한국 전통 식품 분리 Weissella sp. SNUL2 균주 생산 항균물질의 특성과 프로바이오틱스 가이드라인에 따른 평가

학위논문(석사) -- 서울대학교대학원 : 국제농업기술대학원 국제농업기술학과, 2022.2. 김효진.1928년 Fleming의 penicillin 발견 이후, 인류는 세균 감염으로부터 보호받을 수 있게 되었다. 이 발견 이후, 항생제는 비약적인 발전을 이루었고 오늘날에는 없어서는 안 될 필수적인 요소가 되었다. 그러나, 1940년 penicillin의 도입 이후, penicillin 내성균이 발견되기까지 1년밖에 걸리지 않았다. 항생제가 임상에 적용된 80여년 동안, 전 세계적으로 발생하는 항생제 내성은 다양한 세균 감염 치료에 큰 제약인 동시에, 위협이 되었다. 항생제 내성균이 발생하는 주 원인은 항생제의 오남용인데, 항생제 내성은 말 그대로 항생제에 내성을 가진다는 의미이며 임상에서 세균을 사멸시키는 것을 불가능하게 한다는 의미를 가지기도 한다. 때문에 오늘날 항생제 내성을 줄이기 위해 항생제 대체제의 필요성이 끊임없이 대두되고 있고 실제로 많은 연구진들은 항생제를 대체할 만한 물질을 찾기 위해 다양한 연구를 진행하고 있고 후보군중 하나는 probiotics이다. 하지만 현존하는 probiotics는 좁은 범위의 항균성을 갖고 있는 한계점이 있으며 이 때문에 세균 감염 치료목적의 약 보다는 건강 기능 식품으로 사용되는 경우가 많다. 때문에 치료 목적의 probiotics로 개발하기 위해서 넓은 범위의 항균성을 갖는 것이 필수적인 동시에, 생분해성, probiotics guidelines 등의 기준들을 충족해야 한다. 본 논문에서는 이 기준들을 충족하는 균주를 찾아 항생제 대체재 후보로서 제안한다. 챕터 2에서는, 항생제 대체재 후보군으로 사용하고자 하는 균주를 분리하고, 항균물질의 특성을 확인했다. Weissella sp. SNUL2라는 strain을 한국 전통 식품에서 분리했고 16s rRNA sequencing을 통해 동정하였다. 이 균주의 최적온도와 pH를 확인하고, O.D600과 CFU의 상관관계를 확인해서 이 후 실험에 적용했다. 균주를 치료 목적으로 사용하기 위해 가장 중요한 실험인 항균성 실험을 총 70여가지 세균에 대해 진행했으며 그 결과 Weissella sp. SNUL2는 기존의 probiotics 보다 넓은 범위의 항균성을 갖는 것을 확인할 수 있었다. 균주의 배양과정에서, 상등액에 분비되는 항균물질의 특성을 확인하기 위해 효소 실험을 진행했고 항균성 단백질이라는 결과를 얻었다. 항균성 단백질이 어떤 물질인지 추가 정보를 얻기 위해 부분 정제를 진행했고 LC-MS/MS 결과 bacteriocin이라는 항균성 단백질을 생산하는 단백질인 C39 family protein과 세포벽 성분인 peptidoglycan을 분해하는 효소인 peptidoglycan endopeptidase, 두 가지 단백질이 동정되었다. 챕터 3에서는, 한국 전통 식품에서 분리한 Weissella sp. SNUL2라는 균주가 FAO와 WHO의 probiotics guideline을 충족시키는지 확인하기 위해 이 가이드라인에 따라 실험을 진행했다. 먼저 PacBio sequencing을 통한 Weissella sp. SNUL2 전장유전체 정보를 얻었고 그 결과 1개의 염색체와 4개의 plasmid로 구성된 것을 확인했다. 총 2,291개의 CDS가 염색체에 존재한다는 것을 확인했고 이 정보와 toxic metabolite pathway에 기반하여 체내에 악영향을 줄 수 있는 toxic metabolite를 생산하는지 비교, 분석하였다. 앞서 말한 probiotics guideline에 따라 내산성, 내담즙성과 적혈구를 파괴하는 용혈활성 여부 마지막으로 항생제 내성 여부를 확인한 결과 내산성, 내담즙성은 상용화된 probiotics에 상응하는 결과를 보여주었고, 용혈활성은 나타나지 않았다. 항생제 내성은 ampicillin에 대해선 없는 것으로 확인되었지만 streptomycin과 kanamycin에 대해선 내성이 있는 것으로 나왔다. 하지만 이 내성도 Plasmid Finder 2.1이라는 tool을 이용해 외부로부터 얻은 획득성 내성인지, 균주 고유의 특성인 내재성 내성인지 확인 결과 내재성이라는 결과를 얻었다. 한국 전통 식품에서 분리한 Weissella sp. SNUL2 균주가 넓은 범위의 항균성을 갖고 probiotics 기준을 충족하는 결과를 보여준 만큼 미래에 항생제 대체재로서 사용되어 항생제 내성의 위험으로 부터 벗어날 수 있기를 기대한다.After Fleming’s discovery of penicillin in 1928, mankind became more safety from bacterial infection. After this discovery, research on antibiotics began to gain momentum, and the role of antibiotics is still very important to this day. However, it took only one year between the introduction of penicillin in 1940 and the discovery of penicillin-resistant bacteria. The widespread emergence of antibiotic resistance in only 80 years since antibiotics were introduced into clinical practice, is causing treatment failure of various infectious diseases. Antibiotic abuse is the decisive reason for these bacteria to evolve to become resistant to antibiotics. Since these bacteria are literally resistant to antibiotics, it is impossible to kill bacteria during clinical treatment, which is a huge problem. Currently, to reduce antibiotic resistance, the need