邱文英Chiu, Wen-Yen臺灣大學:高分子科學與工程學研究所王晨綱Wang, Chen-GangChen-GangWang2010-05-122018-06-292010-05-122018-06-292009U0001-1707200901362400http://ntur.lib.ntu.edu.tw//handle/246246/183160本研究利用兩種天然耐酸性材料，褐藻酸鈉與醋酸鄰苯二甲酸纖維素(CAP)，以及耐鹼性材料－幾丁聚醣包覆乳酸(Lactobacillus plantarum)，製成乳酸菌複合顆粒及乳酸菌粉。調整包覆製程條件，找尋最佳保護效果，以減少乳酸菌受到胃部酸性環境及腸道膽汁的損害，增進乳酸菌產品效能。 以褐藻酸鈉、幾丁聚醣及氯化鈣製備褐藻酸鈣/幾丁聚醣複合顆粒包覆乳酸菌，探討不同製程條件之複合顆粒對乳酸菌保護效果的影響。實驗結果發現若使顆粒中包覆的乳酸菌濃度過高，反而會造成存活率降低，以30 倍濃縮菌液為最佳包覆乳酸菌數。單層包覆之褐藻酸鈣顆粒，在經pH 2 人工胃液耐酸性測試後，包覆之乳酸菌存活率約為15%，再經褐藻酸鈉二重包覆後，可提升存活率至70%。以包覆法製備複合顆粒能夠有效增加顆粒中幾丁聚醣含量，其中又以使用重量平均分子量6 萬的幾丁聚醣製成複合顆粒有最佳的保護效果，此顆粒再經二重包覆複合顆粒，經pH 2 人工胃液處理兩小時，再於人工膽汁中經一小時後，仍有30%以上的存活率。使用較高濃度的氯化鈣溶液硬化顆粒，也可提升乳酸菌存活率。包覆乳酸菌之複合顆粒於人工腸液中經6~10 小時可將乳酸菌完全釋放，達到提升乳酸菌效能目的。 另一種包覆方法為利用褐藻酸鈉、CAP 及幾丁聚醣製成乳酸菌粉。實驗結果發現CAP 及幾丁聚醣皆可幫助乳酸菌沉降，提升乳酸菌收率。以褐藻酸鈉或CAP於外層包覆製備乳酸菌粉，兩種耐酸性包覆材料皆可有效提升乳酸菌粉耐酸性，其中以褐藻酸鈉包覆有較佳的保護效果，於人工胃液中經二小時後，約有68%的存活率。增加外層CAP 包覆濃度，也可提升菌粉耐酸性保護效果。在耐膽鹽性方面，以幾丁聚醣幫助乳酸菌離心之菌粉有較佳的耐膽鹽保護效果，如外層再以褐藻酸鈉進行包覆，此菌粉經耐膽鹽性測試三小時後，仍有5%以上存率。乳酸菌粉在人工腸液中4~6 小時即可完全釋放，藉此增進乳酸菌效能。In this study, sodium alginate, cellulose acetate phthalate (CAP) and chitosan wereused for encapsulating Lactobacillus plantarum, one species of the lactic acid bacteria(LAB). Survival ratio of the encapsulated LAB in simulated gastric fluid and intestinalenvironments were examined. Improvements of survival ratio were expected from encapsulation of LAB. There were two types of encapsulated products. One was calcium alginate-chitosancomplex beads. The effects of cell load, beads with alginate double coating, different encapsulating methods, different molecular weight of chitosan and concentrations of calcium chloride were investigated. It was noted that raising in cell load could increase the number of survival LAB after 2 hour incubation in simulated gastric condition, but the survival ratio would decrease. If the encapsulated beads were coated with one more alginate layer, viability of encapsulated LAB could further increase. The calcium alginate beads coated with chitosan could significantly add chitosan in the beads, so LAB encapsulated in these beads would have higher viability after incubated inimulated bile solution. The different concentrations of calcium chloride would also affect the survival ratio of encapsulated LAB after gastric and bile test. The encapsulated LAB in all types of beads would release in intestinal environment after about 6 to 10 hours. The other type of encapsulated products was double-layer powder. The inner layer of powder was used CAP or chitosan, and the outer layer was CAP or sodium alginate. The sedimentation abilities of inner layer materials, CAP and chitosan, were investigated, and the survival ratio of encapsulated bacteria was evaluated under simulated gastric and intestinal environments. The result showed CAP and chitosan could also coprecipitate with bacteria, increasing the load of LAB in powder. The increment of concentration of coating CAP could increase the survival ratio after 2 hour incubation in simulated gastric fluid. Moreover, sodium alginate, which was the same as CAP, could also protect encapsulated LAB in gastric environment, and the sodium alginate had better result than CAP. After 2 hour in simulated bile solution, the powder used chitosan as inner layer had better survival results of encapsulated LAB. In addition, the encapsulated LAB could release in simulated intestinal solution after 4 to 6 hours.摘要............................................................................................... Ibstract............................................................................................II錄.............................................................................................. IV目錄...........................................................................................VIII目錄........................................................................................... XI一章 緒論.................................................................................. 1-1 前言.......................................................................................... 1-2 研究目的...................................................................................1二章 文獻回顧............................................................................. 3-1 益生菌......................................................................................... 3-1.1 益生菌定義.............................................................................. 