Anti-influenza virus potential of probiotic Lactobacillus plantarum YML015

국문 초록 (Abstract)

본 연구는 한국 전통 발효식품인 김치에서 분리한 Lactobacillus plantarum YML015 균주의 약리 및 치료 가능성을 평가하였다. MDCK 세포주와 발육란을 이용한 in vitro, in ovo 실험에서 L. plantarum YML015 는 인플루엔자 바이러스 A (IFVA) H1N1 과 저 병원성 조류 인플루엔자 바이러스 H9N2 (조류독감) 에 대하여 상당한 항 바이러스 잠재력을 가지고 있음을 평가하였다. YML015의 항-IFVA (H1N1) 과 항-LPAI (H9N2) 활성을 평가하기 위하여 발육란을 이용한 In ovo 실험과 적혈구응집반응 저해, 세포병원성 저해 실험과 같은 in vitro 실험을 진행하였다. 게다가, 항생제 감수성(MIC)테스트와 용혈 분석을 함으로써 L. plantarum YML015은 자연과 사람에게 안전한 것으로 분류하였다. 서로 다른 온도로 처리된 YML015 무 세포 상등액(CFS)의 열 안정성 실험도 진행하였다. 그 결과, L. plantarum YML015는 포유 동물 세포주 (MDCK 세포)에서 매우 강력한 항-IFVA(H1N1) 및 항-LPAI(H9N2) 활성을 보여주었다. 반면에 NO, SOD, ABTH and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals.와 같은 다양한 scavenging models을 통하여 L. plantarum YML015의 무 세포 상등액 및 에탄올 추출물(EE)의 항산화 활성을 평가하였다. 상업 균주인 Probio65와 Lactobacillus plantarum YML009를 YML015와 비교한 결과, YML015는 여러 라디칼 소거 능력을 가지고 있음을 보였다. 게다가 ORAC, CUPRAC, FRAP 분석을 수행하였으며, YML015의 약리 잠재력을 평가하였다. AAPH에 의해 유도된 세포 반응성 산소 종(ROS)생산의 비율은 L. plantarum YML015를 이용한 것이 더욱 낮았다. 또한 L. plantarum YML015의 CFS 및 EE의 항 염증 가능성을 결정하기 위해, 항 염증 활성은 두 가지 방법으로 측정하였다: 첫 번째로, RAW
264.7 대식세포에 의해 활성화된 지질 다당류(LPS)의 산화 질소(NO)생산 측정. 두 번째로, RAW 264.7 세포의 세포 생존 비율을 MTT분석법으로 측정. 그러나, 항 당뇨 및 함암 전위는 각각 α-glucosidase 억제 분석법으로 평가하였으며 B16F10의 세포 생존 능력은 MTT 분석법으로 평가하였다. 두 분석에서, Lactobacillus plantarum YML015 는 항암 및 항 당뇨병 활성이 다른 두 상업균주보다 우수하게 나타났다. 본 연구의 결과는 Lactobacillus plantarum YML015의 강력한 항 바이러스, 항산화, 항 염증, 항 당뇨병 및 항암 활성에서 약리 및 치료 가능성을 확인하였으며, 다양한 생명을 위협하는 질병 및 건강 장애에서 인간과 동물을 보호하기 위하여 다양한 음식과 약물에 쓰일 것으로 전망한다.

다국어 초록 (Multilingual Abstract)

