博碩士論文 105887602 詳細資訊



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姓名 范明新(Pham Minh Tan)  查詢紙本館藏   畢業系所 生醫科學與工程學系
論文名稱 有益微生物的真菌學和細菌學研究： 在農業和人類健康中的應用
(Mycological and bacteriological studies of beneficial microbes: Applications for Agriculture and Human Health)
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摘要(中) 近來從研究發現微生物可以進行改造，以調節植物抵抗禦環境的防禦機制，並強烈增強植物的生長和健康。木腐蘑菇在木質纖維素培養基中可促進植物生長並有提高農業生產力的潛力，杏鮑菇是存在於自然界的白色腐生性的真菌菌株，研究發現其可持續的在30oC和pH 5的環境下將10 mM L-色氨酸（TRP）轉化為酪氨酸-3-乙酸（IAA），跟其它的杏鮑菇種相比，杏鮑菇更適合在更適合於亞熱帶和熱帶氣候生長。真菌在纖維素存在會使用IPA途徑合成IAA，但是環境中高濃度的外源IAA（10 µg / mL）可能會抑制真菌的生長，IAA在單子葉植物（大米）中，發現會增加側根的數量，而在雙子葉植物（番茄）中，根的長度會增長，通過實驗證明了對線蟲有吸引力和毒性。杏鮑菇有較佳的草酸溶解在菌絲體的區域有氧化物修飾的基質（CaO和ZnO）。
在體外試管雙重培養實驗中發現肺假單尖孢鐮刀菌和霍亂單胞菌有拮抗作用，這些新發現及影響可能為在農業中的應用帶來新的見解。相反，Marasmius palmivorus在台灣的福爾摩沙棕櫚（Arenga engleri）樹木上產生症狀，從而使棕櫚樹分枝桿菌在棕櫚種植園中的危害比以往預期的要大。
近來研究發現人體腸道細菌中檢測到電子產生可能與人體健康密切相關，腸系膜十二指腸菌Leuconostoc mesenteroides可促使亞油酸發酵來當作電子抗氧化機制，連續6週攝入高脂飲食（HFD）會誘導的ROS（活性氧）的產生，脂肪細胞3T3-L1分化過程中，脂質積累的增加與ROS的釋放同時發生，這些可能都與腸系膜十二指腸菌誘導的亞油酸發酵產生的電子有關，提供對脂肪形成過程中脂質積累和ROS產生以及高脂飲食誘導的ROS的抑製作用，此外Cyclophilin A蛋白在腸道中可抑制電子產生的產生，我們的研究結果表明，腸系膜十二指腸菌藉由亞油酸的發酵的通過電子產生抑制ROS和脂質的產生，而電子產生受Cyclophilin A蛋白調節。
膜糖蛋白是嚴重急性呼吸系統綜合症冠狀病毒2（SARS-CoV-2）的最豐富蛋白質，但其在2019年冠狀病毒病（COVID-19）中的作用機制尚未完全。與綠色熒光蛋白 (GFP)的小鼠相比，鼻內接種膜糖蛋白的小鼠在支氣管肺泡灌洗液（BALF）白細胞介素-6增加，白細胞介素-6是細胞因子風暴的標誌。膜糖蛋白誘導的高水平的白細胞介素-6在磷酸二酯酶4（PDE4B）敲除小鼠中顯著降低，證明了PDE4B在白細胞介素-6信號傳導中的重要作用。鼠李糖乳桿菌EH8菌株的菌絲體發酵產生了丁酸，它可以下調巨噬細胞中PDE4B的表達和IL-6的分泌。用菌絲體餵養小鼠可增加鼠李糖乳桿菌的相對豐度。鼠李糖乳桿菌和菌絲體補充兩週後，可大大降低膜糖蛋白誘導的PDE4B表達和IL-6分泌。鼠李糖乳桿菌和菌絲體對膜糖蛋白的益生菌活性在用游離脂肪酸受體2 (Ffar2) 拮抗劑GLPG-0974處理的小鼠中被取消。 Ffar2在腸肺軸中的激活以下調PDE4B-IL-6信號傳導可能為開發包括益生菌在內的治療包括COVID-19中細胞因子風暴的治療方法提供靶點。
摘要(英) Microorganisms have recently been engineered to modulate plant defense mechanisms against environmental stress and strongly enhance plant growth and health. The promising plant-growth promoting capability of a wood-decay mushroom in lignocellulosic media indicates the potential to enhance the agricultural productivity in a sustainable way. A wild native strain of a white rot fungus identified as Pleurotus pulmonarius was found to convert 10 mM L-tryptophan (TRP) to indole-3-acetic acid (IAA) under the optimal growth conditions of 30oC and pH 5. The higher temperature optimum of P. pulmonarius isolated from subtropical environment compared to other Pleurotus species from temperate regions makes it more suitable for application in subtropical/tropical regions. Results of gas chromatography-mass spectrometry indicated IAA synthesis through the IPA pathway when using cellulose as a sole carbon source. However, high concentration of exogenous IAA (10 µg/mL) in the environment could inhibit the fungal growth. The mycelium as well as the culture filtrate promoted the growth and chlorophyll content of seedlings. In a monocotyledonous plant (rice), the number of lateral roots was increased experimentally, whereas in a dicotyledonous plant (tomato), the fungus led to an increased length of shoots and roots. Moreover, the attraction and toxicity against the nematode Caenorhabditis elegans was proven