Freeze-drying of live attenuated Vibrio anguillarum mutant for vaccine preparation

Vibrio anguillarum MVAV6203 is a mutant strain as a candidate of live attenuated vaccine. In vaccine preparation, the freeze-drying conditions of the strain were investigated to improve the survival after freeze-drying, including the protectant, rehydration medium, freezing temperature, and initial cell concentration. Vibrio anguillarum MVAV6203 is sensitive to freeze-drying and the viability was only 0.03% in the absence of protectant. Of the tested protectants, 5% trehalose with 15% skimmed milk gave the highest viability of 34.2%. Higher cell survival was obtained by quick freezing at −80 °C than slow freezing at −20 °C. Initial cell concentration was another important factor, preferable for 1–3 × 1010 CFU/ml. The supplementation of 10% skimmed milk in rehydration medium improved obviously freeze-drying viability. The combination of the optimal conditions achieved 51.4% cell viability after freeze-drying.