Preparation And Characterization Of Endogenous Emulsification Probiotics Microcapsule Freeze-dried Bacteria Powder

As a typical probiotic in the human gut,Lactobacillus acidophilus and Bifidobacteria are widely used in food,medicine and health care products because of its good health.However,due to the poor tolerance of probiotics in the acidic environment of human body and the poor storage stability of probiotics in various microecological preparations,the activity of probiotics and their functional properties are affected.The microencapsulation technology,selecting the right wall material to wrap the core material can significantly improve the survival rate of probiotics in gastric juice,while the liquid microcapsule freeze-dried into a solid form by freeze drying technique can effectively increase the storage stability of probiotic microcapsule,thereby expanding the use of probiotics.Then chosen Lactobacillus acidophilus and Bifidobacterium as an example.(1)TheoptimalgrowthconditionsofLactobacillusacidophilusand Bifidobacterium in mixed culture were investigated.The OD for mixed bacteria was used as an index.Optimization the mixed culture conditions of two bacteria by single-factor test and Response surface experiment,The results showed that the optional conditions were as follows:the pH value was 6.5,concentration ratio of the two bacteria was 1:1 and the inoculation amount was 4.0%,temperature was 37±1?and liquid loading was 200 mL/500 mL.(2)The optimum process conditions for preparing Lactobacillus acidophilus and Bifidobacterium microcapsules were studied.The encapsulation rate of mixed bacteria in microcapsule was used as an index.The results of emulsification method showed that the optional conditions were as follows:wall material/core material ratio was 1:5,the stirring speed was 450 r/min,concentration of glacial acetic acid was 400?L,mass ratio of sodium alginate and calcium carbonate was 1:6,emulsification time was 15 min,the volume ratio between water and oil phase was 1:3 and concentration of emulsifier was600?L.The embedding rate could reach to 87.2%and living Lactobacillus acidophilus and Bifidobacterium were 8.13×10~9 cfu/mL.(3)The optimum process of chitosan as the two coating material of Lactobacillus acidophilus and Bifidobacterium microcapsules was investigated.The encapsulation rate of mixed bacteria in microcapsule,the survival rate of microcapsules in simulated gastric juice and the transmittance of microcapsules in intestinal juice were used as indexes.The condition of two times coating of chitosan by single-factor test and Response surface experiment,the results showed that the optional conditions were as follows:concentration of chitosan solution was 0.60%,pH of chitosan solution was5.00 and film formation time was 15 min.The embedding rate could reach to 85.9%and living Lactobacillus acidophilus and Bifidobacterium were 8.01×10~9 cfu/mL.Compared with the mixed bacteria alginate microcapsules,the microcapsules significantly improved the survival rates of Lactobacillus acidophilus and Bifidobacterium in simulated gastric juice.(4)The protective effects of protective agents on Lactobacillus acidophilus and Bifidobacterium microcapsules during freeze-drying were investigated.The viable counts and the encapsulation rate of microcapsules were used as indexes.The optimum freeze-drying protective agent by single-factor test and orthogonal test,the results showed that the optional conditions were as follows:concentration of trehalose was 2%,concentration of glycerol was 10%,concentration of skim milk was 15%and concentration of L-cysteine was 2%.The embedding rate could reach to 84.5%and living Lactobacillus acidophilus and Bifidobacterium were 7.88×10~9 cfu/mL.(5)The survival rate,acid resistance,enteric solubility and storage stability of the mixed bacteria in the three types of lyophilized bacteria powder(mixed bacteria+protector+secondary embedding group,mixed bacteria+secondary embedding group,mixed bacteria+protective agent)were investigated.The results showed that the survival rate of the mixed bacteria was:mixed bacteria+protective agent+secondary embedding group(84.5%)>mixed bacteria+secondary embedding group(77.6%)>mixed bacteria+protective agent(69.5%).Within 120 minutes of simulated artificial gastric juice,The protective effect of three types of freeze-dried bacterium powder on mixed bacteria in simulated artificial gastric juice:Mixed bacterial+protective agent+secondary embedding group(80.1%)>mixed bacteria+secondary embedding group(74.2%)>mixed bacteria+protective agent(68.9%).The microcapsule technology effectively improved the tolerance of the mixed bacteria to the acidic environment,and the prepared microcapsule had good enteric solubility.Within 60 minutes of simulated artificial intestinal fluid,among the three types of lyophilized bacteria powder,The maximum amount of mixed bacteria in the mixed bacteria+protective agent+secondary embedding group was 7.08×10~9cfu/mL.The storage effects of the three types of lyophilized bacterium powders at 4?and room temperature were different,and the low temperature was more conducive to the storage stability of the mixed bacteria in lyophilized bacterium powders.After storage at 4?for 60 days,the survival rate of the mixed bacteria in the three types of lyophilized bacteria powder was:Mixed bacteria+protector+secondary embedding group(75.3%)>mixed bacteria+secondary embedding group(67.2%)>mixed bacteria+protective agent(58.9%).Microcapsule technology effectively improved the acid resistance of the mixed bacteria,the preparation of microcapsules as lyophilized bacteria powder will help improve the storage stability of probiotics and lay a foundation for expanding the scope of use of probiotics.