微生物作為天然益生菌產生發酵代謝物改善常見皮膚問題

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高馨柔(Hsin-Jou Kao) 查詢紙本館藏

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生物醫學工程研究所

論文名稱

微生物作為天然益生菌產生發酵代謝物改善常見皮膚問題

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至系統瀏覽論文 (2027-9-20以後開放)

摘要(中)

人類皮膚表面蘊含無數種類的微生物定植，利用空間與養分的競爭達到平衡保持健康狀態，當外在壓力或環境改變破壞平衡時便會產生疾病。常見的皮膚問題種類繁多，本研究針對紫外線照射所導致的黑色素累積產生皮膚色素沉著過度的美學問題做了探討，其中以「痤瘡丙酸桿菌發酵產生丙酸改善紫外線B誘導的黑色素合成Propionic acid produced by Cutibacterium acnes fermentation ameliorates ultraviolet B‑induced melanin synthesis」為題，目前市面上經常使用化學美白產品來治療和預防。然而，此類產品相關的安全問題促使尋找天然和無害的替代品。本題旨在評估天然的益生菌通過發酵代謝產物以減少皮膚色素沉著。實驗結果顯示，在痤瘡丙酸桿菌(Cutibacterium acnes, C. acnes)與泊洛沙姆(Pluronic F68, PF68)共培養的發酵培養基中可以檢測到短鏈脂肪酸(Short-chain fatty acids, SCFAs)，其中丙酸(CH3CH2COOH, PA )是濾液中含量最高的脂肪酸，並通過與游離脂肪酸受體2 (free fatty acid receptor 2, FFAR2)顯著抑制細胞內酪氨酸酶(tyrosinase, TYR)活性進而減少多巴陽性黑色素細胞(DOPA-positive melanocytes)。此外，在丙酸對小鼠黑色素瘤细胞(B16F10)存活率試驗中，以4 mM丙酸處理並不會造成黑色素細胞的凋亡。在體內試驗中，注射了C. acnes和PF68混合物的小鼠耳部皮膚組織中也觀察到多巴陽性黑色素細胞和TYR活性降低。總體而言，本研究是一種有效的過度色素沉著解決方案，C. acnes作為益生菌通過使用發酵引發劑PF68介導產生丙酸，藉由丙酸-FFAR2- TYR途徑的參與抑制黑色素的生成，並且不會造成細胞損傷和破壞皮膚微生物組的平衡。這項研究結果有利於未來色素沉著障礙的臨床治療和開發化妝品與美白產品的應用範圍。
另外以「蜂蜜益生菌循環芽孢桿菌轉移的細胞外電子抑制炎症性尋常痤瘡Extracellular Electrons Transferred from Honey Probiotic Bacillus circulans Inhibits Inflammatory acne vulgaris」為題探討蜂蜜中的微生物在人類皮膚疾病上的利用，環狀芽孢桿菌(Bacillus circulans, B. circulans)是被廣泛用作微生物燃料電池(microbial fuel cell, MFC)技術中的產電細菌。這項研究評估了B. circulans是否可以透過葡萄糖發酵來發電並減輕人類皮膚病原體C. acnes的影響。將B. circulans與葡萄糖共培養的培養液通過測量電壓差來檢查發酵產生的電量，並且使用螯合鐵離子能力測定(Ferrozine assay)進行驗證。與對照組相比，添加2%葡萄糖與B. circulans的發酵液中電壓增加到約4 mV。Ferrozine assay結果顯示發酵液中產生電子使反應溶液呈現深棕色。在體外試驗中，葡萄糖與B. circulans共培養提高了發電量並顯著抑制了C. acnes的生長。此外，添加玫瑰黃素顯著降低了B. circulans通過發酵葡萄糖代謝產生的電能，並且在動物實驗中恢復了C. acnes計數和解脂酶誘導的巨噬細胞炎症蛋白2 (Macrophage inflammatory protein-2, MIP-2)水平。總體而言，這項研究揭示了蜂蜜中的益生菌B. circulans通過使用葡萄糖作為益生元來產生電能的分子機制。B. circulans通過黄素單核苷酸(Flavin mononucleotide, FMN)核糖開關和黃素介導的電子轉移干擾C. acnes的生長，從而減少炎症反應。因此，微生物通過碳源發酵的代謝活動從生物質中產生電能可能是一種潛在的抗菌療法並且有利於未來痤瘡皮膚病的臨床治療和護膚品的開發。
本研究透過微生物作為天然益生菌產生發酵代謝物改善常見皮膚問題，C. acnes作為人類皮膚常見微生物透過PF68發酵代謝產生丙酸，改善紫外線造成的黑色素過度沉著；天然蜂蜜中的微生物B. circulans透過葡萄糖發酵產生電子治療抑制炎症性尋常痤瘡。

