD) was employed to maximize spore production of B. subtilis BSC35. OVAT screening identified molasses and yeast extract as the most effective carbon and complex nutrient sources, respectively, while soybean flour and corn steep liquor were the most effective nitrogen sources. Among inorganic salts, MgSO4 and NaCl positively influenced spore formation. PBD analysis confirmed yeast extract, molasses, soybean flour, corn steep liquor, and NaCl as significant contributors to spore yield. CCD-based response surface modeling produced a highly significant regression model (P < 0.0001) with excellent predictive accuracy (R2 = 0.9359, Adj. R2 = 0.8986, CV = 1.08%). A significant interaction between yeast extract and soybean flour indicated a synergistic effect on spore production. Validation experiments using shake flasks and a 5 L bioreactor confirmed the accuracy of the optimized medium, with spore yields closely matching model predictions and demonstrating scalability of the optimized formulation. The optimized medium, composed of cost-effective ingredients compared with conventional TSB, increased spore production from 4.1×107 CFU/mL to 4.71×109 CFU/mL.
In addition, freeze-drying experiments demonstrated that appropriate protectant formulations, particularly combinations of skim milk and sucrose, markedly improved spore survival, supporting the stability and feasibility of BSC35 spores for long-term storage and industrial application. Together, these findings highlight the potential of B. subtilis BSC35 as a scalable, economically viable, and formulation-ready candidate for gut-targeted biocontrol strategies.
Overall, B. subtilis BSC35 spores produce a bacteriocin-like inhibitory substance (BLIS) during germination, showing bactericidal activity against C. difficile and C. perfringens, with production influenced by medium composition and effective under both aerobic and anaerobic conditions. Stepwise medium optimization using OVAT, PBD, and CCD significantly enhanced spore yields, with key nutrients identified and scalability confirmed in shake flasks and a 5 L bioreactor, supporting potential applications for gut-targeted biocontrol.

목차 (Table of Contents)

• 표 목차 iv
• 그림 목차 v
• ABSTARCT vii
• Chapter Ⅰ. Bacillus subtilis BSC35의 혐기성 발아 및 BLIS 생산에 의한 C. difficile과 C. perfringens 억제 1
• I. 서 론 1
• II. 재료 및 방법 3
• 1. 사용 균주 및 배양 조건 3
• 2. Clostridioides difficile의 CFU 측정 3
• 3. 항균 활성 측정을 위한 Well diffusion assay 4
• 4. 액체 배양에서 B. subtilis BSC35 CFS를 사용한 C. difficile 증식 억제 4
• 5. Transmission Electron Microsocpy (TEM) 5
• 6. B. subtilis BSC35의 내열성 포자 준비 6
• 7. 혐기성 조건에서 B. subtilis BSC35 내열성 포자의 발아 및 증식과 CFS의 항균 활성 6
• III. 결 과 7
• 1. Well diffusion assay를 통한 B. subtilis BSC35 CFS의 C. difficile의 증식 억제 확인 7
• 2. Indicator 균주의 농도 및 희석 용액에 의한 Lysis zone 크기 영향 확인 10
• 3. 액체 배양에서 OD600 측정을 통한 B. subtilis BSC35 CFS의 C. difficile 증식 억제 확인 12
• 4. Transmission electron scope (TEM) 확인 15
• 5. Sporulation medium에서 B. subtilis BSC35의 내열성 포자 생산 확인 17
• 6. DSM 배지에서 배양된 B. subtilis BSC35의 항균 활성 확인 19
• 7. B. subtilis BSC35 내열성 포자의 혐기 조건 배양 시 발아 및 증식 확인 21
• 8. 혐기 조건에서 증식한 B. subtilis BSC35의 C. difficile에 대한 항균 활성 확인 23
• IV. 고 찰 25
• Chaper Ⅱ. Bacillus subtilis BSC35의 포자 생산을 위한 배지 조성 최적화 28
• I. 서 론 28
• II. 재료 및 방법 31
• 1. 사용 균주와 방법 31
• 2. B. subtilis BSC35 내열성 포자 수 측정 31
• 3. 포자 생산을 위한 배지 성분 개별 효과 분석(One Variable At a Time; OVAT) 31
• 3.1 배지의 탄소원에 따른 내열성 포자 생산 능력 확인 31
• 3.2 배지의 질소원에 따른 내열성 포자 생산 능력 확인 32
• 3.3 배지의 무기염에 따른 내열성 포자 생산 능력 확인 32
• 4. Plackett-Burman Desgin (PBD)를 통한 주요 영향 배지 성분의 선별 32
• 5. Central Composite Design (CCD)를 통한 배지 성분의 최적 농도 탐색 36
• 6. 분석모델의 검증 39
• 7. 동결건조보호제에 따른 B. subtilis BSC35 포자의 생존율 확인 39
• III. 결 과 41
• 1. One Variable At a Time(OVAT) 실험을 통한 주요 배지 개별 성분의 선별 41
• 1.1 OVAT를 적용한 탄소원 선정 41
• 1.2 OVAT를 적용한 질소원 선정 43
• 1.3 OVAT를 적용한 무기염 선정 45
• 2. Plackett-Burman Design (PBD)를 통한 포자 생성 주요 영향 배지 성분의 선별 47
• 3. Central Composite Design (CCD)에 기반한 반응표면 모델링과 배지 성분 최적 농도 조건 도출 49
• 4. 분석모델의 검증 54
• 5. 동결건조 보호제의 종류, 농도 및 조합에 따른 B. subtilis BSC35 포자 생존율 확인 54
• IV. 고 찰 58
• 참고문헌 60