3-1.2 益生菌種類.............................................................................. 4-2 乳酸菌及其對人體益處............................................................ 4-3 乳酸菌之環境耐性..................................................................... 6-3.1 耐酸性....................................................................................... 6-3.2 耐膽鹽性................................................................................... 7-4 微生物固定化定義及方法......................................................... 8-5.1 褐藻酸鈉結構.......................................................................... 12-5-2 褐藻酸鈉凝膠性質.................................................................. 12-5.3 影響褐藻酸鈣顆粒性質因素................................................ 13-6 幾丁聚醣簡介............................................................................. 14-6.1 幾丁質及幾丁聚醣來源及簡介............................................ 14-6.2 幾丁聚醣凝膠性質.................................................................. 15-6.3 幾丁聚醣應用.......................................................................... 15-7 褐藻酸鈉-幾丁聚醣錯合物....................................................... 17-8 研究目標...................................................................................... 18三章 實驗方法............................................................................. 24-1 實驗菌株..................................................................................... 24-2 實驗藥品..................................................................................... 24-3 實驗儀器..................................................................................... 30-4 實驗流程..................................................................................... 33-4.1 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒............ 33-4.2 以醋酸鄰苯二甲酸纖維素/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉..........................................................................................................33-5 實驗步驟..................................................................................... 33-5.1 種菌培養與活化..................................................................... 33-5.2 以混合法製備褐藻酸鈣/幾丁聚醣包覆乳酸菌複合顆粒34-5.3 以包覆法製備褐藻酸鈣/幾丁聚醣包覆乳酸菌複合顆粒35-5.4 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉................ 36-5.5 活菌體數測定法..................................................................... 36-5.6 掃描式電子顯微鏡(SEM)分析................................................ 37-5.7 元素分析.................................................................................. 37-5.8 介面電位元分析儀分析........................................................ 37-6 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒之耐酸性實驗及探討........................................................................................... 38-6.1 製備人工胃液......................................................................... 38-6.2 包覆乳酸菌之複合顆粒耐酸性實驗................................... 38-7 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒之耐膽鹽性實驗及探討........................................................................................40-7.1 製備人工膽汁......................................................................... 40-7.2 包覆乳酸菌之複合顆粒耐膽鹽性實驗................................41-8 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒於人工腸液放實驗及探討............................................................................... 42-8.1 製備人工腸液.......................................................................... 42-8.2 包覆乳酸菌之複合顆粒於人工腸液釋放實驗....................42-9 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉之耐酸性實驗及探討................................................................................................ 43-10 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉之耐膽鹽性實驗及探討.........................................................................................43-11 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉於人工腸液放實驗及探討................................................................................ 