| Clostridium butyricum is an anaerobic bacillus, widely found in soil, human or animal intestinal. As a probiotic, clinical researchs show that, in the fields of intestinal and digestive tract diseases, Clostridium butyricum can get a remarkable effect; in terms of animal husbandry, as an alternative of antibiotic,it can make good economic and environmental benefits by means of preventing diseases improving feed efficiency, promoting growth and nutrient absorption, reduceing pollution. For further study, Clostridium butyricum will have broader prospect.Clostridium butyricum has ability to resist many adverse conditions in practical application,with good application potential.The purposes of this study are to get high levels of bacteria concentration and bacillus rate. In previous studies, bacteria concentrations were often low, people often used complex medium, or other mediums containing large amounts of insoluble components,this resulted in difficulties in following treatment. This study tries to use clear liquid fermentation, increase the biomass concentration of fermentation, so we can obtain high pure powder after the step of simple treatment.The fermentation medium and conditions were optimized in following four aspects:According single factor experiments,screened carbon nitrogen source and proper density ranges respectively, two level factor of Plackett-Burman was designed, the results confirmed that the significant factors affecting fermentation were pH, yeast powder, nitrogen source M, Based on the result, Set step length, Then the path of Steepest Ascent was used to approach the optimal region, Through response surface design experiments, the optimized medium:glucose 2%(w/v), nitrogen source M 1.3%, yeast extractl.8%, nitrogen source N 1.5%, corn steep powder 0.33%, (NH4)2SO4 0.1%, NaHCO3 0.124%, metal ion C 0.05%, metal ion B 0.05%, metal ionA 0.1%,pH 7.5.Designed single factor experiments, set heat-resistant spores rate as standard, obtained proper density ranges of metal ionA, B and C, conducted combined experiments, the optimized metal ions:metal ion B 0.05%, metal ion C 0.05%, metal ion A 0.1%.Fermentation technics were improved for further more, fed glucose and controlled pH by the way of feeding lye, the optimized fermentation conditions:inoculum 3% to 5%, temperature 37℃, stirring speed 50 r/min,keep the value of pH stable at 6.5 by the way of feeding 20% Na2CO3, fermentation begins at 12 h,16 h,20 h,24 h, feeding the same amount of glucose four times, the total amount of glucose is 2%(w/v),fermentation results showed that bacillus rate was almost 95% under the microscope, the number of bacteria can reach 2.3×109 cfu/ml, compared with fermentation without controlling pH,the result increased 2.6 times,for fermentation without feeding glucose, the result increased 1.16 times.Finally, processed broth using spray drying and freeze-drying respectively, added starch with protecting and adsorbing effects in spray drying, the result was optimum when adding 3% starch,but the survival rate did not exceed 37.6.The freeze-drying, broth was processed as follow:centrifugal separation, washing, suspension with adding cryoprotectant, prefreezing, freeze-drying. The optimized cryoprotectant and proportion: skim milk powder 15% or trehalose 10%, the ratio of fermentation and re-suspension cryoprotectant 1:2, equilibrium time 30 min, the final freeze-dried survival rates can reach 89.7% and 85.6%. |
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