摘要(英)

The human skin surface is colonized by all manner of microorganisms, which maintain a healthy state by balancing the space and nutrients. Exogenous changes like pressure or environmental factors will result in skin disorders or infections. There are many kinds of common skin problems. This study investigated the ultraviolet (UV) irradiation induces melanin accumulation, which caused the aesthetic problem of hyperpigmentation. The first chapter, "Propionic acid produced by Cutibacterium acnes fermentation ameliorates ultraviolet B‑induced melanin synthesis." At present most whitening products on the market are going to have a chemical composition. However, the associated safety concerns of such products have prompted the search for natural and harmless alternatives. This study aimed to identify natural fermentation metabolites to reduce skin pigmentation. The result shows that metabolite propionic acid (PA) was the most abundant fatty acid in the filtrate from PF68 fermentation of C. acnes and reduced the DOPA-positive melanocytes by significantly inhibiting cellular TYR activity via binding to the FFAR2. Moreover, the effects of PA on B16F10 melanoma cells evaluated by BrdU labeling that 4 mM PA treatment did not alter melanocyte proliferation and cellular damage. In in vivo results, the reduced DOPA-positive melanocytes and TYR activity were also observed in mice ear skin tissue injected with a mixture of C. acnes and PF68. Overall, we indicate that this study is an effective solution for hyperpigmentation. C. acnes as a probiotic can produce PA to inhibit melanogenesis through PF68, the fermentation inducer, and suggests the involvement of the PA-FFAR2-TYR pathway. These results are beneficial for the future clinical treatment of pigmentation disorders and to develop cosmetics that increases the range of applications of whitening products.
In the second chapter, we investigate the utilization of microorganisms in honey for human skin diseases, "Extracellular Electrons Transferred from Honey Probiotic Bacillus circulans Inhibits Inflammatory acne vulgaris." B. circulans is widely used as an electrogenic bacterium in MFC technology. This study evaluated whether B. circulans can ferment glucose to generate electricity and mitigate the effects of C. acnes, human skin pathogens. The electricity production of B. circulans was examined by measuring the voltage difference and verified using a ferrozine assay. Compared with the control, the voltage significantly increased to approximately 4 mV in the B. circulans with 2% glucose group. The result of the ferrozine assay shows that fermentation reaction medium formed the dark brown solution through electron production. In vitro results demonstrated that the glucose-B. circulans co-culture enhanced electricity production and significantly supressed C. acnes growth. In addition, the addition of roseoflavin to inhibit flavin production considerably reduced the electrical energy generated by B. circulans through metabolism and, in animal tests, recovered C. acnes count and MIP-2 levels. Overall, this study revealed the molecular mechanism by which the probiotic B. circulans in honey can generate electrical energy by using glucose as a prebiotic. B. circulans reduced the inflammatory response by disrupting C. acnes growth through FMN riboswitch and flavin-mediated electron transfer. Therefore, generating electrical energy from biomass through the metabolic activities of microorganisms may be a potential antimicrobial therapy. These results are beneficial for the future clinical treatment of acne-prone skin disorders and to development of skincare products.
This article focuses on microbes being natural probiotics that produce fermentation metabolites to improve common skin problems. C. acnes is a common microorganism in human skin that can produce PA to inhibit melanogenesis through PF68. B. circulans in honey can generate electrical energy to treat inflammatory acne vulgaris by using glucose.

關鍵字(中)

★ 微生物
★ 益生菌
★ 發酵代謝物
★ 黑色素過度沉著
★ 痤瘡

關鍵字(英)

