| Conjugated linoleic acid (CLA) is a kind of polyunsaturated fatty acid with a variety of biological functions which play the role on anticancer, antiatherosclerosis, losing weight and regulating body's function and so on, and can be used as nutrition health products and strengthening feed additives, which is the nutrition health products with more attention by research institutions at home and abroad. At present, CLA is produced mainly in the method of catalytic summarization in industry, but its CLA product includes other isomers, of which some are harmful to the human body, so, it can't meet the needs of human beings. Many microbes are able to use their enzymes to transform LA into CLA. Compared with chemical synthesis, advantages of microbial synthesis are high purity of the active CLA isomers in production, without toxicity, mild conditions and conducive to the development and application of products. Immobilized cells are a new biological technology which raised in the1970s and by which we can achieve the high cells density culture and can carry out continuous fermentation after one vaccination. It has great potential and value to research CLA biosynthesis with immobilized microorganism cells technology.This paper made lactic acid bacteria UV3-9as starting strain which was screened in our laboratory and is mutagen zed, and made that immobilized lactic acid bacteria produces CLA as research basis. First, the immobilized condition of immobilized lactic acid bacteria were optimized.Second, the fermentation conditions to product CLA of the immobilized lactobacillus were optimized and research the batch fermentation.Then, we made the buffer instead of culture medium to directly transformed into LA for CLA, and optimized the transformation condition, and the batch transformation research. Finally, using the nature of the penicillin we could change of the permeability of the cells, and carried out the study the role of penicillin on increasing the yield of CLA in the fermentation process. The results were as follows:(1)The optimal concentration of lactic acid bacteria immobilized materials for are:SA2%,PVA7%,CaCl23%,H3BO36%. The optimization immobilization conditions were determined:immobilized ball diameter2-3mm, embedding quantity of bacteria cells3%, overnight soaking immobilized processing. After the optimization the biggest CLA output reached35.478μg/mL.(2)The best fermentation way optimized of immobilized lactic acid bacteria UV3-9is:anaerobic and static fermentation in the50mL triangle bottles of, and pre-cultivation of immobilized UV3-9before fermentation. The optimized fermentation conditions are for:the appropriate substrate adding content is0.3%, the optimal fermentation temperature37℃, the optimal initial pH6.5, the optimal fermentation time28h.The CLA production increased to89.986μg/mL after optimization conditions from33.364μg/mL before optimization, and the conversion of LA promoted from1.64%before optimization to4.43%after optimization, is of2.70times of that before optimization.Immobilized lactic acid bacteria can be carried out seven batches fermentation, CLA output volatility is not great, equally above80.476μg/mL, which suggested that immobilized bacteria enzyme activity is stable, but immobilized ball stacked together into pieces, which went against batch fermented continues. The method of soaking in3%CaCl2could improve the batch fermented number to15, and CLA production was stable in the first nine batches and the outputs were all above80.300μg/mL. It is visible that this process way is effective to improve the batches of the immobilized ball.(3)The best condition optimized of immobilized UV3-9directly transformed LA into CLA is:the transformation of medium is0.05mol/L phosphoric acid buffer (pH6.5), the appropriate substrate adding content is0.3%, the optimum transformation temperature39℃, the optimal transformation initial pH6.5, the optimum transformation time28h. After optimization transformation conditions the CLA production increased to111.437μg/mL from103.881μg/mL before optimization, LA conversation rate was increased to5.53%from5.13%before optimization, is of1.71times before optimization. Compared with the fermentation, the composition of the buffer in the method was simple, the environment in the conversion process changed little, and it was more advantageous to the transformation, and low cost, easy to filter the transformation liquid, convenient for batch conversion. CLA production was improved by1.18times of fermentation.Immobilized lactic acid bacteria can be carried out8batches transformation, CLA output volatility is not great, equally above87.222μg/mL, which suggested that immobilized bacteria enzyme activity is stable, but immobilized ball stacked together into pieces, which went against batch fermented continues. The method of soaking in3%CaCl2could improve the batch fermented number to15, and CLA production was stable in the first10batch and the outputs were all above81.269μg/mL. It is visible that this process way is effective to improve the batches of the immobilized ball.It has certain effect to adding penicillin for production of CLA, there were three periods to add penicillin solution:lactic acid bacteria expanding cultivates, immobilized lactic acid bacteria activation and fermentation. The results show that it was the most appropriate to add penicillin after the three experimental stage began5h, the best additives for2mL (25μg/mL), the best way to add penicillin was in two shots to add, CLA yield can reach103.632μg/mL... |
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