High Cell Density Fermentation And Agent Preparation Of Lactobacillus

Lactic acid bacteria have been widely used in food,feed and other industries.It can improve the body metabolism,promote intestinal bacteria proliferation,inhibite the growth of harmful bacteria and enhance immunity of human body.Due to its fragility of resistance to external environment,the storage of lactic acid bacteria agent was difficult and the cost of preparation and application was high.We cultivated Lactobacillus plantarum and several other lactic acid bacterias in high cell density on the basis of the original fermentation medium to explore a preparation routing for both food-grade and feed-grade lactic acid bacteria agents and obtain stable and high yield of lactic acid bacteria agent preparation method at a relatively low cost.The results are as follows:1.The result by single factor experiment showed that the optimal culture conditions for the Lactobacillus plantarum were culture of 24 h at30?,initial pH 6.6 and inoculum concentration 4%.The bacteria OD₅₄₀reached 26.54,the viable count of Lactobacillus plantarum could reach5.26×10¹00 CFU/mL.It promoted the growth of Lactobacillus plantarum when a certain amount of cotton meal and soybean meal were added in the fermentation medium.After the single factor and orthogonal test,we could get the bacteria fermentation liquid containing insoluble solid particles of agricultural byproducts when 1%molasses,3%cottonseed meal and 4%soybean meal were added.The viable count of Lactobacillus plantarum could reach 1.10×10¹²CFU/mL,increased by 20 times compared with the original culture medium.The fermentation liquid could be used for the preparation of feed-grade lactic acid bacteria agents.It also promoted the growth of Lactobacillus plantarum when the soybean meal and cottonseed meal hydrolysate was added to the fermentation medium.The maximum OD₅₄₀40 was 30.23 and the viable count of Lactobacillus plantarum reach 1.10×10¹22 CFU/mL when 3%soybean meal hydrolysate was added,the viable count increased by 11times compared with the original culture medium.In this way,we could obtain bacteria fermentation liquid without insoluble solid particles,which can be used for the preparation of food-grade lactic acid bacteria agents.2.On the basis of cell high density fermentation,we compared seven kinds of protective agents of glucose,sucrose,trehalose,glycerol,maltodextrin,skimmed milk powder and modified starch on different concentrations for the investigation of protective effect on Lactobacillus plantarum based on three different drying methods of freeze-drying,spray-drying and vacuum low temperature drying.Above four preferred protective agtents was usted to the orthogonal experiment.The results showed that the best combination of protective agent was 8%trehalose,6%maltodextrin,6%modified starch and 10%skimmed milk powder in a freeze-drying way.The viable count of Lactobacillus plantarum agent made by freeze-drying could reach 5.93×10¹¹CFU/g,increased about 2times than that before optimization.3.Four kinds of lactic acid bacterias which could grow well on the fermentation medium were screened and the best inoculation proportion was 4%Lactobacillus plantarum1,1%Lactobacillus plantarum2,1%Streptococcus thermophilus,3%Bifidobacterium bifidum.The mixed bacteria OD₆₀₀00 could reach 25.21 and the total viable count could reach1.07×10¹⁰CFU/mL on the condition of culture for 24 h at 30?.Some fruits and vegetables juice were added to the fermentation medium as nutritional factors.It was found that the bacteria biomass OD₆₀₀00 could reach 27.60 and the total viable count could reach 3.16×10¹⁰CFU/mL when 4%watermelon juice was added,increased about 3 times than that before optimization..A comprehensive comparison was carried out for the freeze-drying and spray-drying by viable count,product yield and raw material costs.The result showed that the freeze-drying method was better than spray-drying method for preparation mixed lactic acid bacteria agent,by which the viable count could reach 3.92×10¹¹CFU/g.The bacteria agent prepared by freeze-drying still remained high viable count of 6.53×10¹⁰CFU/g after storing at 4?for 90 days.4.Soybean meal protein feed was fermented by laboratory's homemade mixed lactic acid bacteria agent and the result was compared with the commercially available lactic acid bacteria agent.The result showed that the determination index of soybean meal protein feed fermented by homemade was comparable to that prepared by commercially available lactic acid bacteria agent.The production cost was low and the technology had great application value.Pomace feed had been prepared by fermentation with combination of laboratory's homemade mixed lactic acid bacteria agent and commercial yeast bacterium agent.The best combination of pomace feed ingredients was 70%pomace,15%bran,10%rice bran and 5%strains activated liquid.The viable count of lactic acid bacteria could reach3.82×10⁷CFU/g and the viable count of the yeast could reach 1.98×10⁷CFU/g.Lactic acid content was 9.2 g/100g and soluble protein content was 5.34 g/100g.The recovery rate was over 98%.The fermentation product had a very strong smell of alcohol and sugar after fermentation,which could greatly improve the palatability of feed.