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研究生:張芷婷
研究生(外文):Chih-Ting Chang
論文名稱:探討常見食用乳化劑對於腸道菌相、腸道黏液層與代謝之影響
論文名稱(外文):Investigation into the effects of dietary emulsifiers on gut microbiota, mucus layer and metabolism
指導教授:沈立言沈立言引用關係
口試委員:吳明賢余佳慧湯森林
口試日期:2019-07-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
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:2019
畢業學年度:107
語文別:中文
論文頁數:119
中文關鍵詞:乳化劑腸道菌相腸黏膜代謝食品添加物
DOI:10.6342/NTU201903876
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乳化劑是一類常見的食品添加劑,近年來有研究指出,乳化劑的食用可能與肥胖、代謝症候群的發生或其他慢性炎症疾病發展有關,而越來越多證據顯示,人體腸道菌與宿主之間的交互作用,是肥胖、代謝症候群和腸炎的重要因素。根據先前研究發現,給予小鼠兩種不同乳化劑 carboxymethylcellulose (CMC) 和 polysorbate 80 (P80),可能使腸道黏膜分泌的黏液乳化,導致黏液層(Mucus layer) 變薄,使腸道通透性增加,讓體循環中 lipopolysaccharide (LPS)水平升高,促使腸細胞發炎,可能是導致代謝症候群和慢性腸炎發展的原因之一。然而乳化劑平均曝露量高之脂肪酸甘油酯 (mono-and diglycerides, MDG)、卵磷脂 (lecithin)、脂肪酸蔗糖酯 (sucrose fatty acid esters) 是否對人體產生任何代謝影響,目前仍未清楚。因此,本研究之目的為深入了解乳化劑-MDG, lecithin, sucrose fatty acid esters 對腸道微生物的影響,探討腸道菌是否因為乳化劑的介入而造成失衡,並透過腸道黏液層對宿主造成影響。本實驗採用 C57BL/6 雄性小鼠進行實驗,由於受到乳化劑本身特質影響,本次實驗將會依據親水親油平衡值 (hydrophile-lipophile balance, HLB) 的高低將實驗分為兩個子實驗,子實驗一為透過飲用水介入卵磷脂 (7523 mg/kg bw/day),脂肪酸蔗糖酯 (1110 mg/kg bw/day) 和羧甲基纖維素 (1853 mg/kg bw/day),實驗為 12 週,子實驗二透過客製化飼料介入脂肪酸甘油酯 (40 mg/kg ),在 12 及 24 週後,分別進行犧牲取得血液、臟器及糞便等檢體。子實驗一結果顯示,以 lecithin、sucrose fatty acid esters 和 CMC 介入的小鼠體重略微增加但沒有達到顯著差異,並且與控制組相比實驗組在脂肪重量、腸道通透性、結腸長度及結腸病理切片皆沒有顯著差異,顯示個體並沒有誘發腸炎的表徵。觀察腸道菌與腸黏膜層相對定位及厚度測量,並未發現腸道菌入侵腸黏膜層以及黏膜層變薄之現象。代謝方面與控制組相比 sucrose fatty acid esters 組有胰島素抗性、空腹血糖值與 HOMA-IR 指標升高現象,顯示 sucrose fatty acid esters 有造成代謝功能異常之風險。而腸道菌相分析結果顯示,主座標分析 (PCoA analysis) 可以發現乳化劑實驗組與控制組相比,菌相組成結構相異,而在盲腸內腸道菌相多樣性 (alpha diversity) 和控制組相比並無顯著差異,表示乳化劑的介入並不會造成物種的豐富度與均勻度降低。進一步探究腸道菌相的組成發現在 sucrose fatty acid esters 組中 Akkermansia spp. 顯著增加。子實驗二以 MDG 介入的小鼠在體重與脂肪重量結果顯著低於控制組,在腸道通透性與結腸長度以及血糖代謝試驗與控制組相比皆無顯著差異,顯示個體並沒有誘發腸炎及代謝症候群之表徵。觀察腸道菌與腸黏膜層相對定位及厚度測量,並未發現腸道菌入侵腸黏膜層與黏膜層變薄之現象。腸道菌相分析結果顯示主座標分析 (PCoA analysis) 可以發現乳化劑實驗組與控制組相比,菌相組成結構相異,而在盲腸內腸道菌相多樣性 (alpha diversity) 並無與控制組有顯著差異,表示乳化劑的介入並不會造成物種的豐富度與均勻度降低。本研究之結論為,乳化劑的使用在曝露量 10 倍的條件下並不是造成腸炎與代謝症候群的必要性因子,並且不會透過調節腸道菌組成而利用腸道黏液層對宿主造成影響,但 sucrose fatty acid esters 與 CMC 有造成代謝異常之風險。
Emulsifiers are common food additives used in various kinds of food product. Recent studies reported that two emulsifiers (carboxymethylcellulose, CMC; polysorbate 80, P80) could cause intestinal inflammation and metabolic syndrome by attenuation of intestinal mucus layer as well as increased the gut epithelial permeability to increase the lipopolysaccharide level. Once the high level of lipopolysaccharides in GI tract may induce the inflammation of intestinal cell. One of possible reasons progress the metabolic syndrome and colitis. However, we do not know if other commonly used emulsifiers such as mono- and diglycerides, lecithin and sucrose fatty acid esters could lead to similar health concerns. Thus, the current study is to investigate the effects of emulsifiers on gut microbiota and whether the intervention of the emulsifiers could cause the gut microbiota dysbiosis and via the mucus layer has the impact to host. Because of the property of emulsifiers, the study was divided into two parts. Based on the hydrophile-lipophile balance (HLB) value. In first part, mice experiment was conducted by using fifteen-week-old male C57BL/6 mice fed with chow diet and supplemented by lecithin (7523 mg/kg bw/day), sucrose fatty acid esters (1110 mg/kg bw/day), and carboxymethylcellulose (1853 mg/kg bw/day) in water for 12 weeks. The dosages of intervention are