44四章 結果與討論.......................................................................... 49-1 褐藻酸鈉/幾丁聚醣複合顆粒元素分析結果........................ 49-2 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒之耐酸性探討......................................................................................................... 51-2.1 複合顆粒包覆不同濃度乳酸菌之探討................................ 51-2.2 單層包覆及多層包覆顆粒之探討........................................ 51-2.3 複合顆粒製程方法之探討.................................................... 52-2.4 鈣離子濃度對複合顆粒影響之探討................................... 52-2.5 不同幾丁聚醣分子量對複合顆粒影響之探討.................. 53-3 以褐藻酸鈣/幾丁聚醣包覆製成乳酸菌複合顆粒之耐膽鹽性探討.................................................................................................... 53-3.1 單層包覆及雙層包覆顆粒之探討....................................... 53-3.2 褐藻酸鈣/幾丁聚醣複合顆粒製程方法之探討................ 54-3.3 鈣離子濃度對複合顆粒影響之探討................................... 55-3.4 不同幾丁聚醣分子量對複合顆粒影響之探討.................. 55-4 以褐藻酸鈉/幾丁聚醣包覆製成乳酸菌複合顆粒於人工腸液釋放之探討.........................................................................................56-5 掃描式電子顯微鏡(SEM)分析.................................................... 57-6 乳酸菌及其包覆材料表面電位探討...................................... 58-6.1 pH 值對乳酸菌表面電位影響之探討.................................. 58-6.2 pH 值對CAP 溶液表面電位影響之探討............................... 58-6.3 pH 值對幾丁聚醣溶液表面電位影響之探討..................... 58-6.4 pH 值對褐藻酸鈉溶液表面電位影響之探討..................... 59-7 乳酸菌離心討論及SEM 觀察.................................................... 59-7.1 未經包覆之乳酸菌SEM 觀察................................................. 59-7.2 CAP 對乳酸菌離心效果影響之討論及SEM 觀察................ 59-7.3 幾丁聚醣對乳酸菌離心效果影響之討論及SEM 觀察...... 60-8 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉之耐酸性探討......................................................................................................... 60-8.1 不同CAP 濃度包覆製得乳酸菌粉耐酸性結果討論.......... 60-8.2 以CAP 或褐藻酸鈉包覆製得乳酸菌粉耐酸性結果討論.. 61-9 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉之耐膽鹽性探討......................................................................................................... 62-10 以CAP/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉於人工腸液釋放之探討..........................................................................................63五章 結論....................................................................................... 84考文獻............................................................................................. 87錄...................................................................................................... 96目錄2-1 乳酸菌代謝路徑................................................................. 192-2 細胞固定化技術................................................................. 202-3 以滴入法及乳化法包覆乳酸菌流程圖.......................... 202-4 褐藻酸鈉結構..................................................................... 212-5 褐藻酸鈣及蛋盒結構........................................................ 212-6 幾丁質、幾丁聚醣與纖維素結構圖.............................. 222-7 幾丁質、幾丁聚醣及其衍生物關係圖.......................... 232-8 褐藻酸鈉/幾丁聚醣錯合物之pH 值敏感性示意圖...... 233-1 以混合法製備包覆乳酸菌之褐藻酸鈣/幾丁聚醣複合顆粒流程圖............................................................................................. 453-2 以包覆法製備包覆乳酸菌之褐藻酸鈣/幾丁聚醣複合顆粒流程圖............................................................................................. 463-3 以醋酸鄰苯二甲酸纖維素/褐藻酸鈉/幾丁聚醣包覆製成乳酸菌粉流程圖............................................................................ 473-4 製備褐藻酸鈣顆粒實驗裝置示意圖............................... 484-1 以不同分子量之幾丁聚醣包覆褐藻酸鈣顆粒之SEM 圖654-2 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備單層複合顆粒包覆乳酸菌，其耐酸性測試結果.... 654-3 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備單層複合顆粒包覆乳酸菌，其耐酸性測試結果.... 664-4 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其耐酸性測試結果......664-5 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其耐酸性測試結果..... 