Effects of the Addition of Various Protectants on the Dehydrated Lactobacillus acidophilus CCRC 10695 Strain during Storage

冷凍乾燥廣泛地被使用於菌種保存，但是由於其經過了凍結及在凍結狀態下行真空乾燥的操作，導致了大多數菌體細胞的死亡。嗜酸乳酸桿菌 (Lactobacillus acidophilus) 具有多項有益人體的生理功能，在市售乾燥食品中常有添加，有助人體健康，但是在乾燥食品中之存活率及貯存安定性均差。因此本研究嘗試探討嗜酸乳酸桿菌經由冷凍、真空乾燥及冷凍乾燥處理後之存活率，並決定乾燥後之L. acidophilus在添加lysozyme及oxgall之培養基處理下的敏感性。藉由脫脂乳粉、麥芽糊精及海藻糖等不同保護劑的添加，來瞭解其對經不同乾燥方法處理後之L. acidophilus在三個月之儲藏期限中的保護效果及其β-galactosidase活性變化。 結果顯示L. acidophilus經冷凍及乾燥處理後，存活率以冷凍>冷凍乾燥>真空乾燥，而貯藏穩定性則以冷凍乾燥>冷凍>真空乾燥。冷凍、真空乾燥及冷凍乾燥後之L. acidophilus及其貯藏安定性，添加NFDMS、MD及trehalose作為保護劑的最適添加濃度分別為15%、10%、10%，得知不同保護劑的最適添加濃度不盡相同。結果發現所得效果以脫脂乳粉最佳，其次為麥芽糊精及海藻糖。在真空乾燥及冷凍乾燥後之L. acidophilus貯藏期間的作用上，溫度為重要之影響因子，其中以-40℃貯存之存活率最佳。結果顯示真空乾燥菌體對lysozyme及oxgall的敏感性均較冷凍乾燥高，而又以15% NFDMS的添加對菌體細胞壁之保護效果最佳。無論何種保護劑的添加，由於貯藏期間菌體的死亡分解及環境的不利，致使β-galactosidase活性均隨貯藏時間的增加而減少，而貯藏溫度對β-galactosidase活性下降速率以25℃>4℃>-40℃。Freeze-drying has been applied extensively to the dehydrating preservation of microbial cultures. However, the freezing operation and the vacuum-drying under frozen state may result in the inactivation of the majority of microbial cells. Lactobacillus acidophilus possesses lots of important beneficial functions to human physiology and numerous L. acidophilus containing dried products are now very popular for use as dietary adjuncts for humans and animals. However, low numbers of active L. acidophilus cells were found in the dried products. In this research, survival of L. acidophilus strain due to freezing, vacuum drying and lyophilization was studied and determined their sensitivity by the addition of lysozyme and oxgall. The protective effect of non-fat dry milk solids (NFDMS), maltodextrin (MD), and trehalose on dried L. acidophilus, and the variation of β-galactosidase activity of the dried cultures during 3-month storage were studied. Results showed that the survival of frozen L. acidophilus, rather than vacuum-dried and freeze-dried L. acidophilus, was the best, but the stability of freeze-dried L. acidophilus during sotrage was better than frozen and vacuum-dried L. acidophilus. The most appropriate concentration of L. acidophilus with NFDMS, MD, and trehalose were found to be 15%, 10%, and 10% respectively, and the effect of NFDMS was found to be the best, followed by MD, and trehalose. Storage temperature was an important factor to frozen and dried L. acidophilus, and the best survival was obtained when stored at -40℃. Results showed that the sensitivity of vacuum-dried L. acidophilus in media with lysozyme or oxgall was higher than freeze-dried L. acidophilus, and 15% NFDMS provided the best protection. β-galactosidase activity decreased due to cells lysis during storage and declined slowly when stored at -40℃.目錄 頁次 壹、前言……………………………………………………………1 貳、文獻整理………………………………………………………4 一、關於乳酸菌………………………………………………4 (一)乳酸菌之定義分野………………………………….4 (二)乳酸菌之應用……………………………………….7 (三)乳酸菌在人體健康上的重要性…………………….17 (四)嗜酸乳酸桿菌 (Lactobacillus acidophilus)………...23 二、菌之保存及貯存過程中的影響因子…………………33 三、真空乾燥技術……………………………………………35 四、冷凍乾燥技術……………………………………………37 (一)原理………………………………………………….37 (二)冷凍乾燥機的設備簡介…………………………….37 (三)冷凍乾燥的優缺點………………………………….40 (四)冷凍乾燥的應用範圍……………………………….43 (五)冷凍乾燥的菌株保存……………………………….43 五、冷凍及乾燥對乳酸菌的影響……………………………46 (一)冷凍對乳酸菌的影響……………………………….46 (二)冷凍乾燥對乳酸菌之影響………………………….46 (三)保護劑的添加……………………………………….47 參、研究目的………………………………………………………53 肆、材料與方法……………………………………………………54 一、試驗材料…………………………………………………54 二、研究方法…………………………………………………55 伍、結果與討論……………………………………………………62 一、冷凍、真空乾燥及冷凍乾燥之存活率…………………62 二、冷凍及乾燥後L. acidophilus的貯藏安定性……………65 三、濃度對L. acidophilus貯藏安定性的影響………………69 四、貯藏溫度對真空、冷凍乾燥L. acidophilus貯藏安定性的影響……………………………………………………73 五、保護劑對L. acidophilus貯藏安定性的影響……………76 六、保護劑、溫度及存活率間之影響………………………78 七、貯藏溫度對乾燥L. acidophilus菌體之存活率的影響…86 八、保護劑對L. acidophilus菌體之存活率的影響…………97 九、β-galactosidase活性……………………………………105 陸、結論……………………………………………………………111 柒、中文摘要………………………………………………………113 捌、英文摘要………………………………………………………115 玖、參考文獻………………………………………………………11