properties of kefir manufactured by microencapsulated starter cultures

克弗爾（kefir）為酒精發酵乳，其微生物菌相相當複雜，主要由多種乳酸菌與酵母菌共生而成。研究顯示乳酸菌提供人體多樣機能性功能，為解決因克弗爾粒 (kefir grain) 菌相複雜無法實際商業化大量生產克弗爾發酵乳之問題，本研究使用篩選純化自克弗爾粒之乳酸菌與酵母菌菌株，利用擠出法 (extrusion method) 將各純菌株包覆於褐藻膠中，製作出純菌克弗爾粒，並當成發酵菌元，期望能生產品質穩定，且具保健效用之克弗爾，解決無法商業化大量生產之問題。 本研究使用篩選純化自克弗爾粒之乳酸菌 (Lactobacillus kefiranofaciens H1, Lb. kefiri H2, Leuconostoc mesenteroides H3, Lactococcus lactis H4) 與酵母菌菌株 (Kluyveromyces marxianus Y1, Saccharomyces turicensis Y2, Pichia fermentans Y3)，以三種不同包覆處理的方式，將其包覆於含益菌質之褐藻膠微膠囊內，純菌克弗爾粒以牛奶當作基質，進行連續發酵培養（每次於20 ºC 培養24 h更換牛乳）共28次，測定不同組別在菌數、理化特性及官能品評上與傳統克弗爾之差異。結果顯示，經連續28次繼代培養後，利用純菌克弗爾粒發酵所得之克弗爾與傳統克弗爾有相似的菌數及理化官能特性。然而，以乳酸菌與酵母菌分別進行包覆的純菌克弗爾粒 (組別B) 經連續發酵28次後，其官能品評較佳（P<0.05），並且在純菌微膠囊的生產製造過程上，擁有較為簡化的程序，因此選用組別B進行進一步的分析測定。 利用掃描式電子顯微鏡 (scanning electron microscope) 與聚合酶連鎖反應－變性梯度膠體電泳 (polymerase chain reaction-denaturing gradient gel electrophoresis, PCR-DGGE) 觀察膠囊微生物菌相於連續繼代培養期間菌相之改變，球菌與短鏈的乳酸桿菌會聚集於膠囊表面而長鏈的乳酸桿菌則穩定的生長於膠囊內部；包覆酵母菌的微膠囊無論表面或內部皆有大量酵母菌存在，並且有短鏈乳酸菌圍繞其間。在菌相的測定上，乳酸菌含量以Lb. kefiranofaciens所佔比例最多，達到八成以上的佔有率；酵母菌則以Klu. marxianus為優勢菌種，無論是乳酸菌及酵母菌經過連續28次寄代培養後皆呈現穩定的菌相分布。 以純菌克弗爾粒發酵所得之克弗爾，無論是在理化官能特性或微生物菌相穩定性上，皆與傳統克弗爾有相似的表現，甚至在官能品評與酸度的測定上更能為消費者所接受，因此以純菌克弗爾膠囊替代傳統克弗爾粒進行發酵，能製造出品質穩定的克弗爾，增加乳製品的多樣性。 Kefir is a beverage produced by lactic-alcoholic fermentation of milk using kefir grains. However, kefir grains have a complex microbiological composition that makes it difficult to obtain an optimal and constant starter culture necessary for the production of a quality kefir beverage. Thus, the purpose of this study was to create a mimic kefir grain using encapsulated pure cultures to produce a milk beverage with characteristics similar to those of traditional kefir. In this study, yeast (Klu. marxianus Y1, S. turicensis Y2, P. fermentans Y3) and LAB (Lb. kefiranofaciens H1, Lb. kefiri H2, Leu. mesenteroides H3, Lc. Lactis H4) were entrapped in microcapsules with three different groups. The encapsulated ratio of the strains was based on the distribution of kefir grains. After microencapsulation, both microencapsulated starter and kefir grain were incubated with milk at 20ºC, sieved and transferred to fresh pasteurized milk every 24 h. This procedure was repeated at 28 times. The sensory, microbial and chemical characteristics of microcapsule kefir and traditional kefir were determined. Results indicated that there were similar performance between microcapsule and traditional kefirs in microbial and chemical characteristics during the consecutive cultivations. Among different microcapsule groups, Group B, entrapped all strains of lactic acid bacteria and yeast, respectively in one microcapsule, had better organoleptic property with easy manufacturing processing. Consequently, this group was chosen for the following study. Since kefir quality depends on microflora property, the scanning electronic microscopy