The Preparation Of The Starter Of Lactobacillus Fermentum Grx07 And The Research On The Protection Mechanism Of Freeze-drying

Lactic acid bacteria are popular among consumers for their good fermentation and probiotic characteristics.The market share of products with lactobacillus has exceeded half of dairy products in China by 2017.The development of lactic acid bacteria starter with high activity is practical significance.Not only reduces the dependence on imported bacteria powder,but also protects independent intellectual property rights.In this paper,using Lactobacillus fermentum grx07,the high cell density culture and the mechanism of lyophilization damage protection of grx07 were studied.Moreover,the protective agent,pre-culture conditions and storage conditions of lyophilized lactobacillus were optimized.Finally,the lyophilized powder of grx07 with high number of live bacteria,high activity and long shelf life was obtained.The findings are as follows:Hydrolyzed skim milk culture medium was studied.The effects of hydrolysis degree of skim milk on the growth and metabolism of grx07 were studied.With 15%degreased milk,was the highest protease activity,the highest growth rate,the utilization ratio of lactos and the number of live bacteria were 23 U/mL,1.8g/L·h,77.06%and 1.07×1010 cfu/mL,respectively.Furthermore,carbon,nitrogen sources and buffer salts was were modified and the number of live bacteria could reach to 1.2×1010 cfu/mL.The high cell density culture of lactic acid bacteria was studied.The growth relating factors of grx07 were optimized,including the gas protection,neutralization culture and feed culture.The final process of neutralization culture was as followed:using improved culture medium,feeding nitrogen with stirring the culture,controlling pH value of fermentation broth by adding mixed lye liquid in fermentation process,the final viable bacteria number couled reach to 2.52×1010 cfu/mL.The optimum conditions for feeding strategy were as followed:using the solution with the ratio of carbon to nitrogen 1:1 as the feeding ssolution,the solution was added when the sugar concentration in medium drops to 5 g/L,and maintain sugar concentration of 5-8 g/L in the medium during the fed-batch fermentation.Under this culture condition,the highest number of viable bacteria was 4.2×1010 cfu/mL,which was 2.5 times higher than that of batch culture.The formula and protective mechanism of lyophilization protectant for grx07 were studied.PB test was used to screen out the elements which had significant influence on the survival rate of lyophilization.The formula of protective agent was further optimized by BB experiment.The results were shown that the optimal protective agent was:12.71%skim milk,11.88%sucrose,4.22%sodium glutamate and 2.95%glycerol,and the survival rate of lyophilization reached 94.23%.For enzymes of lactic acid bacteria,lyophilization was revealed no significant effect on the HK and PK contrast to significant effects on ?-Gal,LDH and ATP enzyme.Intracellular Ca2+was lost during the integrity of cell membrane was destroyed and the fluidity and permeability of cell membrane.The optimized protective agent was demonstrated to be a significant protective on the damage of cell membrane.The effects of pre-culture on lyophilization of lactic acid bacteria were studied.Three factors relating to the conditions of pre-culture were optimized by rotation orthogonal optimization,including the ratio of protectant and sludge,the temperature of pre-culture and the time of pre-culture.The ratio of protectant to sludge,the pre-culture temperature and the pre-culture time were separately optimized as 1:1,40 ? and 60 min.Under the optimal conditions,the lyophilization increase rate of grx07-was reached to 31.51%.Pre-culture was demonstrated increase the content of grx07,improve the viability of lyophilized bacteria,increase related enzymes activity and reduce the damage of cell membrane.The effects of storage temperature on grx07 starter were studied.The changes of the moisture activity,the number of active bacteria and the activity of related enzymes were rsearched.The death of the bacteria was accelerated and the enzyme activity was reduced under 20 ? during storage,the LAB was well protected under-20 ?.