Mutation Screening Of High-yield ARA Strain And Its Study Of Technology Parameter Of Extraction

Arachidonic acid is an essential dietary component for human beings,and has various physiological functions and plays an important role in infant nutrition. ARA, widely existing in the animal kingdom,can be isolated from the lipids extracted from pig adrenal gland or pig liver and sardines as well; however, the yield is only 0.2%(w/w) or lower, so it is very dificult to be applied in a industrial scale and the research and application are restricted.It has been focused on the study of ARA fermented by microbe in the whole world. The aim of this study is to selectively breed the high-yield ARA strain, to optimize the technology parameter of extraction and purification for ARA.The study of microwave mutation.Mortierella isabellina3.3410 was used as initial strain, microwave mutation, ultraviolet mutation, protoplast mutation and ultraviolet mutation protoplast were used to mutate, and then the content of the ARA was measured by GC. Ultimum four strains who had the most ARA yield and genetic stability were obtained. Under frequency of microwave mutation at 700W and heating time of 35s,lethality of spores of Mortierella isabellina was 78.3%.After twice microwave mutation and repeatedly screen,a mutation of Mortierella isabellina W35s2-153 was obtained,whose ARA yield was 2.61g/L and were 3.18 times of the control strains.The power of viltalight lamp was 20W,exposure distance was 30cm and exposure time was 80s,lethality of spores of Mortierella isabellina was 76.4%.After twice ultraviolet mutation and repeatedly screen,a mutation of Mortierella isabellina Z80s2-17 was obtained, whose ARA yield was 2.74g/L and were 3.77 times of the control strains.After protoplast mutation, enzyme concentration at 4%,enzymolysis temperature at 30℃and enzymolysis time at 7.5h,the rate of protoplast was 78.4%.After mutation and repeatedly screen,a mutation of Mortierella isabellina Y-69 was obtained,whose ARA yield was 2.92g/L and the concentration in biomass were 3.56 times of the control strains. After ultraviolet mutation protoplast, the power of viltalight lamp at 20W ,exposure distance at 30cm and exposure time at 80s,lethality of spores of Mortierella isabellina was 76.4%,a mutation of YZ-124 was obtained,whose ARA yield was 4.72 g/L and were 5.76 times of the control strains.The process condition optmization of arachidonic acid extraction from microbial oil by supercritical CO2 fluid technology.An orthogonal, rotatable, central composite was employed to determine the optimum conditions for extraction.GC was used to measure the content of the ARA. The results indicated that extraction temperature of 32℃, extraction pressure of 16mPa,extraction time of 101min and 10% methanol as entrainer were the most effective,giving the extraction of 44.6% and purity of 29.34%.The study of ARA purification by urea clathration. An orthogonality experiment was employed to determine the optimum conditions for purification.GC was used to measure the content of the ARA. The results indicated that urea:fatty acid of 4:1, fatty acid:ethanol of 1:12, clathration temperature of -15℃and clathration time of 12h,the purity of ARA was 36.84%.