Study On Freeze-Drying Protectant Selection And Freeze-Drying Technics Construction Of Important Pathogenic Bacteria In Mariculture

Long-term preserved the activities of pathogenic bacteria in mariculture is animportant premise and guarantee for its etiology research. Freeze-drying technology isone of the most effective methods for the preserved of biological materials incurrently. In this research, we preliminary discusses the application of freeze-dryingtechnology in the preservation of5strains of pathogenic bacteria in mariculture, theresults will provide an important reference for the application of freeze-dryingtechnology in the marine bacteria.1. Selection of freeze-drying protectants for PathogensEdwardsiella tarda, Pseudoalteromonas, Vibrio harveyi, Vibrio splendidus andVibrio anguillarum were selected as experimental strains. Different concentrations ofSkim milk, trehalose, mannitol, lactose, fructose, glucose, ascorbic acid and gelatinwere selected as freeze-drying protectants,1.50%NaCl as control. The results showthat: the experimental groups which were took different concentrations of ascorbicacid and gelatin as protectant were not detected the existence of living bacterium.Compared with the control group, A certain concentration of Skim milk andsaccharides can protected the pathogenic bacteria in the process of freeze-drying, Theprotective effect from strong to weak, in order: Pseudoalteromonas: Skim milk,maltose, trehalose, lactose, mannitol, glucose, fructose. Edwardsiella tarda: Skimmilk, lactose, mannitol, trehalose, maltose, glucose, fructose. Vibrio harveyi: mannitol,trehalose, lactose, maltose, fructose, skim milk, glucose. Vibrio anguillarum: lactose,mannitol, skim milk, maltose, glucose, fructose. Vibrio splendidus: mannitol, lactose,skim milk, trehalose.2. Freeze-drying protectant compatibility optimization Skim milk, lactose, trehalose and mannitol were selected as freeze-dryingprotectants. At the same time, a certain amount of ascorbic acid and gelatin was addas antioxidant and excipient.The results show that: The best ratio of protectants forEdwardsiella tarda, Pseudoalteromonas and Vibrio harveyi was obtained By meansof orthogonal analysis, furthermore,Through three times verification tests proved thatthe stability of the protectants was better. The optimum ratio of protectants forPseudoalteromonas and the correspond cell survival rate was:7.5%lactose,5%mannitol,5%trehalose,10%skim milk,0.5%ascorbic acid and0.5%gelatin, from3times Repeated testing, the cell survival rates were greater than32.17%. Edwardsiellatarda:5%lactose,15%skim milk,0.5%gelatin,0.5%ascorbic acid, from3timesRepeated testing, the cell survival rates were about20.0%. Vibrio harveyi:7.5%lactose,5%mannitol,5%trehalose,15%skim milk,0.5%gelatin and0.5%ascorbicacid, from3times Repeated testing, the cell survival rates are around5.0%. The cellsurvival rate of Vibrio anguillarum and Vibrio splendidus was extremely low afterfreeze-drying, there no significant difference between each orthogonal experimentalgroups. Compared with Edwardsiella tarda and Pseudoalteromonas, Three strains ofvibrio had a poorer protective effect to against freeze-drying.3. Optimizing freeze-drying conditions and processPseudoalteromonas was selected as representative strain. The optimum ratio ofprotectants for Pseudoalteromonas was selected as freeze-drying protectant. The saltcontent, rehydration medium, initial cell concentration, The proportion of protectantand bacterium fluid, growth phase were investigated. The results show that: When1.5%NaCl was use to prepare the bacterial suspension, the optimal salinity ofprotectant is0.5%, and the cell survival rate is40.3%. The optimal initial cellconcentration between1-5×109CFU/ml.5-10%skimmed milk gave the bestrehydration effect, sterilized sea water gave the worst effect. When the proportion ofprotectant and bacterium fluid was1:1,1:2,1:3the cell survival rate was35.95%，37.14%，37.15%,It indicated that high concentration of protectant was no significant impact on cell survival rate. Compared with logarithmic phase, stationary phase havea better protective effect.4.The comparison between Freeze-drying and ultra-low temperature preservationThis chapter focuses on the comparison between-80℃ultra-low temperatureand freeze-drying preservation technology. The results show that: In the next half year,the cell survival rates of the ultra-low temperature preserved Edwardsiella tarda andPseudoalteromonas from100%declined to30%gradually.The freeze-drying cellsurvival rates of Edwardsiella tarda from19.83%declined to17.44%, andPseudoalteromonas from33.57%declined to31.22%. It can be seen, compared withultra-low temperature preservation technology, freeze-drying technology keep thebetter stability in the process of long-term preservation. But freeze-drying technologyhave a tedious operation process, larger decline of survival rate before and afterfreeze-drying, freeze-dried products can only be used once. Therefore, ultra-lowtemperature preservation technology have a good preservation effect on thepathogenic bacteria that need Short-term preservation or repeated used.The selection and optimization of freeze-drying protectant of5strains pathogenicbacteria was study. Pseudoalteromonas was selected as representative strain toinvestigate the process conditions in the freeze drying process. And the comparisonbetween-80℃ultra-low temperature and freeze-drying preservation technology wasstudy. The selection and distribution of freeze-drying protectant as well as theoptimization of the feeze-drying process make the freeze-drying technique have abetter applied to the preservation of pathogenic bacteria in mariculture, It will behelpful in etiology research of pathogenic bacteria.