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研究生:鄭婷勻
研究生(外文):Ting-Yun Jheng
論文名稱:乳鐵蛋白與乳鐵蛋白耐受性益生菌菌株併用後之抗菌功效分析
論文名稱(外文):Analysis of the Antibacterial Activities for the Combination of Lactoferrin and Lactoferrin-Resistant Probiotics
指導教授:毛嘉洪
指導教授(外文):Frank Chiahung Mao
口試委員:董光中陳柏文
口試日期:2012-07-02
學位類別:碩士
校院名稱:國立中興大學
系所名稱:獸醫學系暨研究所
學門:獸醫學門
學類:獸醫學類
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:63
中文關鍵詞:乳鐵蛋白益生菌
外文關鍵詞:lactoferrinprobiotic
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乳鐵蛋白是一種天然的蛋白質,主要存在於初乳及乳汁中。乳鐵蛋白是一種具抗菌活性的多功能蛋白,且母乳中的乳鐵蛋白已被發現可以保持腸道中bifidobacteria和lactobacilli成為優勢菌叢;另外,腸道中的益生菌也已被發現可以抑制病原菌的侵入。已有部分研究表明,某些益生菌不會被乳鐵蛋白所抑制;因此,在本實驗中評估了對牛乳鐵蛋白具耐受性的益生菌的抗菌功效。實驗中發現有八株益生菌對於牛乳鐵蛋白及牛乳鐵蛋白水解液具耐受性,分別為:L. rhamnosus (ATCC 7469)、L. reuteri (ATCC 23272)、L. fermentum (ATCC 11739)、L. coryniformis subsp. coryniformis (ATCC 25602)、L. acidophilus (BCRC 14065)、B. longum subsp. infantis (ATCC 15697)、B. bifidum (ATCC 29521)、P. acidilactici (ATCC 8081),其中牛乳鐵蛋白會促進L. fermentum、L. coryniformis subsp. coryniformis及L. acidophilus,而牛乳鐵蛋白水解液不會。在抑制致病菌方面,牛乳鐵蛋白水解液都較牛乳鐵蛋白效果好。接著,實驗取13株益生菌的上清液與牛乳鐵蛋白或牛乳鐵蛋白水解液併用,以評估併用後之抗菌功效。結果發現有3株益生菌上清液(L. fermentum、B. longum subsp. longum及B. animalis subsp. lactis)與牛乳鐵蛋白及牛乳鐵蛋白水解液併用後對於Eschelichia coli (HER1255)、Salmonella Typhi (ATCC19430)、S. Typhimurium (ATCC13311、ATCC14028)、Staphylococcus aureus (ATCC25923)及部分MRSA菌株有部分協同和協同作用。因牛乳鐵蛋白會促進L. fermentum ATCC11739的生長,L. fermentum以活菌狀態與牛乳鐵蛋白併用後,或許可製成抗菌效果較佳的保健產品;因牛乳鐵蛋白水解液的抗菌效果較牛乳鐵蛋白好,且B. longum subsp. longum及B. animalis subsp. lactis會被牛乳鐵蛋白所抑制,因此這兩株益生菌或許可以死菌與牛乳鐵蛋白水解液併用,做為較佳抗菌效果的保健產品。