674-6 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備單層複合顆粒包覆乳酸菌，其經耐膽鹽性測試3 小時結果................................................................................................... 674-7 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備單層複合顆粒包覆乳酸菌，其經耐膽鹽性測試3 小時結果................................................................................................... 684-8 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其經耐膽鹽性測試1 小時結果................................................................................................... 684-9 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其經耐膽鹽性測試3 小時結果................................................................................................... 694-10 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其經耐膽鹽性測試1 小時結果.............................................................................................. 694-11 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其經耐膽鹽性測試3 小時結果.............................................................................................. 704-12 以褐藻酸鈉溶液、0.2M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其於人工腸液中釋放結果....................................................................................................... 704-13 以褐藻酸鈉溶液、0.05M 氯化鈣溶液及不同分子量之幾丁聚醣製備雙層複合顆粒包覆乳酸菌，其於人工腸液中釋放結果........................................................................................................ 714-14 包覆乳酸菌之褐藻酸鈣/幾丁聚醣複合顆粒SEM 圖 (放大4000 倍) ............................................................................................ 724-15 包覆乳酸菌之褐藻酸鈣/幾丁聚醣複合顆粒SEM 圖 (放大12000 倍) ...........................................................................................734-16 未調整pH 值之乳酸菌液電位圖...................................... 744-17 pH 6 乳酸菌液電位圖........................................................ 744-18 pH 5.00 CAP 溶液電位圖.................................................... 754-19 pH 7.00 CAP 溶液電位圖.................................................... 754-20 pH 4.00 幾丁聚醣溶液電位圖........................................... 764-21 pH 6.54 幾丁聚醣溶液電位圖........................................... 764-22 pH 3.50 褐藻酸鈉溶液電位圖........................................... 774-23 pH 5.20 褐藻酸鈉溶液電位圖........................................... 774-24 pH 11.59 褐藻酸鈉溶液電位圖......................................... 784-25 未包覆之乳酸菌SEM 圖...................................................... 784-26 pH 6 乳酸菌及CAP 混合溶液電位圖................................ 794-27 添加CAP 離心之乳酸菌SEM 圖.......................................... 794-28 添加CAP 離心之乳酸菌並添加冷凍乾燥保護劑之SEM 觀察......................................................................................................... 804-29 添加幾丁聚醣離心之乳酸菌SEM 圖................................. 804-30 添加幾丁聚醣離心之乳酸菌並添加冷凍乾燥保護劑之SEM 觀察............................................................................................. 814-31 乳酸菌液加入CAP 離心後，以不同濃度CAP 包覆製得菌粉之耐酸性測試結果...................................................................... 814-32 乳酸菌液加入CAP 或幾丁聚醣離心後，以CAP 或褐藻酸鈉包覆製得菌粉之耐酸性測試結果............................................ 824-33 乳酸菌液加入CAP 或幾丁聚醣離心後，以CAP 或褐藻酸鈉包覆製得菌粉之耐膽鹽性測試結果........................................ 824-34 乳酸菌液加入CAP 或幾丁聚醣離心後，以CAP 或褐藻酸鈉包覆製得菌粉於人工腸液釋放結果........................................ 83錄圖I 幾丁聚醣溶液之濃度-黏度變化關係圖....................... 96錄圖II 乳酸菌顆粒形態SEM 圖.................................................. 97錄圖III 乳酸菌顆粒橫切面形態SEM 圖.................................... 98目錄2-1 益生菌應具備之性質............................................................ 32-2 應用於益生菌產品之微生物種類...................................... 42-3 乳酸菌包覆材料及製程方法.............................................. 103-1 製備褐藻酸鈣/幾丁聚醣複合顆粒實驗條件................... 394-1 褐藻酸鈣/幾丁聚醣複合顆粒元素分析結果................... 504-2 以0.05Ca-[Alg-c-200CS]-Alg 包覆不同濃縮倍數之乳酸菌液，製得之複合顆經耐酸性測試結果................................................. 514-3 乳酸菌液加入CAP 離心後，經不同濃度CAP 包覆製得菌粉，菌粉經耐酸性測試結果.......................................................... 614-4 乳酸菌液加入CAP 或幾丁聚醣離心後，再以CAP 或褐酸酸鈉包覆製得乳酸菌粉，菌粉經耐酸性測試結果........................ 624-5 乳酸菌液加入CAP 或幾丁聚醣離心後，再以CAP 或褐酸酸鈉包覆製得乳酸菌粉，菌粉經耐膽鹽性測試結果................... 63錄表I 以褐藻酸鈣/幾丁聚醣複合顆粒包覆乳酸菌，經耐酸性測試後，存活菌數及存活率結果................................................. 99錄表II 以褐藻酸鈣/幾丁聚醣複合顆粒包覆乳酸菌，經耐膽鹽性測試後，存活菌數及存活率結果............................................ 100application/pdf5431559 bytesapplication/pdfen-US乳酸菌褐藻酸鈉幾丁聚醣醋酸鄰苯二甲酸纖維素包覆lactic acid bacteriasodium alginatecellulose acetate phthalatechitosanencapsulationthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183160/1/ntu-98-R96549007-1.pdf