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研究生:范氏姮娥
研究生(外文):Pham, Thi Hang Nga
論文名稱:丁酸梭菌(Clostridium butyricum)胞外多醣減輕TNBS誘導的斑馬魚小腸結腸炎
論文名稱(外文):Exopolysaccharides extracted from Clostridium butyricum alleviates TNBS-induced enterocolitis of zebrafish
指導教授:陳又嘉陳又嘉引用關係陳與國胡淳怡
指導教授(外文):Chen, Yo-ChiaChen, Yu-KuoHu, Chun-Yi
口試委員:陳玉桂蔡蕙芸
口試委員(外文):Chen, Yu-KueiTsai, Hui-Yun
口試日期:2023-07-03
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:食品科學國際碩士學位學程
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:英文
論文頁數:60
中文關鍵詞:胞外多糖斑馬魚丁酸梭菌抗炎腸結腸炎
外文關鍵詞:ExopolysaccharideZebrafishClostridium butyricumAnti-inflammationEnterocolitis
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最近,丁酸梭菌因其在許多領域的潜力而受到越来越多的關注,包括抗炎、抗應激和許多其他健康益處。然而,針對胞外多糖(EPS)的研究卻十分有限。因此,本研究採用兩種類型的丁酸梭菌(Clostridium butyricum BCRC 80431和Clostridium butyricum CBML2)來萃取EPS。在商品化的強化梭菌培養基中加入不同糖類,以提高EPS的產量,結果發現,在reinforced clostridium medium (RCM)中加入澱粉時,胞外多糖產量最高(4.05±0.11和5.47±0.05毫克/毫升)。單醣組成分析顯示,兩種菌株的胞外多醣都是異多醣,主要由半乳糖和甘露糖組成。從兩種菌株中萃取的EPS具有抗氧化和抗炎活性。BCRC 80431和CBML2的斑馬魚餵食EPS可降低TNF-α表現(5.4倍和27倍),增加IL-10表現(72倍和130倍),表示丁酸梭菌EPS可通過恢復免疫穩態緩解腸道炎症。給予EPS可以明顯減少TNF-α基因的表現,顯示EPS可以經由減少TNF-α的表現和增加IL-10基因的表現來降低發炎。此外EPS還能恢復三硝基苯磺酸(TNBS)誘導的結腸炎,腸道微生物群的平衡,以及促進有益菌的生長和抑制致病菌的生長。動物實驗顯示,兩組餵食EPS的斑馬魚對新環境的焦慮程度都較低。總之丁酸梭菌的 EPS 可以調節細胞激素的產生,恢復斑馬魚腸道微生物多樣性,並減少焦慮行為,這表腸腸道-大腦軸之間存在密切關係。
Recently, Clostridium butyricum has been receiving more and more attention for their potentials in many fields, including anti-inflammation, anti-stress and many other health benefits. However, studies that focus on their exopolysaccharide (EPS) are quite limited. Therefore, in this research, two strains of Clostridium butyricum (Clostridium butyricum BCRC 80431 and Clostridium butyricum CBML2) were used to extract EPS. Supplemental sugars were added to commercial reinforced clostridia medium (RCM) to increase the yield of EPS, and it was found that the highest yield (4.05±0.11 and 5.47±0.05 g/mL) was obtained when starch was added to the RCM. Monosaccharide composition analysis showed that both strains were heteropolysaccharides, mainly composed of galactose and mannose. The EPS derived from both strains demonstrated antioxidant and anti-inflammatory properties. Administration of EPS significantly reduced TNF-α expression (5.4 and 27 times) and increased IL-10 level (72 and 130 times) in BCRC 80431 and CBML2 zebrafish, indicating that EPS of Clostridium butyricum can alleviate gut inflammation by restoring immune homeostasis. In addition, EPS can also restore the balance of gut microbiota in trinitrobenzene sulfonic acid (TNBS)-induced colitis, as well as promoting the growth of beneficial bacteria and suppressing the pathogenic ones. Corresponding to the level of inflammation cytokines, zebrafish from both EPS-treated group show lower anxiety levels towards novel environments. In conclusion, EPSs from C.butyricum can regulate cytokines production, restore gut microbiota diversity in zebrafish as well as reduce anxiety-like behaviors, which indicated a strong relationship of the gut-brain axis.
TABLE OF CONTENTS
摘要 I
ABSTRACT II
ACKNOWLEDGEMENTS IV
TABLE OF CONTENTS V
LIST OF TABLES VII
LIST OF FIGURE VIII
1. INTRODUCTION 1
2. LITERATURE REVIEW 2
2.1 Clostridium butyricum 2
2.2 Exopolysaccharide 3
2.3 Danio rerio 5
2.4 Trinitrobenzenesulfonic acid 7
2.5 Inflammatory bowel disease 8
2.6 Gut microbiota diversity 12
3. MATERIALS AND METHODS 14
3.1 Framework 14
3.2 Material 14
3.2.1 Bacterial strain and Animal model 14
3.2.2 Chemical 15
3.3 Methods 15
3.3.1 Medium optimization for exopolysaccharide production 15
3.3.2 Anti-oxidative properties 16
3.3.3 Monosaccharide composition 17
3.3.4 Anti-inflammation and Anti-anxiety properties in vivo 18
3.3.4.1 Animal and Housing 18
3.3.4.2 TNBS-induced colitis 18
3.3.4.3 Animal behavior experiment 19
3.3.4.3.1 Novel tank test 19
3.3.4.3.2 Light/dark preference test 19
3.3.4.4 Euthanasia 21
3.3.4.5 RNA-free workplace preparation 21
3.3.4.6 RNA extraction 21
3.3.4.7 cDNA synthesis 21
3.3.4.8 Quantitative real-time PCR 22
3.3.4.9 Next Generation Sequencing and Data analysis 24
3.3.4.9.1 Sample preparation and DNA library preparation 24
3.3.4.9.2 Next generation sequencing and Data analysis 24
3.4 Statistical analysis 25
4. RESULTS AND DISCUSSION 26
4.1 Exopolysaccharide extracted from Clostridium butyricum 26
4.1.1 Exopolysaccharide production 26
4.1.2 Monosaccharide composition 28
4.2 Antioxidant activity 31
4.3 Anti-inflammatory properties 32
4.4 Anti-anxiety properties 33
4.4.1 Novel tank test 34
4.4.2 Light/ dark preference test 35
4.5 Gut microflora abundance 35
5. CONCLUSION 40
6. REFERENCES 41
7. INDEX 51
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