Breeding Of β-carotene Over-producing Strains And Primary Studies On Metabolism Regulation

Beta-carotene is a typical kind of carotenoid family; it shows strong ability of free radical scavenging and singlet-oxygen quenching. Beta-carotene has been used for medicine, food colorant, and cosmetic. Blakeslea trispora has the natural ability of producing beta-carotene and has some advantages just like easy to culture, high biomass and higher yield.In this paper, Blakeslea trispora strain (-) in laboratory was treated with ultraviolet radiation, a mutant named UV-9 was selected with the selective pressure of 0.025 %(w/v) lovastatin. Under the unoptimized fermentation condition, the concentration of beta-carotene was 2.06 g/L, increased by 49.3% than that of the original strain.The mutant was genetically stable.With beta-carotene output as the main index , culture medium and cultivation condition was optimized comprehensively considering metabolic features of Blakeslea trispora .The obtained optimal condition was: corn meal 4%, cottonseed oil 6%,soybean flour 3%,KH2PO40.3%, temperature 27℃, medium volume 25mL/250mL,best proportion of (+),(-) 1:49,inoculum concentration 15%,seed age 44h.After incubating for 48h, 0.1%(V/V) Span and 0.15%(V/V) hydrogen peroxide were added into medium. Under this condition, strainUV-9 could produce beta-carotene of 3.35g/L by shaking-flask fermentation, increased by 69.6% than that not optimized.Effects of different metabolic accelerators such as beta-ionone, penicillin, citric acid, isoniazid and citrus molasses were investigated according to single factor experiment. All of which could promote the growth of Blakeslea trispora or the synthesis of beta-carotene in some extent.The effect of beta-ionone was remarkable, the optimal adding time was 44h after incubating.Beta-carotene isomers was separated by C₁₈ reversed phase high performance liquid chromatography, and the ratio of cis-isomer was up to 10.5%.During the process of crystallization preparation, the beta-carotene crystallization contained much trans-isomer while the beta-carotene oil contained much cis-isomers. At last the stability of natural beta-carotene from Blakeslea trispora was studied. Beta-carotene was very unstable under light. It was stable at low temperature and room temperature. Beta-carotene in solutions containing Fe³⁺, Al³⁺ was unstable especially. Antioxidant such as BHT and TBHQ could prevent beta-carotene from oxidation.