Studies on the Enhancement of the Freeze-Tolerance of Lactobacillus reuteri: the Application of Cell Microencapsulation and Trehalose Addition

• Main Authors: Ya-Li Hsu, 徐亞莉
• Other Authors: 金安兒
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/yecjh4

碩士 === 國立中興大學 === 食品暨應用生物科技學系所 === 97 === 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.