Breeding Of Thermophilus Strain Producing L-lactic Acid And Optimization Of Fermentation Conditions

Lactic acid is generally recognized as one of the three big organic acids in the present world. Lactic acid, lactate and the ramification of them are widely applyed in many domains, such as food, medicine, chemical industry. As rapidly developing of poli-lactic acid as biodegrading plastic, the domestic and foreign demand of high optical purity of L-lactic acid is becoming more and more. Thereby, thermophilus strain producing L-lactic acid being fit for fermtation technics has the extremely high commercial value. Thermophilus strain can reduce the quantity of coolong water for the course of the fermtation, so it can reduce the production cost. Thermophilus strain can also reduce the chance of being polluted by other bacterium, and it can make the cycle of fermtation shorter than common bacterium. Besides, thermophilus strain can raise the use efficiency of fermtation pot. Therefore, the study of thermophilus lactic strain is extremely meaningful. Besides, lactic acid bacterium have strict request of medium ingredient and the cost of medium is very high in the course of producing L-lactic, so in the same time of optimizing fermentation we should think over the ingredient and the cost of the medium. It's very necessary for reducing the produce cost of L-lactic acid to filtrate a low-cost fermentation medium.In this paper we got a thermophilus strain producing L-lactic acid from nature, and has carried on the ultraviolet ray mutagenesis and chemistry mutagenesis to it, in order to enhance the capability to produce acid of the strain. And then fermentation culture medium and fermentation condition have been optimized. The last work is enlarging the cultivation, simulating industrialization productioin. The result is as follows:1. The capability of producing acid of bacterium was judged by the size of stransparent ring on detached plate. The thesis has filtrated and got eighty bacterial strains which were initially selected as candidates from pickled vegetable, acidophilus milk and corn steep liquor and so on. According to the fermentation experimentation, the strain that total lactic production is under 20g/L is eliminated. Then the remainder strain was repeatedly selected, and result of HPLC show that only three strain whose L-lactic acd output is obviously high. And the highest output is 34.8g/L. After the conventional appraisal, the strain is rod-shaped, and Gelanshi dyes is positive. The strain has no spore, and is the optional anaerobic fermentation. The contact enzyme experimentation of the strain is positive, and it can not calisthenics. In summary,the strain fall into Brochothrix.According to ultraviolet mutafacient survival percentage curve, 40s is chosen as the quantity of ultraviolet mutafacient, and two strains are selected that output is 48-50g/L. But due to the heredity stability, we choose the strain L36-29 as the strain to continue to mutagenesis. 60 minutes is chosen as the quantity of EMS mutafacient for mutafacient experiment, and overall evaluate fermentation characteristic and heredity stability, at last a strain with good heredity stability is selected. It's output is 73.3g/L, is more 39.8g/L than the primitive strain, and it's named L36-29-1. 2. Determined optimal concentration of medium components of L36-29-1 by single factor experiment: corn saccharification liquid 100g/L, corn steep liquor 30g/L, wheat bran 10g/L, yeast extract 4g/L, MnSO4 0.01g/L and citric acid diammonium 2.0g/L. Then further optimized main influence factors for medium by orthogonal test, variance analysis, determined optimal combination of fermentation medium: saccharification liquid 105g/L, corn steep liquor30 g/L, wheat bran 12 g/L, MnSO4 0.01g/L and citric acid diammonium 2.0g/L.3. Optimized fermentation conditions of L36-29-1 on shaking flask fermentation, determined that : suitable fermentation temperature is 50℃, the inoculum size is 8%, the inoculum age is 14h, the culture medium is 100/250ml, the CaCO3 suitable quantity is 60g/L. Above mentioned optimal conditions basically accorded with fermentation characteristics of L36-29-1, when process amplified by 5L automatic fermentor. Only dissolved oxygen quantity had increased, rotational speed is 250rpm, aeration rate were 1.2L/min, alleviated hypoxia status in fermentation. Ultimate yield increased 1.5g/L,and fermentation period shortened 8h. It showed that in logarithmic growth phase of the strain, enough oxygen in fermentation liquid is adventageous to the growth of strain and yield acid, ultimately shortened fermentation period.