Lactoferrin is a natural protein mainly in the colostrums and milk. It is a multifunctional protein with particularly antibacterial activity. Importantly, lactoferrin in breast milk have been suggested to play a major role to maintain a predominance of bifidobacteria and lactobacilli in the intestinal system. Otherwise, probiotics have been reported to inhibit the invasion of pathogens in the intestine. Interestingly, some previous studies have indicated that some probiotics are lactoferrin-resistant. Thus, in this experiment, the bovine lactoferrin-resistant probiotic were evaluated and harvested. Three probiotics strains, L. fermentum, B. longum subsp. longum and B. animalis subsp. lactis were shown to possess antibacterial activity against pathogens. In these three probiotics, only L. fermentum is lactoferrin-resistant. Moreover, the combinations of bovine lactoferrin or lactoferrin hydrolysate with supernatants from either one of the above probiotics were further determined. These combinations have led to synergy or partially synergy against the pathogens, such as Eschelichia coli (HER1255), Salmonella Typhi (ATCC19430), S. Typhimurium (ATCC13311), S. Typhimurium (ATCC14028), Staphylococcus aureus (ATCC25923) and some MRSA strains. The results showed that L. fermentum can be promoted by bovine lactoferrin, so it may be used as a live microbial food supplement with bovine lactoferrin! Bovine lactoferrin hydrolysate possesses better antibacterial activity than lactoferrin, thus B. longum subsp. longum ATCC 15707 and B. animalis subsp. lactis BCRC 17394 can be uses as a dead microbial food supplement with bovine lactoferrin hydrolysate!

中文摘要...................................................I
ABSTRACT..................................................II
目錄.....................................................III
表目錄.....................................................V
圖目錄...................................................VII
壹、前言...................................................1
貳、文獻探討...............................................2
一.乳鐵蛋白...............................................2
(一)、乳鐵蛋白的結構.....................................2
(二)、乳鐵蛋白的分佈.....................................4
(三)、乳鐵蛋白的功能.....................................7
1.幫助鐵離子的平衡.................................8
2.乳鐵蛋白之抗菌功能...............................8
3.乳鐵蛋白的抗病毒功能............................10
4.乳鐵蛋白的免疫調節功能..........................10
5.乳鐵蛋白的抗發炎反應功能........................11
(四)、乳鐵蛋白水解液 (乳鐵蛋白素).....................11
二、益生菌................................................12
(一)、影響腸道菌群的因素................................13
三、乳酸菌................................................14
(一)、乳酸菌與腸道的關係................................14
(二)、乳酸菌的抑菌功能..................................15
(三)、乳酸菌的其他功能..................................17
四、抗甲氧西林金黃色葡萄球菌..............................17
参、材料與方法............................................19
一、牛乳鐵蛋白............................................19
二、牛乳鐵蛋白水解液......................................19
三、細菌菌株..............................................19
四、篩選不會被牛乳鐵蛋白抑制的益生菌......................24
五、牛乳鐵蛋白抑制致病菌的能力............................24
六、益生菌抑菌活性分析....................................24
七、牛乳鐵蛋白與益生菌上清液的協同試驗....................25
八、篩選不會被牛乳鐵蛋白水解液抑制的益生菌................26
九、牛乳鐵蛋白水解液抑制致病菌的能力......................26
十、牛乳鐵蛋白水解液與益生菌上清液的協同試驗..............26
肆、結果..................................................27
一、篩選不被乳鐵蛋白抑制的益生菌..........................27
二、牛乳鐵蛋白抑制標準致病菌菌株的能力....................29
三、牛乳鐵蛋白抑制MRSA菌株的能力..........................29
四、益生菌上清液抑制標準致病菌菌株........................30
五、益生菌上清液抑制MRSA菌株..............................33
六、牛乳鐵蛋白與益生菌上清液併用之後對抑制標準致病菌菌株的協同效果....................................................38
七、牛乳鐵蛋白與益生菌上清液併用之後對MRSA菌株的協同效果..41
八、篩選不會被牛乳鐵蛋白水解液所抑制的益生菌..............45
九、牛乳鐵蛋白水解液抑制標準致病菌菌株....................46
十、牛乳鐵蛋白水解液抑制MRSA菌株..........................47
十一、牛乳鐵蛋白水解液與益生菌上清液併用後對抑制標準致病菌菌株的影響..................................................48
十二、牛乳鐵蛋白水解液與益生菌上清液併用之後對抑制MRSA菌株的影響......................................................51
伍、討論..................................................55
陸、参考文獻..............................................59



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