Optimization Of Freeze-drying Protective Agent For Active Peptide CC34 Recombinant Pichia Pastori

The objective of this study was to optimize the lyophilized protective agent composition of Pichia Pastoris recombinant bacteria containing active peptide CC34 by response surface methodology,so that the lyophilized powder of Pichia Pastoris recombinant bacteria mediated by active peptide CC34 had the characteristics of stable performance,long shelf life and easy transportation.In this study,three strains with the best protective effect on the survival rate of Pichia Pastoris CC34 were screened by single factor test.The survival rate of recombinant Pichia Pastoris CC34 was established by box-Behnken method with different concentrations of protectants as response surface factors,and the effects of three protectants on the survival rate of recombinant Pichia Pastoris CC34 were analyzed.In addition,the optimal conditions of freeze drying in vacuum freeze drying were studied to prepare the lyophilized powder of Pichia Pastoris recombinant bacteria mediated by active peptide CC34.The freeze-dried powder of recombinant Pichia Pastoris CC34 was stored at different temperature and time to determine its survival rate.The results showed that:(1)The genetic stability of the recombinant Pichia Pastoris showed that the target gene sequence of the recombinant Pichia Pastoris CC34 did not change when it was passed to the fifth generation,but it was still the active peptide CC34 after identification,and some genes mutated in the tenth generation.According to the growth curve drawn,18 h was selected as the collection time for subsequent lyophilized strains test.The optimal centrifugation conditions for the recombinant Pichia Pastoris CC34 were 4 000 r/min,20 min,and the survival rate was93.63%.(2)By optimizing the prefreezing temperature and the mixing ratio of fungus slime and protective agent,the prefreezing temperature was determined as-20℃ for 3 h,and the lyophilized protective agent was added according to the ratio of fungus slime: protective agent of1:2.The selected solvent was used as the protective agent of lyophilized active peptide CC34 recombinant Pichita Pastoris.Finally,univariate screening results showed that skim milk powder concentration of 10%,trehalose of 15%,sorbitol of 20%,sodium glutamate of 3%,sucrose of 10% and glycerol of 9%.Three protective agents with better protective effect in single factor test were selected as the factors of the composite response surface,and the formula with the highest survival rate of lyophilized Pichia Pastoris recombinant peptide CC34 were defatted milk powder of 10.75%,trehalose of 15.70% and sorbitol of 20.19%,and the predicted survival rate was of 77.00% under these conditions.When the balance time of mycelium and compound protectant was 45 min,the highest survival rate of yeast was 81.84%.(3)The lyophilized powder of recombinant Pichia Pastoris CC34 was prepared under the optimal conditions.The storage test showed that the storage effect was better at 4℃,and the survival rate of lyophilized bacteria was(72.69±1.12)% after 90 days of storage.The results showed that the freeze-dried powder had certain storage stability after 90 days of storage.According to the response surface design,the highest survival rate of the recombinant Pichia Pastoris CC34 was obtained as skim milk powder of 10.75%,trehalose of 15.70% and sorbitol of 20.19%,and the survival rate of the recombinant Pichia Pastoris reached 81.84%combined with the above optimal conditions.The survival rate of the recombinant Pichia Pastoris CC34 strain was 72.69% after 90 days of storage in freeze-dried powder at 4℃.