益生菌(Leuconostoc mesenteroides) 細菌對糖尿病小鼠血糖水平的影響

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安貝蘇(Anir Batsukh) 查詢紙本館藏

畢業系所

生醫科學與工程學系

論文名稱

益生菌(Leuconostoc mesenteroides) 細菌對糖尿病小鼠血糖水平的影響
(Effect of probiotic (Leuconostoc mesenteroides) bacteria on the regulation of blood glucose level in diabetic mice)

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摘要(中)

益生菌(Leuconostoc mesenteroides) 細菌對糖尿病小鼠血糖水平的影響

摘要：
本研究的目的是檢查益生菌是否可以調節糖尿病小鼠的血糖水平。從Aaruul（一種蒙古乾凝乳）中分離出10種不同的細菌菌株，並通過16S rRNA測序鑑定。這些鑑定的細菌是金黃色葡萄球菌，芽孢桿菌，單純芽孢桿菌，腸膜明串珠菌，葡聚醣芽孢桿菌，人葡萄球菌，馬尾藻，短小芽孢桿菌，表皮葡萄球菌，嗜水氣單胞菌和腸膜明串珠菌。腸膜明串珠菌可以在沒有冷凍室的室溫，pH值在3到7之間下存活。體外實驗的結果表明，細菌可以使用葡萄糖作為碳源來觸發發酵。已經證明，細菌發酵可以產生短鏈脂肪酸（SCFA），包括丁酸。用鏈脲佐菌素（STZ）注射小鼠顯著增加血液中的葡萄糖，模擬1型糖尿病的表型。通過腹部ICR小鼠注射給予丁酸一周增加胰島素並降低STZ誘導的糖尿病小鼠中的葡萄糖。此外，丁酸的給藥降低了IL-6的水平，IL-6是一種可以促進胰島β細胞功能障礙和異常葡萄糖代謝的細胞因子。我們的研究首次表明，腸膜明串珠菌是一種益生菌，丁酸可作為治療高血糖的有益益生菌代謝產物。

關鍵詞：丁酸，糖尿病，腸膜明串珠，益生菌，短鏈脂肪酸(SCFA)

摘要(英)

Effect of probiotic (Leuconostoc mesenteroides) bacteria on the regulation of blood glucose level in diabetic mice

Abstract:
The aim of this study is to exam if probiotic bacteria can regulate the level of blood glucose in diabetic mice. Ten different bacterial strains were isolated from Aaruul, a Mongolian dried curd, and identified by 16S rRNA sequencing. These identified bacteria are Staphylococcus aureus, Bacillus sp, Bacillus simplex, Leuconostoc mesenteroides, Paenibacillus glucanolyticus, Staphylococcus hominis, Megasphaera massiliensis, Bacillus pumilus, Staphylococcus epidermidis, Enterobacter Xiangfangensis and Leuconostoc mesenteroides. Leuconostoc mesenteroides can survive in the room temperature without a freezer and at pH values in the range from 3 to 7. Results from in vitro experiments indicated that the bacteria can use glucose as a carbon source to trigger fermentation. It has been documented that bacterial fermentation can produce short-chain fatty acids (SCFAs) including butyric acid. Injection of mice with streptozotocin (STZ) dramatically increased the glucose in the blood, mimicking a phenotype of Type 1 diabetes. Administration of butyric acid by injection in abdominal ICR mice for a week increased insulin and decreased glucose in STZ-induced diabetic mice. In addition, administration of butyric acid lowered the level of IL-6, a cytokine which can promote islet β-cell dysfunction and abnormal glucose metabolism. Our study demonstrates for the first time that Leuconostoc mesenteroides is a probiotic bacterium and butyric acid acts as a beneficial probiotic metabolite for the treatment of hyperglycemia.

Keywords: butyric acid, diabetes, Leuconostoc mesenteroides, probiotics, short chain fatty acids (SCFAs)

關鍵字(中)

★ 丁酸
★ 糖尿病
★ 腸膜明串珠
★ 益生菌
★ 短鏈脂肪酸(SCFA)

關鍵字(英)

★ butyric acid
★ diabetes
★ Leuconostoc mesenteroides
★ probiotics
★ short chain fatty acids (SCFAs)

論文目次

CHAPTER 1: INTRODUCTION: 1
1.1 Mongolian traditional dried curd milk: 1
1.2 Background information: 1
1.2.1 Method:1
1.2.2 Minerals:3
1.3 Activities of dried curd: 4
1.3.1 Antibacterial activity: 4
1.3.2 Against influenza: 5
1.3.3 Antiallergic activity: 5
1.3.4 Angiotensin-hypertensive peptide:5
2. Leuconostoc mesenteroides: 5
2.1 Probiotic activity of L.mesenteroides: 7
2.1.1 Anticancer activity: 7
2.1.2 Bacteriocin activity: 7
2.2 Function of probiotic to produce SCFAs: 7
3. Diabetes mellitus: 8
3.1 Type 1 Diabetes mellitus: 9
3.2 Type 2 Diabetes mellitus:10
3.3 Treatments of diabetes: 11
3.3.1 Butyrate in prevention and treatment of diabetes: 11
CHAPTER 2: MATERIALS AND METHODS
1. Apparatus or instruments:13
2. Reagents: 11
1. Probiotic bacterium identification: 15
1.1 Preparation of bacterium sample: 15
1.2 DNA extraction, PCR and 16s RNA sequencing: 15
2. Making dried curd milk using Leuconostoc mesenteroides: 17
2.1 Leuconostoc mesentreoides fermenting cow milk (yogurt): 17
2.2 Making dried curd milk using Leuconostoc mesenteroides 17
3. Probiotic properties (Leuconostoc mesenteroides): 17
3.1 Leuconostoc mesenteroides growth temperature, heat and pH value: 18
3.2 Leuconostoc mesenteroides fermentation: 18
4. Butyric acid detection by HPLC from mice intestine after feeding with L.mesenteroides: 18
4.1 Sample preparation: 18
4.1.1 Animal experiment: 19
4.1.2 Preparation of samples for analysis:19
4.2 Determination of Butyric acid:19
4.2.1 Butyric acid standard stock and sample for HPLC analysis: 19
4.3 Preparation of calibration standard curve: 20
5. Butyric acid treatment in Diabetic induced mice 20
5.1 Blood glucose index:21
5.2 Preparation of the blood sample for insulin and IL-6 analysis: 21
5.2.1 Insulin analysis by ELISA test: 21
5.2.2 IL-6 analysis by ELISA test:22
CHAPTER 3: RESULTS
1. Identification of probiotics: 24
2. Making dried curd with Leuconostoc mesenteroides 25
3. Probiotic properties: 26
3.1 Leuconostoc mesenteroides growth temperature, heat and pH value: 26
3.2 Leuconostoc mesenteroides heat resistance: 27
3.3 Leuconostoc mesenteroides pH value: 28
3.4 Leuconostoc mesenteroides fermentation: 29
3.4.1 Determination of Butyric acid by HPLC: 30
4. HPLC analysis for Butyric acid from mice intestine sample: 31
5. Butyric acid treatment in diabetic induced mice 32
5.1 The blood glucose level of Butyric acid treatment in diabetic mice: 32
5.2 Insulin level of Butyric acid treatment in diabetes mellitus mice: 33
5.3 IL-6 analysis: 34
6. CHAPTER 4: DISCUSSION AND CONCLUSION: 36
7. CHAPTER 5: REFERENCES:38

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指導教授

黃俊銘(Eric Chun-Ming Huang)

審核日期

2019-6-28

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