Study On Cultivation And Cryoprotectant Of Lactobacillus Plantarum

Probiotics are physiological bacteria, which interact with original bacteria that show symbiosis, competition in human intestinal, can be used to regulate flora balance, enhance immunity and promote nutrient absorption. Lactobacillus plantarum is a kind of lactic acid bacteria with a variety of probiotic function. In this study, we used Lactobacillus plantarum L69 with producing of ACE inhibitory peptides from previous selected. The single factor experiment, Plackett-Burman experiment, steepest ascent experiment and Box-Behnken Design response surface method were employed to determine the optimal proliferation medium, culture condition and the ratio of lyoprotectant; the effect of neutralization and fed-batch culture were researched on growth of L69; the storage stability of freeze-dried L69 powder was determined by accelerated tests; the performance of the fermentation of goat milk was studied. The main results are as follows:1. The ratio of basal medium of L69 was screened by single factor, and Plackett Burman experimental was used for screening carbon source/prebiotics, nitrogen source, amino acids, buffer salts, the results showed that the lactose and xylitol, casein peptone, yeast extract, potassium dihydrogen phosphate, sodium acetate, ascorbic acid and methionine have some promoting effect. Especially, methionine, yeast extract powder, dipotassium hydrogen phosphate are the main influence factor. After steepest ascent experiment and Box-Behnken Design experiment and response surface analysis method optimized the multiplication culture medium were:Glucose 20g/L, peptone lOg/L, yeast extract 20g/L, dipotassium hydrogen phosphate 7.65g/L, manganese sulfate 0.04g/L methionine,0.0478g/L, Tween 80 1g/L. The viable counts reached (1.54±0.06)×109CFU/mL. The optimization of L69 culture conditions were inoculation rate 3%, the culture temperature 37?, the initial pH=7.0 of the medium, and the viable cells(3.5×109CFU/mL)was more than the basal medium number of viable cells (3.57×108CFU/mL).2. The viable counts of L69 were 4.0×109CFU/mL and 4.65×109CFU/mL after culturing by NaOH and fed-batch. The viable counts reached 5.1×109CFU/mL by feeding of NaOH supplemented with fresh medium, which was improved 41.7% more than the unoptimized medium(3.6×109CFU/mL).3. The effect of freeze-drying cryoprotectant factors including carbohydrates, amino acids and prebiotics cryoprotector was studied by single factor test. It showed that lactose, galactose, trehalose, fructooligosaccharides, galactose, Xylo, arginine, glycine, proline lyophilized protective effect of L69 were better. Freeze-drying were measured in factorial experiments and response surface method to determin main influencing factors. The ratio of the freeze-dried cryoprotector were:lactose 9%, oligosaccharides 10%, proline 4.8%, galactose 9% and trehalose 6%. The verification test showed that survival rate was (82.88±0.7)%, and the viable counts of freeze-dried L69 was (1.11±0.05)×1011CFU/g, similar to the predicted values (p<0.05). Based on the above cryoprotector, the optimal dose of skim milk was 24%, and the validated survival rate and viable counts of freze-dried L69 were (89.78±0.56)% and (1.94±0.04)×10nCFU/g, respectively.4. Accelerated storage testing (test at 45?,50? and 55?) of freeze-dried L69 powder suggested that the speculated inactivation rate constant of freeze-dried L69 powder under -18? was k-18=8.42x 10-6, and the viable counts reached 1.64×1011CFU/g and 1.38×1011CFU/g after 1 years and 2 years with good storage stability.5. To prepare a DVS fermenting agent, L69 freeze-dried bacteria powder was mixed with goat milk powder, compared with subculture fermentation agent all time points of pH, acidity, viable counts and ACE inhibition rate and researched the feasibility of DVS fermenting agent. The final results were:4.48, 85°T,2.31×109CFU/mL and 68%, respectively. It showed that a good DVS fermentation ability and the fermentable goat yogurt is feasible.The optimized medium and feeding could increase effectively the number of viable cells in the broth and prolong the growth cycle; The use of cryoprotectants, not only can raise survival rate and viable count per unit, but also improve storage stability, which laid the technical foundation for further industrial production.