experimentally. Solubilized oxalates activity of P. pulmonarius on oxide-amended substrates (CaO and ZnO) showed clear zones under the mycelium. A weak antifungal ability against Fusarium oxysporum and Lasiodiplodia hormozganensis was observed in vitro dual cultures. The combination of these capabilities of P. pulmonarius may provide new opportunities for application in agriculture. By contrast, Marasmius palmivorus produced symptoms on Formosa palm (Arenga engleri) seedlings in Taiwan, causing a greater risk by M. palmivorus in palm plantations than hitherto anticipated.
Although several electrogenic bacteria have been identified, the physiological effect of electricity generated by bacteria on host health remains elusive. We found that probiotic Leuconostoc mesenteroides can metabolize linoleic acid to yield electricity via an intracellular cyclophilin A-dependent pathway. Inhibition of cyclophilin A significantly abolished bacterial electricity and the suppressive effect of Leuconostoc mesenteroides on the accumulation of reactive oxygen species (ROS) and lipids during 3T3-L1 adipocyte differentiation. Oral administration of Leuconostoc mesenteroides plus linoleic acid remarkably reduced high-fat-diet (HFD)-induced formation of 4-hydroxy-2-nonenal (4-HNE), a ROS biomarker, and decreased abdominal fat mass in mice. The reduction of 4-HNE and abdominal fat mass was reversed when cyclophilin A inhibitor-pretreated bacteria were administered to mice. Our studies present a novel mechanism of reducing abdominal fat mass by limiting adipogenesis-induced ROS using electricity produced by probiotic bacteria.
Membrane glycoprotein is the most abundant protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but its role in coronavirus disease 2019 (COVID-19) has not been fully characterized. Mice intranasally inoculated with membrane glycoprotein substantially increased the interleukin (IL)-6, a hallmark of the cytokine storm, in bronchoalveolar lavage fluid (BALF), compared to mice inoculated with green fluorescent protein (GFP). The high level of IL-6 induced by membrane glycoprotein was significantly diminished in phosphodiesterase 4 (PDE4B) knockout mice, demonstrating the essential role of PDE4B in IL-6 signaling. Mycelium fermentation of Lactobacillus rhamnosus (L. rhamnosus) EH8 strain yielded butyric acid, which can down-regulate the PDE4B expression and IL-6 secretion in macrophages. Feeding mice with mycelia increased the relative abundance of commensal L. rhamnosus. Two-week supplementation of mice with L. rhamnosus plus mycelia considerably decreased membrane glycoprotein-induced PDE4B expression and IL-6 secretion. The probiotic activity of L. rhamnosus plus mycelia against membrane glycoprotein was abolished in mice treated with GLPG-0974, an antagonist of free fatty acid receptor 2 (Ffar2). Activation of Ffar2 in the gut-lung axis for down-regulation of the PDE4B-IL-6 signalling may provide targets for development of modalities including probiotics for treatment of the cytokine storm in COVID-19.
