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研究生:郭家雯
研究生(外文):Chia-Wen Kuo
論文名稱:嗜酸乳酸桿菌和約氏乳酸桿菌的微膠囊化
論文名稱(外文):Microencapsulation of Lactobacillus acidophilus and Lactobacillus johnsonii
指導教授:金安兒金安兒引用關係
口試委員:賴麗旭曾政鴻
口試日期:2014-06-26
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:2014
畢業學年度:102
語文別:中文
論文頁數:162
中文關鍵詞:Lactobacillus acidophilusLactobacillus johnsonii微膠囊化技術二流體噴霧凝結法擠壓法反應曲面法海藻糖
外文關鍵詞:Lactobacillus acidophilusLactobacillus johnsoniimicroencapsulationtwo-fluid spray-coagulationextrusionresponse surface methodology (RSM)trehalose
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乳酸菌屬於益生菌的一種,具有許多保健功效,可定殖於腸道中並且維持腸道正常菌相。但是乳酸菌藉由口服攝取進入人體後,必須通過胃和腸道等嚴苛的環境,因此本研究利用反應曲面法探討以二流體式噴霧凝結法搭配混合凝膠製備微膠囊化 Lactobacillus acidophilus,提高菌體對外界環境的耐受性,針對不同的褐藻酸鈉濃度、果膠濃度、氯化鈣濃度等三個因子,探討其對膠球菌體存活率的影響,並找出最適操作條件,以進行模擬消化液連續作用之耐受性試驗、β-半乳糖苷酶活性試驗、產酸測定、蛋白質分析、掃描式電子顯微鏡觀察等。此外,利用不同菌液處理方式和不同的膠球發酵條件,以擠壓法製備微膠囊化Lactobacillus johnsonii,並添加海藻糖以保護菌體,再進行相關研究之探討。結果顯示,褐藻酸鈉濃度對膠球菌體存活率具有最顯著的影響,其次為果膠濃度,在褐藻酸鈉濃度 2.5%、果膠濃度 1.5%、氯化鈣濃度 0.1M 的條件下製備膠球,並搭配 2%幾丁聚醣具有最高的存活率,而且在模擬消化液連續作用之耐受性試驗、β-半乳糖苷酶活性試驗、產酸測定、蛋白質分析、掃描式電子顯微鏡觀察等結果顯示 微膠囊化在模擬胃腸液的環境中可以達到保護 L. acidophilus 的效果。,
在擠壓法製備微膠囊化 L. johnsonii 方面,經過不同處理條件作用後,未濃縮菌液和濃縮菌液製成的膠球經過表面發酵後可增加菌體包含率,添加海藻糖有助於提升菌體包含率,而且在模擬消化液連續作用之耐受性試驗、β-半乳糖苷酶活性試驗、產酸測定、蛋白質分析、掃描式電子顯微鏡觀察等結果顯示,微膠囊化在模擬胃腸液的環境中可以有效地保護 L. johnsonii。
Lactic acid bacteria are kinds of probiotics. They had many health benefits and could colonize in the human intestine. However, lactic acid bacteria must pass
through the stomach and the intestine by oral consumption into the body. Therefore,response surface methodology (RSM) was used to study by using two-fluid spray-coagulation with mixed gels to prepare microencapsulated Lactobacillus acidophilus in order to enhance its tolerance to the external environment. It was used to study the effects of sodium alginate concentration, pectin concentration, and calcium chloride concentration on the survival rate of L. acidophilus, and to determine
the optimum conditions. The microcapsules prepared by the optimum conditions were treated with simulated digestive juices and some tests were studied, including survival
rate assay, β-galactosidase activity, acidification power test, protein analysis, and scanning electron microscopic observation. Furthermore, we used different cell
suspensions and different microcapsule fermentation to study by using the extrusion to prepare microencapsulated Lactobacillus johnsonii and adding trehalose to protect
L. johnsonii. Results showed that when sodium alginate concentration, pectin concentration, and calcium chloride concentration were 2.5%, 1.5%, and 0.1M,respectively, and combining with 2% chitosan, the highest survival rate could be obtained. The results of survival rate assay, β-galactosidase activity, acidification power test, protein analysis, and scanning electron microscopic observation all showed that microencapsulation could achieve the effect of protecting L. acidophilus from the simulated gastrointestinal fluids. In addition, the results of using extrusion to prepare microencapsulated L. johnsonii showed that the entrapment efficiency of microcapsules prepared by un-concentrated and concentrated cell suspensions could increase after surface fermentation, and adding trehalose could increase the entrapment efficiency of microcapsules. The results of survival rate assay,β-galactosidase activity, acidification power test, protein analysis, and scanning electron microscopic observation all showed that microencapsulation could
effectively protect L. johnsonii from the simulated gastrointestinal fluids.
