| Probiotics with healthcare function has been the main diet assistant. As a preferred probiotic (?)strain, Lactobacillus has a wide application prospect and market potential. To rapidly developing an efficient preparation technology of active agent of Lactobacillus and laying a foundation for production practice, it’s of great significant for promoting the industrialization process of Lactobacillus agent and improving the industrial development of Probiotics. Lactobacillus acidophilus and Lactobacillus planetarium were chosen as research objects in this paper. It’s aimed to study on the enrichment cultivation, cell concentration and freeze-drying technology to prepare compound Lactobacillus agent with high viability, and then test the quality and storage stability of Lactobacillus agent. The high efficient proliferation mediums and compound freeze-drying protectants were selected in paper, and the large-scale culture of Lactobacillus were also explored and analyzed. The results were showed as following:(1) Through the study on the growth characteristics of Lactobacillus acidophilus and Lactobacillus planetarium in individual culture and mixed culture, individual culture was chosen for subsequent research.(2) The single-factor-experiment results of proliferation medium for Lactobacillus acidophilus showed:the optimum buffer salts were CaCO3and K2HPO4, the optimum trace element was MgSO4, the optimum growth factor was tomato juice. By Plackett-Burman design, the significant factors were glucose, CaCO3and tomato juice, which the degree of influence was CaCO3> glucose>tomato juice. According to the steepest ascent experiment and Box-Behnken design, the optimal medium of Lactobacillus acidophilus contained the following ingredients:3.2%glucose,0.84%CaCO3,33%tomato juice,1%peptone,1%beef extract,0.5%yeast powder,0.2%K2HPO4,0.058%MgSO4. The concentration of Lactobacillus acidophilus reached4.78×109cfu/mL by using this proliferation medium.(3) The single-factor-experiment results of proliferation medium for Lactobacillus planetarium showed:the optimum buffer salts were CaCO3, the optimum trace element was MgSO4, the optimum growth factor was tomato juice. By Plackett-Burman design, the significant factors were peptone, CaCO3and tomato juice, which the degree of influence was CaCO3> tomato juice> peptone. According to the steepest ascent experiment and Box-Behnken design, the optimal medium of Lactobacillus planetarium contained the following ingredients:2.45%peptone,0.985%CaCO3,37.3%tomato juice,2%glucose,1%beef extract,0.5%yeast powder,0.058%MgSO4. The concentration of Lactobacillus planetarium reached2.94x109cfu/mL by using this proliferation medium.(4) The culture temperature, inoculation and initial pH were studied on the effect of growth, the results indicated:the optimal culture conditions for Lactobacillus acidophilus were cultivate temperature37℃, inoculation4%, initial pH6.6of medium; the optimal culture conditions for Lactobacillus planetarium were cultivate temperature34℃, inoculation4%, initial pH6.4of medium.(5) Results of growth curves in enrichment cultivation showed:the highest bacterial counts of Lactobacillus acidophilus was6.15×109cfu/mL, the best harvesting time was21h; the highest bacterial counts of Lactobacillus planetarium was4.91×109cfu/mL, the best harvesting time was21h.(6) Results of the large-scale culture of Lactobacillus in fermentation tank indicated:the highest bacterial counts of Lactobacillus acidophilus was5.84×109cfu/mL and the highest bacterial counts of Lactobacillus planetarium was4.66×109cfu/mL,while on the condition that the initial liquid volume was30L, fermentation process was stuffied and stirring speed is50r/min.(7) In the study of the effect of centrifugal rotational speed and centrifugal time on centrifugal yield of Lactobacillus, the optimum centrifugal conditions were decided:the centrifugal yield of Lactobacillus acidophilus could reach above95%when centrifugated at8000r/min for10min; the centrifugal yield of Lactobacillus planetarium could reach above97%when centrifugated at4000r/min for10min.(8) Through the study on the effect of freeze-drying protectants on survival rate of Lactobacillus, the single-factor-experiment results showed:the optimum single-factor freeze-drying protectants of Lactobacillus acidophilus were30%skim milk,20%trehalose,20%glucose; the optimum single-factor freeze-drying protectants of Lactobacillus planetarium were10%skim milk,10%peptone,5%glucose.(9) The compound freeze-drying protectants of Lactobacillus were determined by orthogonal experiment. Results showed:the ingredients of compound freeze-drying protectants of Lactobacillus acidophilus were35%skim milk,15%glucose and15%trehalose, which the degree of protection effect was skim milk> glucose> trehalose; the ingredients of compound freeze-drying protectants of Lactobacillus planetarium were12.5%skim milk,10%peptone and2.5%glucose, which the degree of protection effect was peptone>skim milk>glucose.(10) The ratio of compound freeze-drying protectants and bacterial mud, the initial pH of compound freeze-drying protectants were studied on the effect of survival rate of Lactobacillus, the results indicated:for Lactobacillus acidophilus, the best ratio of compound freeze-drying protectants and bacterial mud was2.5:1, optimum pH was6.5, in this condition, the freeze-dried survival rate could reach75.63%and the living bacterial counts of lyophilized agent was1.03×1011 cfu/g; for Lactobacillus planetarium, the best ratio of compound freeze-drying protectants and bacterial mud was2:1, optimum pH was6.5, in this condition, the freeze-dried survival rate could reach86.64%and the living bacterial counts of lyophilized agent was1.12×1011cfu/g.(11) The main quality indicators of lyophilized agent were tested, the results were as follows: the agent was yellow powder with slightly milk flavor and sour, which the living bacterial counts was1.06×1011cfu/g, and contained water2.7%, protein33.7%, fat1.4%, sugar25.3%. Escherichia coli were not detected.(12) Through the study on the storage stability of Lactobacillus agent, results showed:the living bacterial counts of lyophilized agent were on the decline during storage, the survival rate which stored at4℃was far higher than that of room temperature. Stored with vacuum seal at4℃was best for the storage stability of Lactobacillus agent, which the living bacterial counts of lyophilized agent was6.48×1010cfu/g and survival rate was61.13%after3months. |
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