and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were also used to observe kefir starters in group B and grains. The SEM showed that the short-chain lactobacilli and lactococci were occupied on the surface and the long-chain lactobacilli were in the inner of LAB microcapsules. For yeast, high density of yeast cells was entrapped on crack of the surface and the interior of the microcapsules with surrounded by the short-chain lactobacilli. The distribution of the species of LAB and yeasts in kefirs from grains and microcapsules showed that Leu. mesenteroides and K. marxianus were the predominating microflora in both kefirs. There was no difference among cultivations. In conclusion, kefir produced by microencapsulated starter, the basic microbiological, physicochemical and sensory characteristics of traditional kefir were reproduced with less acidity, which might increase the acceptability for kefir drink. This alternative procedure eliminating the problems arising from the use of kefir grains make possible the production of a standard product in the dairy industry.口試委員會審定書謝錄………………………………………………………………………I目錄……………………………………………………………………IV目錄……………………………………………………………………V文摘要…………………………………………………………………VI文摘要…………………………………………………………………IXamp;#32214;言………………………………………………………………………XI、文獻檢討……………………………………………………………1 一、 克弗爾及克弗爾粒…………………………………………1 (一) 克弗爾粒菌元之研究…………………………………1 (二) 克弗爾發酵乳之製作…………………………………9 二、微生物菌相及菌種鑑定……………………………………9 (一) 16S rDNA 的獨特性及其運用………………………11二) 聚合酶連鎖反應－變性梯度膠體電泳（polymerase链 反应-使改变本性倾斜的凝胶体电泳, PCR-DGGE）.… 12 (三) 乳酸菌鑑別之研究……………………………………13 三、 微膠囊於乳製品上之應用研究……………………………14 (一) 微膠囊包覆技術及其特性之介紹………………………14 (二) 益生菌微膠囊之製作……………………………………21 (三) 微膠囊固定化對乳酸菌生長之影響……………………22 (四) 益生菌微膠囊在乳製品上的應用………………………23、材料與方法…………………………………………………………26 一、實驗材料………………………………………………………26 (一) 乳酸菌與酵母菌之菌種來源……………………………26 (二) 微膠囊組成基質…………………………………………26 (三) 菌種及培養基……………………………………………26 (四) 試藥………………………………………………………26 (五) 實驗儀器及器材…………………………………………27 二、實驗方法……………………………………………………29 (一) 乳酸菌元與酵母菌元之活化……………………………29 (二) 菌體之收集……………………………………………29 (三) 純菌克弗爾微膠囊製備………………………………29 (四) 傳統克弗爾之製備……………………………………30 (五) 微生物菌數測定………………………………………30 (六) 不同微膠囊克弗爾粒製成之發酵乳理化特性比較…………………30 (七) 不同微膠囊克弗爾粒製成之發酵乳官能特性探討…………………32 (八) 連續培養之菌相穩定性探討…………………………………………33、結果與討論………………………………………………………………………42 一、純菌克弗爾微膠囊與傳統克弗爾粒製成克弗爾之理化官能特性比較…42 (一) 酸度與pH值……………………………………………………………43 (二) 黏度表現………………………………………………………………43 (三) 胞外多醣體之生成……………………………………………………44 (四) 克弗爾中乳酸菌之生長………………………………………………44 (五) 克弗爾中酵母菌之生長………………………………………………45 (六) 品評試驗之比較………………………………………………………46 二、純菌克弗爾微膠囊與傳統克弗爾粒之顯微構造…………………………55 (一) 傳統克弗爾粒與純菌克弗爾微膠囊於發酵前之比較………………55 (二) 傳統克弗爾粒與純菌克弗爾微膠囊經過連續14次發酵後之比較…56 (三) 傳統克弗爾粒與純菌克弗爾微膠囊經過連續28次發酵後之比較…57 三、純菌克弗爾微膠囊與傳統克弗爾粒製成克弗爾之風味物質分析…………62 (一) 乳酸含量測定……………………………………………………………62 (二) 乙醇含量測定……………………………………………………………62 四、純菌克弗爾微膠囊與傳統克弗爾粒製成克弗爾菌相穩定性探討…………66 (一) 連續培養期間乳酸菌菌相穩定性………………………………………66 (二) 連續培養期間酵母菌菌相穩定性………………68、結論………………………………………………………74、參考文獻…………………………………………………77、作者小傳…………………………………………………8