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研究生：	劉芮君 Liu, Rui-Jun
論文名稱：	葡萄籽萃取物對細菌生長及葉酸參與之單碳循環的影響 The impact of grape seed extract on the growth of bacteria and folate-mediated one-carbon metabolism
指導教授：	傅子芳 Fu, Tzu-Fun
學位類別：	碩士 Master
系所名稱：	醫學院 - 醫學檢驗生物技術學系 Department of Medical Laboratory Science and Biotechnology
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	36
中文關鍵詞：	葡萄籽萃取物、細菌、葉酸、單碳循環
外文關鍵詞：	grape seed extract, bacteria, folate, one-carbon metabolism
相關次數：	點閱：69 下載：0
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葡萄籽萃取物（Grape seed extract）富含多種維生素和類黃酮，已被證實具有多種有益的生物活性，如抗氧化、抗菌及抗發炎作用。葡萄籽萃取物被認為是一種重要的營養食品，已被大量生產以做食用。在實驗室之前的研究中發現，葡萄籽萃取物透過抑制二氫葉酸還原酶（一種對核苷酸生物合成和蛋白質代謝至關重要的葉酸代謝酶且是許多抗生素的常見靶標）來抑制金黃色葡萄球菌的生長。然而，尚不清楚服用葡萄籽萃取物是否會抑制益生菌的生長並影響腸道微生物群。在本研究中，我們研究了葡萄籽萃取物對具有臨床意義的細菌生長的影響，特別是耐甲氧西林金黃色葡萄球菌（Methicillin-resistant Staphylococcus aureus, MRSA）和胚芽乳酸桿菌（Lactobacillus plantarum）。 MRSA是一種常見的病原體，易導致皮膚感染且因其抗藥性使得治療上有困難。胚芽乳酸桿菌則是常用以製作許多發酵食品和益生菌補充劑中的重要益生菌。我們觀察到葡萄籽萃取物對於金黃色葡萄球菌，MRSA和胚芽乳酸桿菌的生長抑制有明顯差異。總葉酸含量分析顯示，在三種實驗的細菌中，胚芽乳酸桿菌含有最低的總葉酸含量。高效液相色譜分析單一葉酸的含量發現，在葡萄籽萃取物存在下，四氫葉酸（Tetrahydrofolate一種主要葉酸衍生物）在金黃色葡萄球菌和MRSA中的含量顯著降低，在胚芽乳酸桿菌中則消失。利用資料庫分析及比較三種細菌參與之單碳循環的酵素，發現了單碳循環中葉酸酶組成有些差異，這可能是導致金黃色葡萄球菌和胚芽乳酸桿菌對於葡萄籽萃取物介導的不同抗菌作用。為了評估葡萄籽萃取物對於體內腸道微生物群中的影響，開始嘗試通過口服灌食法在成年斑馬魚中建立胚芽乳酸桿菌的生長，並且獲得了令人鼓舞的初步數據。我們得出結論，葡萄籽萃取物雖然在先前的研究被認為是殺菌的，但並未影響胚芽乳酸桿菌的生長。此外，斑馬魚有極大潛力成為研究腸道微生物群的動物模式。

