| Lycopene, a member of carotenoids, is an important natural pigment with the physiological function such as antioxidation, anti-aging, and immunity enhancement. At present, lycopene market is still in the developing stage, which is reflected in the relatively high price and the increasingly expanding market demand. The high price of natural extraction is as a result of the low concentration of the pigment in fruits and the complex purification process due to the numerous carotenoids present in the raw materials. Synthetic lycopene, although cost-effective, has the drawback of not being a product identical to the natural one. In recent decades, the low cost and high efficiencyof microbial synthesis has gained more and more attention. Till now, Blakeslea trispora is the sole strain which has achieved industrial production. In this study, an efficient screening system was constructed in order to obtain mutants with high-yield of lycopene, after which the processes of fermentation and extraction were optimized. Furthermore, the relationship between intracellular fatty acid composition and lycopene production was studied. The major contents and results of this studyare summarized as follows:(1) B.trsipora NRRL 2896(-) was treated with N+ ion implantation, NTG, or ARTP biological breeding system, and further isolated on the screening plates supplemented with lovastatin and crude extracts of trisporic acid. After several rounds of screening, three mutants with higher yield of lycopene and biomass were isolated. Among these mutants, mutant I5 obtained by N+ ion implantation showed a maximum lycopene production(0.97 g·L-1). Compared with parent strain, the transcriptional levels of genes hmg R, car RA and car B in mutant I5, involved in carotene biosynthesis, increased by 123, 80 and 75%, respectively.(2) A BP network with the topology of 5-12-1 was used to build the network model between fermentation medium composition and lycopene production. After genetic algorithm optimization, lycopene production increased by 31.6%. The optimum medium composition are as follows(per liter): 41.2 g corn flour, 8.93 g corn steep liquor, 26.5 g soybean oil, 1.39 g KH2PO4, and 0.46 g Mg SO4. Lycopene concentration reached 29.5 mg·g-1 when 90 mg·L-1 2-methylimidazole was added at 24 h. A nonsynchronous inoculation process, in which the(+) mating type was inoculated after the(-) mating type has been grown for a certain period of time, replaced traditionally synchronous inoculation process. The lycopene concentration with nonsynchronous inoculation was 31% higher than that with synchronous inoculation. The optimum inoculation interval was 24 h with an inoculation ratio of 1:2(+/-, v/v).(3)The exogenous plant oils enhanced the total content of intracellular lipids and the desaturated degree of reserve lipid due to the alteration of fatty acid composition, especially in neutral lipids. As a result, lycopene production was enhanced because more lycopene could be deposited in storage lipid thus reducing the feedback inhibition of free lycopene. Among the oils used in this study, soybean oil whose fatty acid composition were similar to the mycelium exhibited the best stimulating effect on lycopene formation. In oil-enriched substrates, the supply of NADPH, ATP and carbon for carotenoid synthesis were enhanced. Consequently, the biosynthesis of lycopene was stimulated.(4) The geometry of impeller significantly affected lycopene fermentation. Three-blade propeller agitator showed a best result among the impellers used in this study. B. trispora mycelium was more sensitive to shear force in growth stage than lycopene producing stage. The optimum stirring process was 200 r·min-1 in prophase of fermentation and 350 r·min-1 after 48 h. In varied stirring rate process, the dissolve oxygen and volumetric oxygen transfer coefficient were increased, which is beneficial to the oxygen intake of mycelium. The optimum aeration rate was 1.25 vvm. Lycopene production would be decreased with a too high or too low aeration rate. As a result, lycopene production reached 1.30 g·L-1 in a 30-L stirred tank reactor.(5) Saponification and ultrasonication were the effective methods to improve the extraction rate of lycopene. The optimum condition of saponification are as follows: the concentration of KOH solution was 0.6 mol·L-1; the treating time was 30 min; and the temperature of saponification was 30 ℃. The optimum extraction process are as follows: extracting solvent was ethyl acetate; the ultrasonic power output was 300 W; the ratio of solid to liquid was 1:250(v:v); and the extraction time was 15 min. After two extraction stages, the extraction rate of lycopene reached 92.2%. Silica gel was used as filler in column chromatography. After gradient elution with cyclohexane, cyclohexane:dichloromethane(6.5:1,v:v), and dichloromethane, γ-carotene and β-carotene were thoroughlyremoved from lycopene extraction. |
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