Lactobacillus reuteri 冷凍耐受性提升的探討： 細胞微膠囊化及海藻糖添加的利用

Abstract

Lactobacillus reuteri 為常用於保健產品製造的格蘭氏陽性菌，對於人體生理具有多項保健的功效，能產生抗菌物質(reuterin)，故視為動物腸道中的重要菌種之ㄧ，在宿主健康的維護上扮演重要角色。ㄧ般利用冷凍貯藏的方式，保存食品中的活菌數，然而冷凍可能會對菌體造成傷害，因此可結合細胞微膠囊化技術及添加保護劑以減少菌體在冷凍貯藏時之傷害。本研究利用褐藻酸鈣來對L. reuteri 進行細胞微膠囊化處理，同時分別添加不同濃度(1％、2％、3％)的海藻糖作為冷凍保護劑，再放置於不同凍結溫度下(-20℃、-40℃、-60℃、-80℃、-196℃)來進行貯藏，探討其對L. reuteri在冷凍貯藏中的保護效果，以提升菌體之存活率、活性與貯藏安定性，並比較不同處理 間的差異。結果顯示，細胞經微膠囊化處理後能有效提高菌體冷凍貯藏的存活率，保護效果較佳。在凍結貯藏溫度方面，溫度對菌體的存活率的影響並無ㄧ致性，但菌體於-60℃貯藏有得到一較低的死滅速率常數，即添加2%海藻糖並經微膠囊化處理之菌體在-60℃冷凍貯藏下有較好的存活率，保護效果較佳。不同濃度海藻糖的添加，對於菌體的死滅皆有減緩的效果，且於2%的添加濃度可以獲得較小的死滅速率，亦即最高存活率。综合微膠囊化技術、不同冷凍貯藏溫度及不同濃度冷凍保護劑的添加對菌株存活率所得之結果，發現經微膠囊化處理於冷凍溫度-60℃貯藏並添加2%海藻糖，可以獲得最小死滅速率常數，此即為菌體較佳之貯藏條件。在β-半乳糖苷酶酵素活性方面，發現此酵素活性會因為菌體的死滅而減少。由細胞膜脂肪酸組成分析結果，可發現各處理組之不飽和脂肪酸含量皆增加且不飽和脂肪酸/飽和脂肪酸(U/S)比例亦增加。而蛋白質分析結果顯示各處理組皆無新的胞內蛋白質生成。利用掃描式電子顯微鏡觀察發現L. reuteri呈短桿狀(約1 μm)， 且有時幾乎接近球狀，而細胞表面也出現有皺縮的現象，推測可能是菌體在經冷凍乾燥或臨界點乾燥時受到壓迫。

Lactobacillus reuteri is a gram-positive bacterium that commonly used in manufacturing health products, and possesses lots of important physiological functions beneficial to human body, and produces antibacterial material (reuterin). L. reuteri is considered a major organism of the human gastrointestinal tract and plays very important roles in the maintenance of host's health. In order to achieve the effective viable cell numbers in food, freezing storage is the most frequently used technique to preserve the cells. However, freezing will cause damage to the cell, so the cell microencapsulation technology and the addition of protectants are frequently used to reduce the cell damage during frozen storage. In this study, Ca-alginate was applied to L. reuteri for cell microencapsulation, and different concentrations (1％, 2％, 3％) of trehalose were added as cryoprotectants at the same time for the storage test at different frozen temperatures (-20℃, -40℃, -60℃, -80℃, -196℃) in order to study their protection effects on the enhancement of cell survival, activity and storage stability of L. reuteri during frozen storage and effects of different treatments were also compared. Results indicated that cell microencapsulation could effectively increase the cell survival during frozen storage, and the good protection effect was obtained. Wth respect to frozen temperatures, results indicated that there is no consistency between the temperatures and the cell survival existed. But -60℃ frozen storage could obtain a lower death rate constant among various frozen temperatures used, and microencapsulated L. reuteri with 2％ trehalose addition during storage at -60℃ could obtain the highest survival. Regarding trehalose addition, various concentrations of trehalose addition could reduce cell death. 2％ addition of trehalose could obtain the lowest death rate constant and the highest survival. It was found that 2％ trehalose addition combined with cell microencapsulation and -60℃ frozen temperature could obtain the lowest death rate constant and were found to be the best conditions among all the treatments. Concerning β-galactosidase activity, results indicated that freezing kills many of the cells and causing loss of enzymatic activity. Results of the cellular membrane fatty acid composition analysis showed that the concentration of unsaturated fatty acids and the ratio between unsaturated and saturated fatty acids (U/S) all increased in all treatments. In SDS-PAGE analysis, no new intracellular protein was found. The scanning electron microscopic images of L. reuteri were short rod-shaped (approximately 1 μm in length), and sometimes nearly spherical, and then the cell surfaces were also appeared to be shrinked and wrinkled. Extrapolating from this trend, it''s probable that cells were subjected to the suppression of freeze drying or critical point drying.