Enhancing the Viability and Storage Stability of Lactobacillus plantarum CJLP133 via High-Efficiency Production and Novel Encapsulation Technology = 고효율 생산 및 신규 캡슐화 기술을 통한 락토바실러스 플란타룸 CJLP133의 생존율 및 저장 안정성 증진

국문 초록 (Abstract)

프로바이오틱스는 상당한 건강상의 이점을 제공하지만, 가공, 저장 및 위장관(GI) 스트레스로 인한 낮은 생존율로 인해 산업적 적용이 심각하게 제한됩니다. 본 학위 논문은 고부가가치 균주인 Lactobacillus plantarum CJLP133의 생존율과 저장 안정성을 향상시키기 위해 포괄적인 통합적 접근법(Integrated Approach)을 목표로 하였습니다. 이 접근법은 (1) 내재적 스트레스 내성을 가진 강건한 세포(robust cells)의 고효율 생산 프로토콜을 확립하고, (2) 최적화된 신규 캡슐화 시스템을 개발하는 두 가지 핵심 전략으로 구성됩니다.
첫째 생리적 안정화, 강건한 세포 생산 프로토콜을 확립하였습니다(제2장). 표준 MRS 배지에 강력한 항산화제인 0.1%(w/v) L-cysteine(시스테인)을 보충한 결과, 세포 수율(cell yield)이 약 3.0배( ~ 1.2 x 10^10 CFU/mL로) 증가하였습니다. 더욱 중요한 것은, 이 Cys-Cells는 캡슐화 전임에도 불구하고 40 °C에서 4주 후 2배 이상 높은 생리적 열안정성(생존율 77.4% vs 38.0%)을 나타내어, 세포 자체가 강건해졌음을 증명하였습니다.
둘째, 신규 캡슐화 시스템을 개발하고 검증하였습니다. 제형 스크리닝(제3장)에서, 소이 펩톤(Soy Peptone), 트레할로오스, 말토덱스트린을 함께 포집한 알긴산 제형(Bead F)이 동결건조 후 가장 높은 생존율(88.7%)을 보였으며, 이는 수크로오스(51.6%)나 소르비톨(68.4%) 같은 전통적 동결보호제보다 유의하게 우수했습니다. 이 최적의 Bead F 캡슐을 제4장에서 검증한 결과, 12주간 40 °C 저장 안정성이 우수하였고(비캡슐화 세포 1.68 log 감소 vs 1.03 log 감소), 모의 위장관(SSDP) 환경에서 획기적인 보호 효과(비캡슐화 세포 2.43 log 감소 vs 0.46 log 감소)를 나타냈습니다.
결론적으로, 본 연구는 (1) 시스테인 보충 배양을 통한 생리적 안정화와 (2) 소이 펩톤 기반 캡슐화를 통한 물리적 보호라는 통합적 접근법의 두 가지 핵심 전략을 성공적으로 규명하고 확립하였습니다. 본 연구는 L. plantarum CJLP133의 안정성과 기능적 전달을 극대화하는 과학적 근거와 산업적 활용 방안을 제시합니다.

다국어 초록 (Multilingual Abstract)

Probiotics offer significant health benefits, but their industrial application is severely limited by low viability due to processing, storage, and gastrointestinal (GI) stresses. This dissertation aimed to enhance the viability and storage stability of the high-value strain, Lactobacillus plantarum CJLP133, through a comprehensive Integrated Approach. This approach was designed to (1) establish a high-efficiency production protocol for robust cells with physiological stress tolerance, and (2) develop a novel encapsulation system with optimized extrinsic protection.
First, the robust cell production protocol was established (Chapter 2). Supplementing the standard MRS growth media with 0.1% (w/v) L-cysteine, a potent antioxidant, resulted in a ~3.0-fold increase in viable cell yield (to 1.2 x 10^10 CFU/mL). Notably, these Cys-Cells demonstrated over 2-fold higher physiologically enhanced thermal stability (77.4% vs 38.0% survival) after 4 weeks at 40 °C, proving enhanced cellular robustness prior to encapsulation.
Second, a novel encapsulation system was developed and validated. In the formulation screening (Chapter 3), an alginate-based formulation (Bead F) co-encapsulated with Soy Peptone, trehalose, and maltodextrin yielded the highest post-freeze-drying survival (88.7%), significantly outperforming traditional cryoprotectants like sucrose (51.6%) and sorbitol (68.4%). This optimal Bead F capsule was then validated in Chapter 4. It provided superior storage stability at 40 °C for 12 weeks (1.03 log loss vs. 1.68 log for free cells). Critically, it demonstrated exceptional protection in simulated stomach-duodenum passage, showing only a 0.46 log reduction, whereas free cells experienced a 2.43 log loss.
In conclusion, this dissertation successfully establishes and validates the two core strategies of the integrated approach: (1) Cysteine-supplemented cultivation for physiological modulation, and (2) Soy Peptone-based encapsulation for extrinsic protection. This work provides a scientifically-grounded and industrially-viable pathway to maximize the stability and functional delivery of L. plantarum CJLP133.