for alternative antibiotics is necessary. Therefore, many researchers are studying to find substances that can replace antibiotics, and one of the alternative antibiotics to solve the problem is probiotics. However, existing probiotics have a narrow range of antibacterial properties, so they are mostly used as health functional foods rather than for the purpose of treating bacterial infections, which is considered the biggest weakness. Thus, to develop into therapeutic probiotics, it is essential to have a broad antibacterial spectrum, and at the same time, several criteria must be met. The criteria that aim for are as follows: Broad spectrum antibacterial properties; Biodegradable; Probiotics Guidelines. Here, we try to find a strain that meets this criterion and propose it as one of the antibiotic alternative candidates. In Chapter 2, strains that can be used as an alternative to antibiotics were isolated and various characteristics of antibacterial substances were confirmed. The strain, Weissella sp. SNUL2, was isolated from Korean traditional fermented food was identified through 16s rRNA sequencing. The optimum temperature and pH of the strain were confirmed, and the correlation between O.D600 and CFU was confirmed and applied to subsequent experiments. In the study of probiotics for therapeutic purposes, the most important antibacterial test was conducted on a total of 70 strains, and as a result, it was confirmed to have broad spectrum antibacterial properties than previous known probiotics. To know the characteristics of the antibacterial substance secreted in the supernatant, an enzyme test was conducted, and as a result, it was confirmed that it was a proteinaceous substance. Partial purification was performed to further subdivide this antibacterial protein, and as a result of LC-MS/MS analysis, two proteins were found: C39 family protein that produces bacteriocin, a well-known antibacterial protein, and a peptidoglycan endopeptidase that degrades peptidoglycan, a cell wall component. Chapter 3 confirmed whether the Weissella strains met the probiotic conditions according to the FAO and WHO probiotic guidelines. Weissella sp. SNUL2 performed whole genome sequencing through PacBio sequencing and acquired genetic information for one chromosome and four plasmids. There were 2,291 CDSs on one chromosome, and as a result of comparing them with the toxic metabolite pathway, several related genes could be identified. Weissella sp. SNUL2 was determined to have both acid tolerance and bile tolerance, in addition, as a result of the hemolytic activity, it was confirmed that hemolytic activity that destroys red blood cells did not occur, and although it has antibiotic resistance to streptomycin and kanamycin, using the tool called Plasmid Finder 2.1, it was confirmed as a characteristic of bacteria its selves, not resistance acquired from outside thus Weissella sp. SNUL2 met all probiotic trials. As it is a strain with broad range of antibacterial properties, it is thought that it can be used as probiotics for treatment purposes in the future. In addition, it