Optimization of Protectant, Salinity and Freezing Condition for Freeze-Drying Preservation of Edwardsiella tarda  

作　　者：YU Yongxiang ZHANG Zheng WANG Yingeng LIAO Meijie LI Bin XUE Liangyi 

机构地区：[1]College of Marine Sciences, Ningbo University [2]Laboratory for Marine Fisheries Science and Food Production Processes, Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences

出　　处：《Journal of Ocean University of China》2017年第5期831-839,共9页中国海洋大学学报（英文版）

基　　金：the National Natural Science Foundation of China (No. 31302206);Special Research Funds for Independent Innovation and Scientific & Technology Achievements Transformation of Shandong Province (No. 2014ZZCX06205);Agriculture Seed Improvement Project of Shandong Province

摘　　要：Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose(8.0%), skim milk(12.0%), sodium citrate(2.0%), serum(12.0%) and PVP(2.0%). Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of Na Cl was 10.0, 30.0 and between 5.0 and 10.0 g L^(-1) for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at-80℃ or-40℃ for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate(79.63%–82.30%) of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy(SEM) image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.Novel preservation condition without ultra-low temperature is needed for the study of pathogen in marine fishes. Freeze-drying is such a method usually used for preservation of terrigenous bacteria. However, studies using freeze-drying method to preserving marine microorganisms remain very limited. In this study, we optimized the composition of protectants during the freeze-drying of Edwardsiella tarda, a fish pathogen that causes systemic infection in marine fishes. We found that the optimal composition of protectant mixture contained trehalose（8.0%）, skim milk（12.0%）, sodium citrate（2.0%）, serum（12.0%） and PVP（2.0%）. Orthogonal and interaction analyses demonstrated the interaction between serum and skim milk or sodium citrate. The highest survival rate of E. tarda was observed when the concentration of Na Cl was 10.0, 30.0 and between 5.0 and 10.0 g L^-1 for preparing TSB medium, E. tarda suspension and protectant mixture, respectively. When E. tarda was frozen at-80℃ or-40℃ for 6 h, its survival rate was higher than that under other tested conditions. Under the optimized conditions, when the protectant mixture was used during freeze-drying process, the survival rate（79.63%–82.30%） of E. tarda was significantly higher than that obtained using single protectant. Scanning electron microscopy（SEM） image indicated that E. tarda was embedded in thick matrix with detectable aggregation. In sum, the protectant mixture may be used as a novel cryoprotective additive for E. tarda.

关 键 词：tarda pathogen citrate preservation preserving contained preparing thick additive suspended 

分 类 号：S941[农业科学—水产养殖]