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、文獻回顧 .............................................. 3
一、益生菌與乳酸菌 ........................................ 3
(一)益生菌(probiotics)之定義 .............................. 3
(二)益生菌所具備之特性 .................................... 3
(三)人類腸道菌相分布 ...................................... 7
(四)益生菌之益生功效 ...................................... 7
(五)乳酸菌(lactic acid bacteria, LAB)之定義與特性 ........ 16
(六)乳酸菌之分類及代謝途徑................................ 17
(七)乳酸菌在食品工業上的應用 .............................. 20
(八)嗜酸乳酸桿菌(Lactobacillus acidophilus) .............. 20
(九)約氏乳酸桿菌(Lactobacillus johnsonii) ................ 23
二、細胞固定化技術(cell immobilization technology)........ 25
(一)定義與分類 ........................................... 25
(二)微膠囊化技術(microencapsulation)...................... 28
三、海藻糖(trehalose) .................................... 47
四、反應曲面法(response surface methodology, RSM) ........ 51
(一)簡介 ................................................ 51
(二)操作與特點 ........................................... 52
(三)應用 ................................................ 53
參、研究目的 ............................................. 56
肆、材料與方法 ........................................... 57
一、實驗材料與儀器 ....................................... 57
(一)材料 ................................................ 57
(二)儀器 ................................................ 62
二、實驗方法 ............................................. 63
PartⅠ利用二流體式噴霧凝結法製備混合凝膠微膠囊化 L. acidophilus ......................................................... 63
(一)實驗架構 ............................................. 63
(二)實驗菌株之製備 ....................................... 63
(三)反應曲面法試驗設計 ................................... 63
(四)利用二流體式噴霧凝結法製備微膠囊化菌株................. 65
(五)菌體存活率測定 ....................................... 66
(六)菌株受傷程度測定 ..................................... 66
(七)反應曲面法分析及最適化 ............................... 67
(八)模擬消化液作用後之耐受性試驗........................... 67
(九)模擬消化液作用後之 β-半乳糖苷酶活性試驗 ............... 69
(十)模擬消化液作用之產酸測定 .............................. 69
(十一)模擬消化液作用之蛋白質分析........................... 71
(十二)掃描式電子顯微鏡(scanning electron microscope, SEM)觀察 ......................................................... 71
(十三)統計分析 ........................................... 71
PartⅡ利用擠壓法製備微膠囊化 L. johnsonii.................. 75
(一)實驗架構 ............................................. 75
(二)實驗菌株之製備 ....................................... 75
(三)利用蠕動幫浦以擠壓法製備微膠囊化菌株................... 77
(四)菌體存活率測定 ....................................... 77
(五)菌株受傷程度測定 ..................................... 77
(六)模擬消化液作用之耐受性試驗 ............................ 77
(七)模擬消化液作用後之 β-半乳糖苷酶活性試驗 ...................................................... 79
(八)模擬消化液作用後之產酸測定 ............................ 79
(九)模擬消化液作用後之蛋白質分析........................... 79
(十)掃描式電子顯微鏡(scanning electron microscope, SEM)觀察........................................................79
(十一)統計分析 ........................................... 79
伍、結果與討論 ........................................... 80
PartⅠ利用二流體式噴霧凝結法製備混合凝膠微膠囊化 L. acidophilus ......................................................... 80
一、利用反應曲面試驗設計探討不同試驗條件對菌體存活率之影響 ......................................................... 80
二、驗證試驗 ............................................. 90
三、模擬消化液作用之耐受性試驗 ............................ 92
四、β-半乳糖苷酶活性試驗 ................................. 97
五、產酸能力分析 ........................................ 101
六、蛋白質分析 .......................................... 107
七、掃描式電子顯微鏡觀察 ................................ 109
PartⅡ利用擠壓法製備微膠囊化 L. johnsonii................. 111
一、探討不同的膠球製備方法對菌體包含率之影響 .............. 111
二、模擬消化液作用之耐受性試驗 ........................... 116
三、β-半乳糖苷酶活性試驗 ................................ 122
四、產酸能力分析 ........................................ 125
五、蛋白質分析 .......................................... 131
六、掃描式電子顯微鏡觀察 ................................ 131
陸、結論 ............................................... 136
柒、參考文獻 ............................................ 139
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