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Studies on the Preparation of Microencapsulated Lactic Acid Bacteria by Using Spray-Coagulation Method and Its Freeze-Drying
利用水凝膠作為包埋基質之微膠囊化技術，在加工處理過程中可有效提升乳酸菌的存活率及活性，目前常用的方法包括擠壓法與乳化法，兩種方法各有其優缺點。前者具有低成本、操作簡單之優點；後者則具備可產生粒徑較小的微膠囊及可大量生產之優勢，但是複雜度及成本高。為了改良傳統擠壓法微膠囊化技術的缺點，本研究擬採用二流體式噴頭，將菌體、低溫保護劑和褐藻酸鈉之混合液，以噴霧方式噴出滴入氯化鈣溶液中進行凝結，以開發出能以低成本且操作簡易的方式，大量生產出粒徑較小之乳酸菌微膠囊的技術。Lactobacillus reuteri能於人體腸道中生存，具有許多益生功效，加以本研究室對其有多年之研究經驗，因此本研究擬採用其作為被包埋之菌株。另一方面，冷凍乾燥被廣泛應用於菌株的保存上，但因其操作成本高且耗費時間長，故申請人開發出間歇式遠紅外線冷凍乾燥法來改善上述缺點，再配合上低溫保護劑及微膠囊化對菌株之保護效果，在菌株保存上不但能有效達到保存之目的，而且又可降低時間與金錢的成本。綜合上述因素，本研究擬應用反應曲面法，針對經二流體式噴頭噴霧凝結之微膠囊化L. reuteri及其冷凍乾燥進行為期三年之一系列探討。第一年擬利用反應曲面法，配合四因子三層階的試驗設計，探討在分別以三種不同氯化鈣濃度進行凝結的情況下，針對以二流體式噴頭製備菌株微膠囊化樣品時，不同空氣流速、菌株與褐藻酸鈉混合液流速、膠濃度、低溫保護劑濃度等四種因子間之作用進行探討，嘗試找出在三種不同氯化鈣濃度下凝結時，分別能獲得最高菌體存活率之最適操作條件；第二年則擬對前一年找出之三組最適操作條件，進行模擬腸胃液測試，並分別進行菌株活性及菌體所受影響等性質之探討，進而得出一組最適操作條件；第三年則擬利用間歇式遠紅外線冷凍乾燥法，進一步針對由第二年所找出之最適條件樣品，進行微膠囊化菌體的冷凍乾燥，配合上三因子三層階之試驗設計，利用反應曲面法針對遠紅外線加熱溫度、加熱間歇率、預冷凍溫度等三種因子間之作用進行探討，並針對根據存活率結果所得之最適樣品進行與第二年相同之試驗，以瞭解間歇式遠紅外線冷凍乾燥的作用效果。
Microencapsulation technique which uses hrdrogel as entrapment matrix could enhance survival and activity of lactic acid bacteria during processing. The popular methods applied nowadays could be classified into two groups, i.e. extrusion and emulsion. The former has the advantages of low cost and simplicity, and the latter could produce smaller beads and is easy to scale up but the cost is much higher. In order to improve the conventional extrusion microencapsulation technique, this study would apply a two-fluid nozzle system to spray the mixture of strains, cryoprotectant and sodium-alginate into calcium chloride solution to make the coagulation. The goal is to develop an easy way to produce a large number of microcapsules with smaller particle sizes efficiently at low cost. Lactobacillus reuteri could colonize in human intestines and possesses lots of important physiological functions beneficial to human health, and our laboratory has studied it for several years. Therefore, this study would use L. reuteri for microencapsulation. On the other hand, freeze-drying (FD) is a popular method used in the culture preservation of microorganisms. However, a major problem of FD is its long drying time, which in turn leads to high energy consumption and high capital costs. Intermittent far-infrared radiation freeze-drying (Intermittent FIR-FD) which was developed by our laboratory is a technique which could solve those problems. Furthermore, this method combined with the addition of cryoprotectant and microencapsulation of strains could not only provide protection effect for culture preservation but also reduce the cost of time and money. Finally, response surface methodology (RSM) would be employed to analyze the effects of spray-coagulation and intermittent FIR-FD on the microencapsulated L. reuteri, and a three-year project would be proposed. In the first year, a four-variable and three-level design, analyzed by RSM, will be used to study the effects of feed flow rate, air flow rate, gel concentration, cryoprotectant concentration on the survival of L. reuteri when coagulated with three different calcium chloride concentrations respectively, and determine the optimum conditions. In the second year, based on those optimum conditions found with three different calcium chloride concentrations in the first year, the simulated gastric and intestinal fluids test will be performed, and the activity and variation of the strains obtained by those conditions will be researched. The most optimum one will therefore be determined. In the third year, intermittent FIR-FD will be used to study the dehydrated properties of the strain obtained by the most optimum condition found in the second year. A three-variable and three-level design, analyzed by RSM, will be applied to study the effects of FIR heating temperature, intermittence, prefreezing temperature on the survival of the strain, and the optimum conditions of intermittent FIR-FD will be determined. The activity and variation of the dehydrated strain obtained by the optimum intermittent FIR-FD conditions will be researched.
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