based on human daily exposure of these emulsifiers reported in the literature. The results of the first part showed the body weight of mice treated with emulsifiers were slightly increased without significant difference. There was no significant finding on fat mass, intestinal permeability, colon length and histopathology analysis as compared with the control group. It means no clue for inducing symptom of colitis. Observation the bacteria localization and measurement the mucus layer thickness, there is no microbiota encroachment and mucus layer thinning. However, emulsifier treatments (excluding lecithin) impaired glycemic control as assessed by fasting blood glucose concentration and serum insulin concentration. Our data suggest that sucrose fatty acid esters and CMC might induce metabolic disorder. In addition, 16S rRNA metagenomic analysis showed the gut microbiota compositions were altered by emulsifiers in cecum content. Significantly, we found that Akkermansia spp. Are much higher in sucrose fatty acid esters group. However, no significant differences were observed in alpha diversity. Another part of experiment was supplementation with mono- and diglycerides (40 mg/kg) in customized feeding. After intervention for 12 and 24 weeks, mice were sacrificed. Organs and blood were collected for subsequent analysis. In this part, the body weight and fat mass in the mono- and diglycerides group were significantly decreased. The results of gut permeability test, colon length and OGTT test which were no significant differences were observed between groups. Our data suggested there is no potential for colitis and metabolic syndrome. Analyzed bacteria localization at the surface of the intestinal mucosa and measured the thickness of mucus layer. The emulsifier-treated mice did not show microbiota encroachment and mucus layer thinning. The 16S rRNA metagenomics analysis revealed that mono- and diglycerides dramatically altered microbiota composition, which assessed by principal coordinate analysis. However, mono- and diglycerides administration did not reduced the alpha diversity. Our results suggest that common dietary emulsifiers can shape the microbiome composition, but did not cause mucus layer thinning, not necessarily cause metabolic disorder or intestinal inflammation in mouse model but exist potential risk for glucose metabolic disorder.
中文摘要 I
Abstract III
目錄 V
圖次 VIII
表次 I
縮寫表 I
第一章、前言 1
第二章、文獻回顧 2
第一節 腸道菌 2
一、 簡介 2
二、 飲食對於腸道菌與宿主之影響 3
三、 腸道菌與乳化劑之關係探討 4
第二節 乳化劑 5
ㄧ、 簡介 5
二、 目前已被研究之乳化劑與腸道菌之關聯性 5
三、 本次實驗所使用之乳化劑 7
第三節 腸道黏液層 8
ㄧ、 簡介 8
二、 功能 9
三、 腸道黏液層、腸道菌與飲食之關係探討 11
第三章、研究假說與目的 12
第一節 研究假說 12
第二節 研究目的 12
第四章、實驗架構 13
第一節 子實驗一 13
第二節 子實驗二 14
第五章、實驗材料與方法 15
第一節 實驗材料 15
一、 乳化劑 15
二、 實驗藥品 15
三、 實驗設備及儀器 16
四、 溶液配置 18
第二節 實驗方法 19
一、 乳化劑劑量設計 19
二、 乳化劑飼料 20
三、 動物實驗 21
第三節 統計分析 26
第六章、實驗結果 27
第一節 子實驗一 27
一、 小鼠體重、脂肪組織重量、瘦體組織重量 27
二、 結腸長度、腸通透性檢測與血清中 LPS 測定 27
三、 結腸組織病理切片檢測 28
四、 腸道菌與腸道黏液層共定位與距離觀測 28
五、 口服葡萄糖耐受性測驗結果 28
六、 空腹血糖、空腹胰島素濃度與 HOMA-IR 指標分析 28
七、 血清生化值分析 29
八、 菌相分析 29
第二節 子實驗二 30
ㄧ、 小鼠體重、脂肪組織重量、瘦體組織重量 30
二、 結腸長度、腸通透性檢測與血清中 LPS 測定 30
三、 結腸組織病理切片之檢測 31
四、 腸道菌與腸道黏液層共定位與厚度檢測 31
五、 口服葡萄糖耐受性測驗結果血清生化值分析 31
六、 空腹血糖、空腹胰島素濃度與 HOMA-IR 指標分析 31
七、 血清生化值分析 32
八、 菌相分析 32
第七章、討論 33
第一節 子實驗一 33
一、 小鼠體重變化 33
二、 誘導腸炎討論 33
三、 誘導代謝症候群討論 33
四、 菌相分析 34
五、 CMC組討論 35
第二節 子實驗二 37
一、 小鼠體重與脂肪組織變化 37
二、 誘導腸炎討論 37
三、 誘導代謝症候群討論 38
四、 血清生化值 38
第八章、結論 39
第九章、未來研究方向 41
第十章、實驗結果圖表 42
第一節 子實驗一 42
第二節 子實驗二 63
第三節 與先前文獻結果比較腸道菌分析 85
第十一章、參考文獻 90
第十二章、附錄 96
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