Characterization of Haloimpatiens sporogenes sp. nov., a Postbiotic Candidate Isolated from Traditional Korean Fermented Anchovy, Exhibiting Antioxidant and Immunoenhancing Activities

다국어 초록 (Multilingual Abstract)

Rod-shaped, Gram-positive, spore-forming, motile, and strictly anaerobic bacterium, designated FM7315ᵀ, was isolated from Myeolchi-jeot, a traditional Korean fermented anchovy. Phylogenetic and phylogenomic analyses based on 16S rRNA gene and whole- genome sequences revealed that the strain FM7315T represents a novel species within the genus Haloimpatiens. The genome size of strain FM7315ᵀ was 3.05 Mbp, with a G + C content of 29.7 mol%. Strain FM7315ᵀ grew at 20–37 °C, 0–2% (w/v) NaCl, and pH 5.0– 8.0. The major cellular fatty acids and polar lipids were C₁₄:₀, C₁₆:₀, C₁₈:₁ ω9c, Summed Feature 3 (C₁₆:₁ ω7c/C₁₆:₁ ω6c), and Summed Feature 8 (C₁₈:₁ ω7c/C₁₈:₁ ω6c); and diphosphatidylglycerol, phosphatidylglycerol, glycolipid, two aminophospholipids, and five unidentified lipids, respectively. Therefore, strain FM7315ᵀ is proposed as Haloimpatiens sporogenes sp. nov. (FM7315ᵀ = KCTC 25939ᵀ = JCM 37574ᵀ). Genome-based functional analysis revealed that strain FM7315ᵀ possesses a genome enriched in metabolic genes and harbors a complete gene cluster responsible for vitamin B₁₂ biosynthesis. Phenotypic characterization further showed that FM7315ᵀ exhibits greater cell surface hydrophobicity and auto-aggregation capacity than Lacticaseibacillus rhamnosus GG. However, the strain did not survive under simulated gastrointestinal conditions, indicating limited potential as a conventional probiotic. In contrast, postbiotic fractions obtained through ultrasonication displayed strong antioxidant activity, and metabolic profiling confirmed the production of vitamin B₁₂ and short-chain fatty acids (SCFAs), which contribute to epithelial barrier homeostasis. Additionally, production of putrescine—one of the major polyamines—was observed, and this metabolite is implicated in supporting epithelial homeostasis and maintaining intestinal barrier integrity. In vitro immune evaluations showed that FM7315ᵀ lysate exhibited no cytotoxicity up to 400 µg/mL and elicited a measurable increase in macrophage cytokine expression relative to untreated controls, indicating a mild immunostimulatory response without compromising cell viability. Genome-based safety assessment demonstrated the absence of acquired antimicrobial resistance genes, virulence factors, and mobile genetic elements, and in vitro assays confirmed that hemolytic, β- glucuronidase, and gelatinase activities, as well as biogenic amines, were not detected. Collectively, these findings indicate that Haloimpatiens sporogenes FM7315ᵀ is not only a novel taxon but also a promising next-generation postbiotic candidate with functional potential.

목차 (Table of Contents)

• Chapter Ⅰ. General Introduction 1
• Chapter Ⅱ. Isolation and characterization of novel bacteria from
• Myeolchi-jeot, a Korean traditional fermented anchovy 3
• 1. Introduction 3
• 2. Materials and Methods 4
• 2.1. Isolation and 16S rRNA gene-based identification 4
• 2.2. 16S rRNA gene-based phylogenetic comparison 5
• 2.3. Genome characterization 5
• 2.4. Phenotypic characterization 7
• 2.5. Chemotaxonomic characterization 8
• 3. Results 9
• 3.1. 16S rRNA gene-based phylogenetic comparison 9
• 3.2. Genome characteristics 12
• 3.3. Phenotypic characteristics 22
• 3.4. Chemotaxonomic characteristics 26
• 4. Discussion 30
• Chapter Ⅲ. Postbiotic functionality and safety assessments of
• Haloimpatiens sporogenes FM7315T 33
• 1. Introduction 33
• 2. Materials and Methods 35
• 2.1. Tolerance to simulated gastrointestinal conditions of Haloimpatiens
• sporogenes FM7315T 35
• 2.2. Genome-based prediction of functional potential and safety
• characteristics 36
• 2.2.1. Functional gene profiling 36
• 2.2.2. Genome-based safety assessments 36
• 2.3. In vitro Postbiotic functional properties of Haloimpatiens sporogenes
• FM7315T 37
• 2.3.1. Preparation of bacterial strains 37
• 2.3.2. Evaluation of cell-surface properties 37
• 2.3.3. Radical scavenging activities 38
• 2.3.4. Functional metabolites analyses 38
• 2.3.5. Preparation of cell line 39
• 2.3.6. Cell viability 39
• 2.2.7. RT-qPCR analysis of immune-related gene expression 40
• 2.4. Safety Assessment 41
• 2.5. Statistical Analysis 42
• 3. Results 43
• 3.1. Simulated gastrointestinal tolerance of Haloimpatiens sporogenes
• FM7315T 43
• 3.2. Genome-based prediction of functional potential and safety
• characteristics 45
• 3.2.1. Functional genome properties of strain FM7315T 45
• 3.2.2. Genome-based safety assessments 47
• 3.3. In vitro postbiotic functional properties of Haloimpatiens sporogenes
• FM7315T 50
• 3.3.1. Cell-surface properties of strain FM7315T 50
• 3.3.2. Radical scavenging activities of strain FM7315ᵀ 52
• 3.3.3. Functional metabolites of strain FM7315T 55
• 3.3.4. Cell viability 59
• 3.3.5. RT-qPCR Analysis of Immune-Related Gene Expression 61
• 3.4. Safety assessment of strain FM7315T 63
• 4. Discussion 66
• Conclusion 77
• Reference 79
• 국문 요약 89