목차 (Table of Contents)

• Abstract Ⅰ
• Contents Ⅲ
• List of Tables Ⅵ
• List of Figures Ⅵ
• Chapter Ⅰ. Literature review 1
• Abstract 2
• 1.1. Probiotics: Health Benefits and Industrial Significance 4
• 1.2. The Viability Challenge: A Critical Hurdle for Probiotic Efficacy 6
• 1.3. Encapsulation: A Primary Strategy and Its Inherent Limitations 8
• 1.4. Emerging Perspectives: An Integrated Approach from Production to Stabilization 10
• 1.5. Research Objectives 12
• 1.6. References 13
• Chapter Ⅱ. Optimization of Medium Composition for High-Concentration Culture and Enhanced Stress Tolerance of Lactobacillus plantarum CJLP133 17
• Abstract 18
• 2.1. Introduction 20
• 2.2. Materials and Methods 22
• 2.2.1. Bacterial Strain and Culture Conditions 22
• 2.2.2. Evaluation of Cysteine-Supplemented Cultivation 22
• 2.2.3. Evaluation of Cellular Thermal Stability 23
• 2.2.4. Statistical Analysis 24
• 2.3. Results 25
• 2.3.1. Effect of Cysteine on High-Cell-Density Culture 25
• 2.3.2. Effect of Cysteine Cultivation on Cellular Thermal Stability 26
• 2.4. Discussion 28
• 2.4.1. Mechanism of High-Cell-Density Culture: Redox Modulation and Metabolic Flux 28
• 2.4.2 Robust Cells: Membrane Phase Transition and Enzyme Protection 29
• 2.4.3 Industrial Feasibility and Sustainability: Comparative Advantage of L-cysteine 30
• 2.5. Conclusion 31
• 2.6. References 32
• Chapter Ⅲ. Screening and Optimization of Cryoprotectants to Improve the Viability of Microencapsulated Lactobacillus plantarum CJLP133 during Freeze-Drying 35
• Abstract 36
• 3.1. Introduction 37
• 3.2. Materials and Methods 39
• 3.2.1. Materials 39
• 3.2.2. Bacterial Culture and Growth Conditions 39
• 3.2.3. Bacteria Encapsulation in Calcium Alginate Beads 40
• 3.2.4. Encapsulation Efficiency (EE) 41
• 3.2.5. Viability of Encapsulated Cells During Freeze-drying 41
• 3.2.6. Statistical Analysis 42
• 3.3. Results 43
• 3.3.1. Encapsulation Efficiency (EE) 43
• 3.3.2. Survival Rate after Freeze-Drying 44
• 3.4. Discussion 46
• 3.4.1. Mechanism of Encapsulation Efficiency Improvement via Osmotic Equilibrium 46
• 3.4.2. Physicochemical Protection: Water Replacement and Glass Formation 47
• 3.4.3. Synergistic Superiority of Soy Peptone: Multifunctional Cryoprotection 47
• 3.4.4. Strategic Shift from Liquid EE to Powder Yield: The Osmotic Balancing Strategy 49
• 3.5. Conclusion 49
• 3.6. References 51
• Chapter Ⅳ. Validation of the Protective Efficacy of Optimized Encapsulated Lactobacillus plantarum CJLP133 under High-Temperature Storage and Simulated Stomach-Duodenum Passage Conditions 56
• Abstract 57
• 4.1. Introduction 59
• 4.2. Materials and Methods 61
• 4.2.1. Preparation of Experimental Groups 61
• 4.2.2. Storage Stability Assessment 61
• 4.2.3. Survival in Simulated Stomach-Duodenum Passage (SSDP) 62
• 4.2.4. Statistical Analysis 62
• 4.3. Results 63
• 4.3.1. Storage Stability at 40 °C 63
• 4.3.2. Survival in Simulated Stomach-Duodenum Passage (SSDP) 64
• 4.4. Discussion 66
• 4.4.1. Mechanism of Storage Stability: Vitrification and Sacrificial Antioxidants 66
• 4.4.2. Mechanism of GI Protection: Acid-Induced Shrinkage and Buffering 67
• 4.4.3. Rationale and Industrial Implications: Enhancing Consumer Convenience in Food Systems 68
• 4.5. Conclusion 69
• 4.6. References 70
• Chapter V. General Conclusion and Future Directions 73
• 5.1. Conclusion 74
• 5.2. Future Directions 77