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研究生:呂秀清
研究生(外文):Chanita Chotirosvakin
論文名稱:自飲用母乳嬰兒的糞便及母乳中篩選出具有免疫調節能力的比菲德氏菌
論文名稱(外文):Screening Immunomodulatory Probiotics of Bifidobacterium strains Isolated from Breast-Fed Infant Feces and Breast Milk
指導教授:林美吟林美吟引用關係
指導教授(外文):Meei-Yn Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
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:2011
畢業學年度:99
語文別:英文
論文頁數:84
中文關鍵詞:比菲德氏菌嬰兒糞便母乳細胞激素附著性酸及膽鹽耐受性
外文關鍵詞:Bifidobacteriainfant fecesbreast milkcytokinesadhesionacid and bile tolerance
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比菲德氏菌 (Bifidobacterium) 為飲用母乳嬰兒腸道中的主要菌株,而這群菌株在飲用母乳嬰兒的糞便中也是優勢族群,並認為在調節宿主免疫系統及有益維持腸道健康。本研究主要目的為,從飲用母乳嬰兒的糞便及母乳中篩選出 20 株具有刺激人類周邊血液單核球細胞 ( human peripheral blood mononuclear cells, hPBMCs) 分泌細胞激素的比菲德氏菌。本研究結果指出,不同菌屬及不同菌株的比菲德氏菌對於誘發細胞激素的產生會有不同的影響,且比菲德氏菌的劑量與誘導細胞激素的產生具有相關性。B. adolescentis DB-2458 及 B. longum subsp. infantis GB-1496 於濃度比 1:30 (hPBMC: bacteria cells) 時,能夠有效的誘導 IFN-γ secretion (Th1 cytokine) 產生,並且降低 Th2 細胞激素 IL-5 及 IL-13 的分泌;B. adolescentis DZN092 、 B. adolescentis DZN365 (濃度比1:3, 1:0.3)、B. adolescentis DB-2458 (濃度比 1:30, 1:3) 及 B. longum HB-762 (濃度比1:30) 能夠有效的誘導 TNF-α 及 IL-6 分泌,能夠促進傷口感染時的發炎反應;然而,B. adolescentis DZN092 (濃度比 1:30, 1:3, 1:0.3)、 B. adolescentis DZN365 (濃度比 1:30, 1:0.3)、 B. longum GK - 22 (濃度比 1:30) 及 B. longum GL-78 (濃度比 1:0.3) 能夠誘導高濃度 IL-10 及 TGF-β (抗發炎細胞激素) 的釋放,並且降低 TNF-α 及 IL-6 (促發炎細胞激素) 產生,能使免疫反應趨向平衡,而達到抗發炎的作用。本研究並進一步的比較其免疫調節能力及標準化的檢測益生菌規範,將 B. adolescentis DZN092、B. adolescentis DB-2458、B. longum subsp. infantis GB-1496、B. longum HB-762、B. longum GK-22 及 B. longum GL-78 進行耐酸及耐膽鹽的測試,結果顯示這些菌株可以於 pH 值 2-4 及 0.3% 膽鹽的條件下生長;在附著性的試驗中,所有的菌株皆具有附著的能力。因此本篇研究結論出,篩選出的菌株可以用於治療,並可將其應用於功能性食品中以有益於健康。

Bifidobacterium strains were well known as a predominant component of the intestinal flora in breast-fed infant feces and breast milk. This bacterial group plays a vital role in modulating host immunity and maintaining the gut health. In this study, the eighteen strains of Bifidobacterium strains which were isolated from infant feces and breast milk were screened for their ability to induce cytokines production by peripheral blood mononuclear cells (hPBMCs). The results indicated that not only different strains but also different species have different effect on the stimulation of all tested cytokines production. Moreover, the results also revealed that capacity of cytokine induction was dose-dependent. B. adolescentis DB-2458 and B. longum subsp. infantis GB-1496 at a concentration ratio 1:30 (hPBMCs: bacterial cells) which were potent inducers for IFN-γ secretion (Th1 cytokine) and induced low level of IL-5 and IL-13 (Th2 cytokines) were viable choices to down regulate Th2 cytokines. B. adolescentis DZN092, B. adolescentis DZN365 (concentration ratio 1:3, 1:0.3), B. adolescentis DB-2458 (concentration ratio 1:30, 1:3) and B. longum HB-762 (concentration ratio 1:30) which strongly induced TNF-α and IL-6 were promoted for the inflammation to initiate the healing process due to the infection. While B. adolescentis DZN092 (concentration ratio 1:30, 1:3, 1:0.3), B. adolescentis DZN365 (concentration ratio 1:30, 1:0.3), B. longum GK - 22 (concentration ratio 1:30) and B. longum GL-78 (concentration ratio 1:0.3) which highly induced both of IL-10 and TGF-β (anti-inflammatory cytokines) releases and induced low level of TNF-α and IL-6 (pro-inflammatory cytokines) could exert immune balance toward anti-inflammatory response. Based on comparison of immunomodulating activity and general criteria tests for probiotic, B. adolescentis DZN092, B. adolescentis DB-2458, B. longum subsp. infantis GB-1496, B. longum HB-762, B. longum GK-22 and B. longum GL-78 were acid and bile resistant strains. They could survive under acidic condition at pH 2-4 and bile condition (0.3% bile salts). For adhesion ability, all of test strains were adhesive strains. In conclusion, the selected strains were viable choices for therapeutic use and could be applied in desirable functional food due to their specific health benefits.

中文摘要................................................. i
ABSTRACT................................................iii
LIST OF CONTENTS................................................v
LIST OF TABLES..........................................viii
LIST OF FIGURES.........................................ix
LIST OF ABBREVIATIONS...................................xii
INTRODUCTION............................................1
LITERATURE REVIEW.......................................3
1. Bifidobacterium microflora of the human intestinal tract ........................................................3
2. The intestinal microflora of the breast-feeding infant ........................................................4
3. Genus Bifidobacterium................................5
4. Bifidobacterium and health benefits..................7
5. T cell differentiation and cytokines.................9
6. Criteria for selection as probiotics.................12
OBJECTIVES..............................................17
1. Research Objective...................................17
2. Research Map.........................................18
MATERIALS AND METHOD....................................19
1. Materials............................................19
1.1 Bifidobacterium strains and Cells...................19
1.2 Chemical substances.................................19
1.3 Test Kits...........................................21
1.4 Equipments..........................................21
2. Medium and solution preparation......................21
3. Methods..............................................23
3.1 Bacterial strains and culture condition.............23
3.2 Bacterial growth curve..............................24
3.3 Immunomodulatory of Bifidobacterium strains.........24
3.4 Sensitivity of Bifidobacterium strains to artificial gastrointestinal conditions.............................28
3.5 In vitro adhesion assay.............................29
3.6 Statistic...........................................30
RESULTS AND DISUSSIONS..................................31
1. Results..............................................31
1.1 Effect of Bifidobacterium test strains on cytokines production..............................................31
1.2 Sensitivity of Bifidobacterium strains to artificial gastrointestinal condition..............................35
1.3 Adhesion ability of Bifidobacterium strains to Caco-2 cell....................................................37
2. Discussions..........................................38
2.1 Effect of Bifidobacterium test strains on cytokines production..............................................38
2.2 Sensitivity of Bifidobacterium strains to artificial gastrointestinal condition..............................44
2.3 Adhesion ability of Bifidobacterium strains to Caco-2 cell....................................................48
CONCLUSION..............................................66
REFERENCES..............................................67
APPENDIXS...............................................79


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