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研究生:鄭淑芬
研究生(外文):Su-Fen Teh
論文名稱:褐藻酸鈣微膠囊化及海藻糖添加對Lactobacillus reuteri冷凍安定性之影響
論文名稱(外文):Effects of calcium-alginate microencapsulation and trehalose addition on the freezing stability of Lactobacillus reuteri
指導教授:金安兒金安兒引用關係
指導教授(外文):V. An-Erl King
口試委員:賴麗旭曾政鴻
口試日期:2011-06-28
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:中文
論文頁數:110
中文關鍵詞:Lactobacillus reuteri微膠囊化技術冷凍保護劑海藻糖褐藻酸鈣
外文關鍵詞:Lactobacillus reuterimicroencapsulationcryoprotectanttrehaloseCa-alginate
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Lactobacillus reuteri為來自人體的天然益生菌種,具有許多益生功效,不僅可以吸附腸道的黏膜,存活於人體的腸道中,而且可產生抗菌物質,抑制病原菌。不過往往乳酸菌經人攝取後,受到人體消化道內胃酸、膽鹽的作用,到達下消化道之存活菌數並不高,因而無法吸附定殖以便發揮其益生效用。為了增加乳酸菌在惡劣的環境中的存活率,所以本研究利用褐藻酸鈣對L. reuteri進行微膠囊化處理,再添加2%海藻糖作為冷凍保護劑,然後放置於-60℃下來進行貯藏,探討其對L. reuteri在冷凍貯藏中的保護效果,並對其在腸道中吸附性的影響,以及在模擬人體胃腸道中不良環境因子之抵抗性(分別是以pH 2、3、4之酸液及0.1%、0.2%、0.4%之膽鹽溶液進行試驗),與對腸炎沙門桿菌(Salmonella enteritidis)之抑菌能力等進行研究,希望L. reuteri可藉由海藻糖加上微膠囊的保護下,除了在冷凍貯藏中之存活率可以提升外,同時也能提高菌體在惡劣環境下的耐受能力,以便增加菌體在經過人體腸胃道的存活率。結果顯示,微膠囊化技術及海藻糖能有效提高菌株在冷凍貯藏後的存活率,而微膠囊化技術有助於提升菌體在冷凍後對膽鹽的耐受力,但卻無法提升菌體對酸的耐受力。在產酸試驗方面,微膠囊化技術及海藻糖可以提高在冷凍儲藏後L. reuteri的產酸能力,表示其能夠有效保護冷凍後菌體的活性。在抑菌圈實驗中,乳酸菌上清液皆對腸炎沙門桿菌有抑菌功效。隨著冷凍儲藏時間的增加,乳酸菌上清液的抑菌效果有顯著性減弱的趨勢(p<0.05)。其中以經微膠囊化處理及添加了海藻糖的乳酸菌在經過冷凍儲藏後,其抑制腸炎沙門氏菌生長的能力較其他組別的好,並且對於Caco-2細胞也有較佳的吸附力。此外,L. reuteri不具侵入性,對人體健康安全無害。

Lactobacillus reuteri is a species of microbes which is naturally present in the animal stomach and intestine microflora could benefit the human health. L. reuteri produces reuterin that can inhibit the growth of a broad range of pathogenic microbes. However, a major problem is low viability of lactic acid bacteria when arriving at the lower digestive tract due to the gastric acid and bile salts existed in the gastrointestinal system. In order to increase the viability of lactic acid bacteria at the harsh environments, microencapsulation could be used to improve probiotic survival against severe environmental factors. In this study, Ca-alginate was applied to L. reuteri for microencapsulation and trehalose 2% (w/v) was used as a cryoprotectant for protecting the cell during -60°C frozen storage so as to study their protection effect on the enhancement of cell survival during frozen storage and their adhesion in the intestine. We also focused on the resistance of lactic acid bacteria against adverse environmental factors (pH 2, 3, 4 and 0.1%, 0.2%, 0.4% bile salt) and their antibacterial ability on Salmonella enteritidis. Results showed that microencapsulation techniques and trehalose not only protect L. reuteri from freezing injury but also maintain their activity for the production of acid. Microencapsulation could enhance bile salts tolerance of L. reuteri but unable to improve on acid tolerance. Besides, both microencapsulation techniques and trehalose enable to increase the inhibition of L. reuteri against Salmonella enteritidis and adherence to Caco-2 cells after frozen storage. In the well diffusion assay, the supernatant of L. reuteri could inhibit the growth of Salmonella enteritidis. As the frozen storage time increased, the antibacterial activity of L. reuteri supernatants significantly decreased (p <0.05). In addition, L. reuteri did not show any invasion effect and is safe for human health.

中文摘要 i
英文摘要 ii
目錄 iv
表目次 vii
圖目次 viii
壹、前言 1
貳、文獻整理 2
一、腸胃道微生物的組成 2
(一)人體腸胃道菌相的分佈 2
(二)腸胃道微生物與宿主之關係 4
二、益生菌 6
(一)益生菌(probiotics)的簡介 6
(二)益生菌的益生效果 9
三、乳酸菌簡介 14
(一)乳酸菌的定義與分類 15
(二)乳酸菌的代謝特性 16
(三)乳酸菌在食品加工上的應用 17
(四)洛德乳桿菌(Lactobacillus reuteri) 20
(五)乳酸菌對沙門氏菌的抑菌效果 22
四、乳酸菌的冷凍耐受性 23
(一)冷凍對微生物的影響 24
(二)海藻糖(trehalose) 27
五、微膠囊於乳製品上之應用研究 32
(一)微膠囊固定化技術 32
(二)微膠囊固定化對乳酸菌生長之影響 38
(三)益生菌微膠囊在乳製品上的應用 38
(四)褐藻膠(alginate) 38
六、篩選乳酸菌菌株的先決條件之相關研究 40
(二)模擬腸道吸附性之研究 42
(三)乳酸菌的安全評估 43
參、研究目的 44
肆、材料與方法 45
一、實驗材料與儀器 45
(一)材料 45
(二)儀器 47
二、實驗方法 48
(一)實驗架構 48
(二)實驗菌株的製備 48
(三)實驗樣本的製備 48
(四)凍結儲藏實驗 50
(五)菌體存活率測定 50
(六)產酸能力測定 52
(七)耐酸耐膽鹽實驗 52
(八)乳酸菌培養基上清液抑制腸炎沙門桿菌生長實驗 54
(九)乳酸菌培養基上清液之抑菌成份分析 55
(十)乳酸菌的吸附性實驗 56
(十一)乳酸菌的安全性評估 59
(十二)電子顯微鏡觀察 59
(十三)統計分析 62
伍、結果與討論 63
一、菌株之存活率 63
二、產酸能力分析 66
(一)不同處理的菌體對產酸能力的影響 66
(二)冷凍儲藏對產酸能力的影響 68
三、耐酸耐膽鹽研究 68
(一)耐酸實驗和耐膽鹽實驗結果 70
四、乳酸菌培養基上清液抑制腸炎沙門氏菌生長實驗 74
(一)不同處理之乳酸菌培養基上清液之pH值和乳酸產量的比較 75
(二)培養基上清液中和實驗結果 75
(三)不同處理之乳酸菌培養基上清液對沙門桿菌抑菌效果的比較 79
(四)培養基上清液熱穩定性實驗結果 79
(五)培養基上清液經蛋白酶處理之結果 79
五、乳酸菌的吸附性實驗 83
六、菌體初步安全性評估的結果 87
七、電子顯微鏡觀察 87
陸、結論 91
柒、參考文獻 92


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