Screening And Optimization Of Lacticaseibacillus Paracasei PC646 Anti-Cryoprotectant

Vacuum freeze-drying technology is a common method for the production of lactic acid bacteria preparations,because bacterial cells are easily damaged or even dead during the freeze-drying process,and after freeze-drying,it is manifested as reduced survival rate,cell activity and cell membrane function,and decreased intracellular enzyme activity.Therefore,before lyophilization,it is necessary to add an appropriate amount of protective agent to reduce the degree of cell damage,but there are many types of anti-cryoprotectants,and the effects of different effects with different strains are not the same,at present,there are more studies on screening protective agents at home and abroad,but there is no research on Lacticaseibacillus paracasei PC646 anti-cryoprotectant and the explanation of the mechanism of action of protectants on the bacteria from the cellular level.In this thesis,Lacticaseibacillus paracasei PC646 was used as the research object,and the main research content was to screen and optimize its protective agent.Optimize the mixing ratio of bacteria and protective agents;The survival rate,acid production rate,optimal storage temperature and survival rate during storage of the control group,skim milk group and compound protective agent group before and after lyophilization were studied,and the effects of different protective agent groups were compared.The mechanism of action of protectors was explored by measuring the changes of cell damage,intracellular reactive oxygen species,malondialdehyde,cell membrane fluidity,intracellular and extracellular p H gradient,and intracellular key enzyme activities in three different groups of protectors before and after lyophilization.Taking the lyophilized survival rate of Lacticaseibacillus paracasei PC646 as an evaluation index,the main influencing factors of carbohydrates,salts,antioxidants and compound protective agents were screened by P-B design,and the anti-cryoprotectant formula was optimized by the response surface method,namely trehalose 5.318%,skim milk 14.06%,sodium glutamate 1.013%,glutathione 1.025%,the maximum survival rate was 83.66%,and the verified survival rate was 82.39%,which was similar to the predicted value.At this time,it was about 78%and 20%higher than that of the control group and skim milk,respectively,and the survival rate was significantly improved,and the above indicators were improved to varying degrees,and the effect of lyophilized protectant was in the compound protectant group>skim milk group>control group.The best effect is when the mixing ratio of bacteria to protective agent is 1:1.2.The results of the above cell damage and other indicators were measured by flow cytometry,and the results showed that the effect of the complex protective agent group was better than that of other groups,mainly because trehalose can maintain cell membrane and protein molecular structure in high-temperature,cold and dry environments,and has antioxidant effects.Sodium glutamate and skim milk can reduce the mechanical damage caused by ice crystals to cell membranes during the freezing process;At the same time,trehalose,skimmed milk,sodium glutamate,glutathione can remove oxygen free radicals in cells and reduce the content of reactive oxygen species and malondialdehyde in cells;Under the action of protective agents,the activities of intracellularβ-galactosidase,Na⁺-K⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase were increased compared with the control group,which proved that the cell membrane was protected to different degrees under the action of the protective agent,and the best protection effect was still the complex group protector.