is expected to be used as a substitute for antibiotics, thereby avoiding the risk of antibiotic resistance.Abstractⅰ Contents ⅲ List of Tables viii List of Figures x List of Abbreviations xiii Chapter 1 Research background 1 1. Antibiotics 1 2. Antibiotics resistance 1 3. The risk of antibiotic resistance bacteria 2 4. Antibiotics prescribed diseases 10 5. Candidates of antibiotics alternative 10 6. Bacteriocin 12 6.1 Classification of bacteriocin 12 6.2 Synthesis of bacteriocin 13 6.3 Mechanism of bacteriocin 14 7. Overall objective 16 Chapter 2. Antibiotic properties of Weissella sp. SNUL2 isolated from Korean traditional fermented food 1. Introduction 18 2. Materials and methods 19 2.1. Sample isolation 19 2.2. Fermentation and growth conditions 21 2.3. Antibacterial test against 60 bacteria with microplate 21 2.4. Antibacterial test against E. coli 22 2.5. Antibacterial test against pathogens 22 2.6. Antibacterial test against beneficial microbes 25 2.7. Minimum inhibition concentration (MIC) test 27 2.8. Hourly antibiotic substance production 28 2.9. Antibiotic substance assay 29 2.10. Kirby-Bauer disk diffusion assay 30 2.11. Partial purification 2.11.1. Ethyl acetate extraction 31 2.11.2. Gel filtration 31 2.11.3. Bradford assay 32 2.11.4. UV assay with gel filtrated CFS 33 2.11.5. Kirby-Bauer disk diffusion assay with gel filtrated CFS 33 2.11.6. Ion-exchange chromatography 34 2.11.7. Kirby-Bauer disk diffusion assay with Ion-exchanged CFS 35 2.12. SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 36 2.13. Gel extraction 37 2.14. Liquid chromatography tandem mass spectrometry (LC-MS/MS) 38 3. Results 39 3.1. Sample isolation 39 3.2. Fermentation and growth conditions 40 3.3. Antibacterial test against 60 bacteria with microplate 45 3.4. Antibacterial test against E. coli 48 3.5. Antibacterial test against pathogens 52 3.6. Antibacterial test against beneficial microbes 55 3.7. Minimum inhibition concentration (MIC) test 58 3.8. Hourly antibiotic substance production 62 3.9. Antibiotic substance assay 64 3.10. Kirby-Bauer disk diffusion assay 66 3.11. Partial purification 3.11.1 Ethyl acetate extraction 69 3.11.2. Gel filtration 69 3.11.3. Ion-exchange chromatography 73 3.12. SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) 75 3.13. Liquid chromatography tandem mass spectrometry (LC-MS/MS) 77 4. Conclusion 87 Chapter 3. Characteristics of Weissella sp. SNUL2 according to ‘Probiotics guidelines’ 1. Introduction 88 2. Materials and methods 90 2.1. Cell viability test 90 2.1.1. Acid tolerance test 90 2.1.2. Bile tolerance test 90 2.2. Whole genome sequencing 91 2.2.1. DNA extraction 91 2.2.2. Genomic sequencing and assembly 92 2.3. Toxic metabolite 93 2.4. Hemolysis 94 2.5. EFSA MIC cut-off value of antibiotics 94 3. Results 95 3.1. Cell viability test 95 3.1.1. Acid tolerance test 95 3.1.2. Bile tolerance test 96 3.2. Genome sequencing and assembly 99 3.3. Toxic metabolite 101 3.4. Hemolysis 103 3.5. EFSA MIC cut-off value of antibiotics 104 4. Conclusion 107 5. Discussion 108 References 109 Abstract in Korean 114 Acknowledgements 116석