The goals of the study were to isolate a Lactobacilli strain Lactobacillus plantarum YLM015 from Korean traditional fermented food, “Kimchi” and to evaluate its pharmacological and therapeutic potential as a beneficial probiotic. Research evaluated that L. plantarum YML015 had significant anti-viral potential against influenza virus A (IFV) H1N1 and avian influenza virus H9N2 (Bird flu) when tested using MDCK cell line in vitro, and embryonated eggs in ovo assays. A total of 1200 Lactobacilli strains were isolates and screened. Among them, isolate L. plantarum YML015 was selected for further studies base d on its potent anti-viral, antioxidant, anti-inflammatory, anti-diabetic and anti-skin cancer efficacy. Further, isolate YML015 was characterized as Lactobacillus plantarum based on 16S rRNA gene sequencing analysis and by API 50 CHL Kit biochemically. In ovo assay with embryonated eggs and in vitro assay such as hemagglutination inhibition assay and cytopathogenic reduction assay were performed to evaluate anti-IFV (H1N1) and anti-AIFV (H9N2) activity of YML015. Additionally, L. plantarumYML015 was classified for its non-resistance nature as safe for human as confirmed by the antibiotic susceptibility (MIC) test and hemolysis assay. Heat stability test was also experienced by using different heat- treated cell free supernatant (CFS) samples of YML015. As a result, L. plantarum YML015 showed highly potent anti-IFV (H1N1) and anti- AIFV (H9N2) activities in vitro in mammalian cell line (MDCK cell). On the other hand, the antioxidant activity of cell free supernatant (CFS) and ethanol extract (EE) of L. plantarum YML015 was evaluated in vitro using various
scavenging models including NO, SOD, ABTH and 1,1-diphenyl-2- picrylhydrazyl (DPPH) radicals. The results indicated that comparing with commercial strain Probio65 and Lactobacillus plantarum YML009, YML015 displayed significant scavenging ability on several radical scavenging models. Furthermore, ORAC, CUPRAC, and FRAP assays were performed and pharmacological potential of YML015 was evaluated. Percentage of cellular reactive oxygen species (ROS) production induced by AAPH was lower by using L. plantarum YML015. Furthermore, to determine anti-inflammatory potential of CFS and EE of L. plantarum YML015, anti-inflammatory activity was measured in two ways: first, by measuring nitric oxide (NO) production in lipopolysaccharide (LPS) activated RAW 264.7 macrophages, and second, with the percentage of cell viability of RAW 264.7 cell followed by MTT assay. However, anti-diabetic and anti-cancer potentials was evaluated by α- glucosidase inhibitory assay and cell viability of B16F10 by MTT assay, respectively. In both assays, it was proved that Lactobacillus plantarum YML015 exhibited excellent anticancer and anti-diabetic activities rather than other two commercial strains. Findings of this study confirm pharmacological and therapeutic potential of Lactobacillus plantarum YML015 in terms of its potent anti-viral, antioxidant, anti-inflammatory, anti-diabetic and anticancer properties along with its probiotic nature which makes it a candidate of choice for using in various food and pharmacological preparations to protect humans and animals from various life-threatening disease and health disorder.

목차 (Table of Contents)

• CHAPTER 1 Introduction 1
• 1. Probiotics and Lactic Acid Bacteria 1
• 2. Influenza Virus H1N1 2
• 3. Influenza Virus H9N2 3
• 4. Influenza Virus and Lactic Acid Bacteria 4
• 5. Anti-oxidant and lactic Acid Bacteria 6
• 6. Anti-inflammation and Lactic Acid Bacteria 8
• 7. Antidiabetic, Anti-Cancer and Lactic Acid Bactria 10
• 8. Conclusion 102
• 9. Reference 103
• CHAPTER 2 In vitro and in ovo antiviral potential of a probiotic strain Lactobacillus plantarum YML015 isolated from Korean fermented food Kimchi against highly pathogenic influenza virus A H1N1 12
• 1. Abstract 12
• 2. Introduction 12
• 3. Materials and methods 15
• 3.1 Isolation and identification of Lactobacillus
• plantarum YML015 15
• 3.2 Characterization of Lactobacillus plantarumYML015 15
• 3.2.1 Biochemical characterization 15
• 3.2.2 Heat stability test 16
• 3.2.3 Antibiotic susceptibility assay 16
• 3.2.4 Hemolytic phenomenon assay 18
• 3.3 Culture of MDCK cell 19
• 3.4 Virus culture 19
• 3.5 Antiviral potential of L plantarumYML015 20
• 3.5.1 Cytopathogenic reduction a 20
• 3.5.2 Hemagglutination inhibition assay 21
• 3.6 In ovo antiviral assay 22
• 4. Results 23
• 5. Discussion 27
• 6. Conclusion 31
• 7. Reference 31
• CHAPTER 3 Evolutionary antiviral activity of potential probiotic strain Lactobacillus plantarum YML015 isolated from Korean fermented Vegetable against Bird flu (H9N2) 44
• 1. Abstract 44