Breeding Of Carotenoids Production Strain And Study Of Its Fermentation Conditions

The original strain Arthrobacter globiformis RC-1(conserved in our laboratory) wastreated by traditional mutation methods, and an ideal strain M-3was selected out finally. Weaimed to determine the technologies for cell disruption and the methods of extracting andpurifying pigments, and also the qualitative analysis of pigments were investigated. Theoptimal carotenoids fermentation conditions by the strain M-3were studied. The amount ofcarotenoids was increased largely in the presence of oxidative stressors. We analyzed thecomponents of amino acids and fatty acids of dry cells, and the possibility of using asfunctional additives in the future was investigated. At last, the effects of severalenvironmental factors on the stability of carotenoids in extracts were studied, and weconfirmed the appropriate storage conditions for carotenoids extracts. The work and resultsare as follows:(1) The original strain RC-1was treated by UV and DES treatment gradually, and thedeath rate of70%-80%was used as processing conditions. Then the ideal strain was selectedout on the special diphenylamine containing plate, and the diameter of bacterial colony andthe depth of its color were used as screening conditions. At last, the ideal strain M-3wasobtained through the first screening and the second screening with shake flask fermentation.Total carotenoids concentration was increased from12.43mg·L-1to26.32mg·L-1before theoptimization of fermentation conditions finally.(2) Investigations on the methods of cell disruption, pigments extraction, qualitativeanalysis were carried out. Several methods of cell disruption were investigated, and the freezethaw method was selected. By comparing the results of several different polar solvent forextracting carotenoids, the mixture of absolute ethanol and acetone (2:1, by volume) was usedas the optimal extracting agent. Further purification of carotenoids was performed by gradientelution on silica gel column. Based on the results of TLC, HPLC, LC-MS and scanningspectra, the major components of carotenoids in cells were identified as β-carotene,anhydrorhodovibrin, spirilloxanthin, and OH-spirilloxanthin, respectively.(3) One-factor-at-a-time approach was used for optimization of major media componentsand response surface method was further applied to determination of optimum values ofprocess variables for maximum carotenoids production. It was found that the optimumcombination for the total carotenoids concentration was sugarcane molasses40.00g·L-1, cornsteep liquor50.00g·L-1, KH2PO41.00g·L-1, MgSO4·7H2O0.75g·L-1, at100r·min-1,30°Cand initial pH of7.5. When cells were grown under aerobic conditions in500mL baffledconical flask with50mL of the nutrient medium for60h, the maximum carotenoidsconcentration of49.43mg·L-1(increased by87.80%) was demonstrated. Detailed studiesshowed the final caroteoids concentration of73.34mg·L-1(increased by48.37%) wasobserved in the presence of addition of2mL ethanol at40h.(4) At last, we analyzed the components of amino acids and fatty acids of dry cells, andthe possibility of using as functional additives in the future was investigated. Also, the optimalstorage conditions for carotenoids extracts were confirmed.