Optimization Of The Preparation Process Of Recombinant Lactobacillus Casei Spray-dried Formulations

For the industrial promotion of recombinant Lactobacillus casei with vaccine function,the traditional storage method of lactic acid bacteria should be improved to form a dry powder product with living bacteria.The production process should be improved and form an optimum recombinant L.casei dry powder product processes.Recombinant pLA-PoRV-VP7/L.casei was used as the research object to prepare live bacterial products in this study.The spray-dried recombinant L.casei with higher number of living microbe and higher living bacteria rate was prepared by optimizing the process parameters of spray drying and the screening of thermal protective agents,and it was compliance with safety standards.First of all,the growth curve was drawn according to the fermentation of the recombinant L.casei,and the fermentation broth with a large number of bacteria in the log phase was selected for subsequent experiments.After that,the spray drying process parameter ranges,including addition amount of whey protein,the inlet air temperature,the air input and the charging rate,were determined by single factor experiment,and orthogonal experiments were used to optimize the process parameters.Under the optimal parameter conditions,the resulting product was called spray-dried preparation.Subsequently,seven of the 13 anti-thermal protective agents were preliminarily screened through single factor experiment.The Plackett-Burman design and the steep ascent experiment were applied to determine the best composite protective agents.The resulting product prepared with the composite protective agents was called compound spray-dried preparation.Finally,the main quality indexes,surface morphology,storage stability,heat resistance and the survival rate in the in vitro simulated digestion fluid of two different spray-dried preparations were investigated.The results showed that the excessive whey protein during spray drying can not improve the survival rate of coated recombinant L.casei.There was mutual influence among the three process parameters of inlet air temperature,air input and the charging rate in the process of spray drying.The optimal addition amount of whey protein,inlet air temperature,air input and the charging rate were 20%,120?,75%and 10 mL/min respectively.Tryptone,sucrose,trehalose,maltodextrin,glycine and glycerin as anti-thermal protective agents were significantly different from the control group in the single factor experiment.Trehalose and glycerin had a significant effect on the survival rate of the microorganism(P<0.05).The final composite protective agent was determined to be 10%whey protein+6% trehalose+8 mL/L glycerol.The optimal storage condition was ensured to be the vacuum-packed spray-dried preparations at 4?.After 5 months of storage at room temperature,the number of viable bacteria was still kept above 10~7 cfu/g,which satisfied the minimum daily intake prescribed in the national standard.It proved that the spray drying process of this paper could effectively prolong the storage time of the recombinant L.casei.The main quality indexes of both the spray-dried preparation and the compound spray-dried preparation comformed the national standard,and the recombinant plasmids were stable and not lost during the storage period.The morphology of the compound preparation was observed to be more round and smooth under SEM.The two spray-dried preparations improved the survival rate of recombinant L.casei after high temperature treatment for 30 min and in vitro simulated digestion treatment for 6 h,and the compound spray-dried preparation showed higher stability in the treatments.The optimum spray drying process of recombinant pLA-PoRV-VP7/L.casei in this paper was:inlet air temperature 120?,air input 75%,the charging rate 10 mL/min,10%whey protein+6%trehalose+8 mL/L glycerin as anti-thermal protective agent.The recombinant L.casei dry powder product prepared by this process possessed increased the number of living microbe and the living bacteria rate,and could effectively resist the negative influence of external environment.The research in this paper provided a technical basis for the industrial production of recombinant L.casei spray-dried preparations.