論文目次

摘要 VII
ABSTRACT IX
誌謝 XI
圖目錄 XV
縮寫說明 1
第壹章痤瘡丙酸桿菌發酵產生丙酸改善紫外線B誘導的黑色素合成 3
一、緒論 3
1-1-1 研究背景 3
1-1-2 研究動機與目的 6
二、研究內容與方法 7
1-2-1 研究架構 7
1-2-2 微生物特性 8
1-2-2-1 C. acnes 8
1-2-3 C. ACNES與PF68共培養之相關試驗 8
1-2-3-1 C. acnes藉由PF68產生發酵反應後，發酵菌液進行酚紅指示劑分析 8
1-2-3-2 C. acnes與PF68共培養後，發酵菌液進行GC-MS分析 8
1-2-3-3 PF68對C. acnes之MBC試驗 8
1-2-4 體外試驗 9
1-2-4-1 黑色素瘤细胞培養 9
1-2-4-2 PA對黑色素瘤细胞乙醯化離胺酸表達試驗 9
1-2-4-3 PA抑制小鼠黑色素瘤細胞中TYR基因表達試驗 9
1-2-4-4 PA、乙酸(Acetic acid, AA)於小鼠黑色素瘤細胞中對黑色素生成之影響 10
1-2-4-5 PA影響黑色素瘤细胞存活率試驗 10
1-2-4-6 FFAR2基因敲除後PA影響黑色素瘤细胞TYR活性測試 10
1-2-5 體內試驗 10
1-2-5-1 C. acnes藉由PF68發酵於小鼠耳朵抑制黑色素生成試驗 10
1-2-5-2 PA於小鼠耳朵抑制黑色素生成試驗 11
1-2-5-3 FFAR2 siRNA阻斷FFAR2於小鼠耳朵對PA抑制黑色素生成試驗 12
1-2-5-4 西方墨點法(Western blot)檢測FFAR2 siRNA阻斷FFAR2於小鼠耳朵試驗 12
1-2-6 統計分析 13
三、結果與討論 15
1-3-1 C. ACNES與PF68共培養之相關試驗 15
1-3-1-1 C. acnes藉由PF68產生發酵反應後，發酵菌液進行酚紅指示劑分析 15
1-3-1-2 C. acnes與PF68共培養後，發酵菌液進行GC-MS分析 15
1-3-1-3 PF68對C. acnes之MBC試驗 16
1-3-2 體外試驗 17
1-3-2-1 PA對黑色素瘤细胞乙醯化離胺酸表達試驗 17
1-3-2-2 PA抑制小鼠黑色素瘤細胞中TYR基因表達試驗 17
1-3-2-3 PA、AA對小鼠黑色素瘤細胞中黑色素生成之影響 18
1-3-2-4 PA影響小鼠黑色素瘤细胞存活率試驗 18
1-3-3-1 C. acnes藉由PF68發酵於小鼠耳朵抑制黑色素生成試驗 20
1-3-3-2 PA於小鼠耳朵抑制黑色素生成試驗 20
1-3-3-3 FFAR2 siRNA阻斷FFAR2於小鼠耳朵對PA抑制黑色素生成試驗 21
1-3-3-4 FFAR2 siRNA阻斷FFAR2於小鼠耳朵後，PA對TYR活性測試 22
1-3-3-5 Western blot檢測FFAR2 siRNA阻斷FFAR2於小鼠耳朵試驗 23
四、結論 24
一、緒論 25
2-1-1 研究背景 25
2-1-2 研究動機與目的 28
二、研究內容與方法 30
2-2-1 研究架構 30
2-2-2 微生物特性 31
2-2-2-1 B. circulans 31
2-2-3 B. CIRCULANS與葡萄糖共培養之相關試驗 31
2-2-3-1 B. circulans藉由葡萄糖產生發酵反應 31
2-2-3-2 B. circulans與葡萄糖共培養後，發酵菌液進行MFC系統的產電量分析 31
2-2-3-3 Ferrozine assay 31
2-2-4 體外試驗 31
2-2-4-1 B. circulans藉由葡萄糖發酵產生電子抑制C. acnes生長 32
2-2-5 體內試驗 32
2-2-5-1 玫瑰黃素抑制B. circulans藉由葡萄糖發酵產生電子，影響C. acnes生長 32
2-2-5-2 解脂酶誘導促炎性細胞因子MIP-2表現 32
2-2-5-3 玫瑰黃素抑制B. circulans藉由葡萄糖發酵產生電子，影響C. acnes解脂酶誘導促炎性細胞因子MIP-2表現 32
2-2-5-3-1 ELISA 32
2-2-6 統計分析 32
三、結果與討論 34
2-3-1 分離蜂蜜中微生物進行16S RRNA測序 34
2-3-2 B. CIRCULANS與葡萄糖共培養之相關試驗 34
2-3-3-1 B. circulans藉由葡萄糖產生發酵反應 34
2-3-3-2 B. circulans與葡萄糖共培養後，發酵菌液進行MFC系統的產電量分析 35
2-3-3-3 Ferrozine assay 36
2-3-3-4 玫瑰黃素抑制B. circulans藉由葡萄糖發酵產生電子 36
2-3-4 體外試驗 37
2-3-4-1 B. circulans藉由葡萄糖發酵產生電子抑制C. acnes生長 37
2-3-5 體內試驗 38
2-3-5-1 玫瑰黃素抑制B. circulans藉由葡萄糖發酵產生電子，影響C. acnes生長 38
2-3-5-2 C. acnes解脂酶誘導促炎性細胞因子MIP-2表現 40
2-3-5-3 玫瑰黃素抑制B. circulans藉由葡萄糖發酵產生電子，影響C. acnes解脂酶誘導促炎性細胞因子MIP-2表現 40
四、結論 41
五、總結 43
參考文獻 44
發表文獻 48
附件一 49

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指導教授

陳純娟

審核日期

2022-9-28

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