關鍵字(中) ★ 微生物
★ L-色氨酸
★ 3-乙酸
★ 福爾摩沙棕櫚
★ 現人體腸道細菌		 關鍵字(英) ★ Microorganism
★ L-tryptophan
★ 3-acetic acid
★ Formosa Palm
★ Human intestinal bacteria
論文目次 TABLE OF CONTENTS

CHINESE ABSTRACT i
ENGLISH ABSTRACT iii
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vii
LIST OF FIGURES xii
LIST OF TABLES xx
CHAPTER I INTRODUCTION 1
1.1 Plant biotic and abiotic stress 1
1.2 White rot fungi 1
1.2.1 Pleurotus pulmonarius 2
1.2.2 Marasmius palmivorus 2
1.3 Effect of ligninolytic mushroom on plant growth and health 3
1.3.1 Plant probiotic 3
1.3.2 Pathogenic Marasmiaceae on Formosa palm (Arenga engleri) 3
1.3 Human gut prebiotic from mushroom 4
1.4 Oxidative stress-related obesity 4
1.5 Leuconostoc mesenteroides 5
1.6 Cyclophilin A (CypA) 6
1.7 Electron production by bacteria 6
1.8 Coronavirus disease 2019 (Covid-19) 7
1.9 Cytokine storm in COVID-19 pneumonia 7
2.0 Gut microbiota treatment in COVID-19 pneumonia 8
CHAPTER II THE PLANT GROWTH-PROMOTING POTENTIAL OF THE MESOPHILIC WOOD-ROT MUSHROOM PLEUROTUS PULMONARIUS 9
2.1 Introduction 9
2.2 Materials and methods 10
2.2.1 Fungal strains 10
2.2.2 DNA isolation and amplification of 5.8S-ITS rDNA regions 10
2.2.3 Phylogenetic analysis 11
2.2.4 Fungal growth in lignocellulosic medium 11
2.2.5 IAA production by P. pulmonarius in CMC medium 12
2.2.6 IAA estimation 12
2.2.7 Gas chromatography–mass spectrometry analysis of IAA produced by P. pulmonarius 13
2.2.8 Effects of exogenously tested IAA on P. pulmonarius growth 13
2.2.9 The effect of culture crude extract on rice coleoptile elongation 13
2.2.10 The effect of growing mycelium and culture crude extract on rice and tomato seedlings 14
2.2.11 Measuring chlorophyll content 14
2.2.12 Statistical analysis 14
2.3 Results 15
2.3.1 Identification and phylogenetic analyses of the test fungi 15
2.3.2 Lignocellulosic substrate utilization test 16
2.3.3 Triggered-IAA production by TRP in P. pulmonarius 18
2.3.4 Effects of exogenously tested IAA on P. pulmonarius growth 20
2.3.5 Tryptophan-dependent auxin production by P. pulmonarius 20
2.4 Discussion 25
CHAPTER III ANTAGONISM AGAINST SOIL NEMATODES AND PLANT PATHOGENS AND TEST OF OXIDE SOLUBILIZATION IN A SUBTROPICAL WOOD-DECAY MUSHROOM 29
3.1 Introduction 29
3.2 Materials and methods 30
3.2.1 Strains 30
3.2.2 Chemotaxis and nematicidal assay 30
3.2.3 In vitro antagonism of P. pulmonarius against potential plant pathogens 31
3.2.4 Oxide solubilization 31
3.2.5 Statistical data analysis 32
3.3 Results 32
3.3.1 Attraction and toxicity of P. pulmonarius to C. elegans 32
3.3.2 In vitro antagonism of P. pulmonarius against potential plant pathogens 33
3.3.3 Oxide solubilization 34
3.4 Discussion 34
CHAPTER IV FIRST REPORT OF THE OIL PALM DISEASE FUNGUS MARASMIUS PALMIVORUS FROM TAIWAN CAUSING STEM ROT DISEASE ON NATIVE FORMOSA PALM ARENGA ENGLERI AS NEW HOST 37
4.1 Introduction 37
4.2 Materials and methods 38
4.2.1 Collection and morphology 38
4.2.2 DNA isolation and amplification of 5.8S-ITS rDNA region 39