Grape seed extract (GSE) is enriched of numerous vitamins and flavonoids, possessing multiple beneficial bioactivities such as anti-oxidant, anti-bacteria and anti-inflammation. GSE has been considered an important nutritional supplement and consumed by general publics in large quantity daily. We had shown in our previous studies that GSE inhibited the growth of Staphylococcus aureus by inhibiting dihydrofolate reductase, a folate enzyme crucial to nucleotides biosynthesis and protein metabolism, as well as a common drug target of many antibiotics. However, it remains unclear whether taking GSE will inhibit the growth of probiotics and affect gut microbiota. In this study, we investigated the impact of GSE to the growth of bacteria with clinical significance, specifically Methicillin-resistant Staphylococcus aureus (MRSA) and Lactobacillus plantarum. MRSA is a common pathogen causing skin infection and difficult to treat due to its drug resistance. L. plantarum is an important probiotic found in many fermented food product and probiotic supplement. We observed apparent difference in the growth inhibition mediated by GSE and anti-folate reagents among S. aureus, MRSA and L. plantarum. Total folate content analysis revealed that L. plantarum has the lowest content of total folate among the three examined bacteria strains. HPLC analysis on individual folate showed that the content of tetrahydrofolate (THF), one major folate derivative in S. aureus and MRSA but lacking in L. plantarum, was significantly decreased in the presence of GSE. The folate-mediated one-carbon metabolic pathways and the enzymes involved in these three studied bacteria were analyzed and compared in Silico. Several differences in the components of folate enzymes in one-carbon metabolism were identified, which might contribute to the different antibacterial effects mediated by GSE between S. aureus and L. plantarum. To evaluate the impact of GSE in gut microbiota in vivo, the attempt to establish the growth of L. plantarum in adult zebrafish by oral gavaging was initiated and encouraging preliminary data were obtained. We conclude that GSE, although considered bactericidal, did not affect the growth in L. plantarum. In addition, there is potential to use zebrafish as in vivo model for studying gut microbiota.

Table of Contents

中文摘要	I
Abstract	II
Acknowledgements	III
Table of Contents	IV
Figures	VI
Abbreviations	VII
I.	Introduction	1
1	Folate	1
2	Structure of folate	1
3	Folate in bacteria	3
4	Grape seed extract	3
5	Lactobacillus plantarum	4
6	Zebrafish	5
7	Gastrointestinal tract of zebrafish	5
8	Research about probiotic with zebrafish	6
II.	Rationale and Specific Aims	7
III.	Materials and Methods	8
1.	Materials	8
2.	Fish lines and maintenance	8
3.	Bacterial strain	8
4.	Folate extraction and folate content analysis	9
5.	Disk diffusion antifolate sensitivity test	10
6.	Bacteria growth curve analysis	10
7.	Zebrafish embryo toxicity test	10
8.	Infection of zebrafish with L. plantarum	10
9.	Bacterial DNA extraction and PCR analysis	11
10.	Statistical analysis	11
IV.	Results	12
1.	Inhibition effect of GSE on Staphylococcus aureus and MRSA	12
2.	Different antibacterial effect of GSE on different gut bacteria	12
3.	The growth of L. plantarum was not affected by GSE and L. plantarum may produce folate	13
4.	GSE influenced the folate metabolism in L. plantarum	13
5.	GSE changed the folate composition in bacteria	14
6.	Folate synthesis in Silico: L. plantarum are unable to produce folate in the absence of pABA	14
7.	One-carbon metabolism in Silico	15
8.	DHFR structure of L. plantarum ATCC 14917 is similar to S. aureus	15
9.	L. plantarum can secrete folate in to the culture medium	16
10.	Additional acetate induced the folate content in L. plantarum	16
11.	Embryo toxicity test	16
12.	L. plantarum infection model with adult zebrafish	17
V.	Conclusion	18
VI.	Discussion	19
VII.	Reference	22
VIII.	Figures	26





Figures

Figure 1. The antimicrobial effect of GSE on S. aureus and MRSA.	26
Figure 2. Antibacterial effect of GSE on different gut bacteria	27
Figure 3. Different inhibition effect of antifolate or folic acid on E.coli, S. aureus, and L. plantarum	28
Figure 4.The impact of GSE on the total folate content in E. coli, S. aureus, and L. plantarum	29
Figure 5. Folate composition in bacteria	30
Figure 6. General de novo biosynthetic pathway of folate with indication of the enzyme present in L. plantarum WCFS1 according to KEGG	31
Figure 7. One-carbon metabolism pathway with indication of the enzyme present in (A) L. plantarum WCFS1 and (B) S. aureus according to KEGG	32
Figure 8. Prediction of L. plantarum dihydrofolate reductase structure and compared with DHFR of S. aureus.	33
Figure 9. Secretion of folate into medium and additional acetate in medium change the folate content in L. plantarum	34
Figure 10. Toxicity text of GSE on zebrafish embryo	35
Figure 11. Oral gavaging wild-type adult zebrafish with L. plantarum	36
                                    

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校內：2024-12-31公開
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