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| Research on the Formula and Lyophilization Process of Vibrio alginolyticus Cryoprotectant |
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| DOI：10.16768/j.issn.1004-874X.2024.06.012 |
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| Abstract: |
| Abstract: 【Objective】The study aims to screen the formula of vacuum freeze-drying protective agent for Vibrio
alginolyticus, optimize the freeze-drying process conditions, and explore the influence of freeze-drying on the virulence and drug sensitivity of the strains, with a view to providing a theoretical basis for the application of freeze-drying in the preservation
of the strains.【Method】With Vibrio alginolyticus HY9901 as the research object, the vacuum freeze-drying technology was
used to freeze-dry the strains. Through single factor test and orthogonal test, the effects of six single cryoprotectants (L-glutamate
sodium, trehalose, skim milk, glucose, mannitol and glycine) on the activity of the strains were investigated, and the optimal
formula of cryoprotectants was selected. The influences of four factors, namely, concentration of bacterial solution, pre-freezing
time, pre-freezing temperature and rehydration medium, on the freeze-drying process of strains were studied, and the freezedrying process conditions for Vibrio alginolyticus were optimized. At the same time, storage test, LD50 test and antimicrobial
susceptibility test were used to investigate the changes in the activity of lyophilized strains and their of biological characteristics.
【Result】There were significant differences in the protective effects of 6 cryoprotectants on lyophilized Vibrio alginolyticus,
and the effects from strongest to weakest showed as: L-glutamate sodium, trehalose, skim milk, glucose, mannitol and glycine.
The optimum freeze-drying compound formula of cryoprotectants was trehalose 50 g/L+ L-glutamate sodium 50 g/L+ skim
milk 50 g/L, with a survival rate of 62.12%, which was significantly different from that of single cryoprotectant. The optimal
conditions of freeze-drying process were as follows: bacterial solution concentration 1×108
-109
CFU/mL, pre-freezing time 3
h, pre-freezing temperature -80 ℃ , rehydration medium PBS or 50 g/L skim milk, under which the activity of the strains could
reach the maximum of 63.72%. There was no significant difference in the activity of the strains during 60 days of storage under
optimal freeze-drying conditions. The LD50 values of the strains before and after freeze-drying were 1.22×108
CFU/mL and
1.20×108
CFU/mL, respectively, with no significant difference in LD50. There was no significant difference in the diameter of
the antimicrobial inhibition zone before and after freeze-drying, and the strains were sensitive to florfenicol, chloramphenicol,
ciprofloxacin and other drugs. They were moderately sensitive to neomycin and kanamycin, and resistant to drugs like amimcana
and piperacillin.【Conclusion】The freeze-drying formula for Vibrio alginolyticus HY9901 is trehalose 50 g/L+ L-glutamate
sodium 50 g/L+ skim milk 50 g/L, and the freeze-drying process conditions are: bacterial solution concentration 1×108
-
109
CFU/mL, pre-freezing time 3 h, pre-freezing temperature -80 ℃ , rehydration medium PBS or 50 g/L skim milk. The fomula
and process for this strain are reasonable and feasible, with good storage and stability of biological characteristics. |
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