4.2.3 Phylogenetic analysis 39
4.2.4 Pathogenicity tests on palm seedlings 39
4.3 Results 40
4.3.1 Molecular identification 40
4.3.2 Morphology 42
4.3.3 Pathogenicity tests on palm seedlings 43
4.4 Discussion 45
CHAPTER V LEUCONOSTOC MESENTEROIDES MEDIATES AN ELECTROGENIC PATHWAY TO ATTENUATE THE ACCUMULATION OF ABDOMINAL FAT MASS INDUCED BY HIGH FAT DIET 47
5.1 Introduction 47
5.2 Materials and methods 48
5.2.1 Ethics statement 48
5.2.2 Bacterial culture 49
5.2.3 Electricity detection 49
5.2.4 Real-time polymerase chain reaction (PCR) 49
5.2.5 Ferrozine assays 50
5.2.6 Gas chromatography mass spectrometry (GC-MS) analysis 50
5.2.7 Bacterial adhesion assay 50
5.2.8 Cell culture and differentiation 51
5.2.9 Oil red O staining 51
5.2.10 Measurement of intracellular ROS 52
5.2.11 High-fat-diet (HFD) fed mice 52
5.2.12 Western blotting 52
5.2.13 Statistical analysis 53
5.3 Results 53
5.3.1 Electricity and SCFAs were produced by L. mesenteroides EH-1 plus linoleic acid 53
5.3.2 Adipocyte differentiation was attenuated by fermentation media of L. mesenteroides and butyrate 55
5.3.3 The formation of ROS and 4-HNE was suppressed by L. mesenteroides EH-1 57
5.3.4 Cyclophilin A mediated electricity production of L. mesenteroides EH-1 59
5.3.5 Cyclophilin A was essential for bacterial adhesion and reduction of the formation of 4-HNE and abdominal fat depots 61
5.4 Discussion 63
CHAPTER VI GUT PROBIOTIC LACTOBACILLUS RHAMNOSUS ATTENUATES PDE4B-MEDIATED INTERLEUKIN-6 INDUCED BY SARS-COV-2 MEMBRANE GLYCOPROTEIN 68
6.1 Introduction 68
6.2 Materials and Methods 69
6.2.1 Ethics statement 69
6.2.2 Cloning, expression and purification of SARS-CoV-2 membrane glycoprotein 69
6.2.3 Detection of IL-6 in BALF 70
6.2.4 Real-time polymerase chain reaction (RT PCR) 70
6.2.5 Mycelium preparation 70
6.2.6 Mycelium fermentation of L. rhamnosus EH8 71
6.2.7 Quantification of L. rhamnosus 71
6.2.8 Cell culture 71
6.2.9 Detection of butyric acid by high performance liquid chromatography (HPLC) 72
6.2.10 Inhibition of Ffar2 72
6.2.11 Statistical analysis 72
6.3 Results 72
6.3.1 PDE4B is essential for induction of IL-6 by SARS-CoV-2 membrane glycoprotein 72
6.3.2 Mycelium fermentation of L. rhamnosus EH8 mitigates SARS-CoV-2 membrane glycoprotein-induced IL-6 74
6.3.3 Butyric acid was produced by mycelium fermentation of L. rhamnosus EH8 and reduced IL-6 secretion and PDE4B expression in macrophages 75
6.3.4 Ffar2 mediates the effect of mycelium fermentation of L. rhamnosus EH8 on reduction of PDE4B and IL-6 77
6.4 Discussion 78
CHAPTER VII CONCLUSION 81
CHAPTER VIII OUTLOOK 84
BIBLIOGRAPHY 86
APPENDIX 1 LIST OF CONFERENCES 115
APPENDIX 2 LIST OF PUBLICATIONS 116
Appendix 2.1 Journal papers 116
Appendix 2.2 Manuscript in preparation 116
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指導教授 黃俊銘 羅南德(Chun-Ming Huang Roland Kirschner) 審核日期 2020-12-29
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