| Docosahexaenoic acid (DHA) is one of the ω-3polyunsaturated fatty acids. DHA hasimportant biological and physiological functions, including development of the brain andintelligence, protection of eyes, prevention of cardiovascular disease and improvement ofbody immunity. Therefore, DHA has been widely used in medicine, food, feed and otherindustries, and more and more attention has been paid to the production and preparationprocess of DHA. Traditionally, DHA is extracted from deep sea fish oil. Due to the lowconcentration of DHA yield in fish oil as well as the complex composition of fish oil, to findand use marine microbial fermentation production of DHA has gradually become a researchhotspot. Schizochytrium is a promising microorganism in producing DHA as it grows fast andhas high content of DHA, simple composition of PUFAs and is harmLess to human. In thepresent study, Schizochytrium limacinum SR21was selected to produce DHA, and the mainresearch contents and results are as follows:1) According to the research results of the oil extraction process, acid-heat process wasused to break the cell wall and chloroform-methanol was chosen as the extraction solvent toextract oil, namely acid-heat-organic solvent process. After experimental optimization, the oilyield obtained was52.56%using this method, increased by14.91%compared with that of thecontrol (45.74%). The standard curve of strain Schizochytrium limacinum SR21wasestablished for rapid determination of lipid content by phosphoric acid-vanillin reaction andacid-heat-organic solvent process, and the total lipid content of strain Schizochytriumlimacinum SR21was quickly estimated according to this standard curve. The phosphoricacid-vanillin reaction was also used in rapid screening high-lipid-yield mutants in thesubsequent mutagenic work.2) The effect of exogenous additives such as biotin, citric acid, malic acid and lovastatinon the DHA fermentation by the strain Schizochytrium limacinum SR21was also studied.Results showed that exogenous additives could improve the DHA content in intracellular fattyacids by improving the production of DHA and changing the distribution of fatty acids incells. According to orthogonal experiments，the optimal combination of exogenous additiveswas as following:0.3mg/L biotin,1g/L malic acid and0.4mg/L lovastatin. After theaddition of exogenous additives by optimal combination, the production of total fat was28.93g/L, which was68.88%of dry cell weight; DHA production reached up to11.55g/L,improved by71.87%compared to the original strain which was6.72g/L, and the percentagecomposition in fat raised from31.93%to39.92%。3) Breeding of high-producing strain was performed with Schizochytrium limacinumSR21as the original strain. Firstly, the optimal concentrations of iodoacetic acid and malonicacid on regeneration agar plates was determined at low temperature (15℃). Then, using thebacterial production of oil and DHA as screening indexes, ARTP, UV and ARTP-UVmutagenesis were conducted, followed by phosphoric acid-vanillin reaction preliminaryscreening and GC secondary screening. Finally, a high-yield mutant AU-1was obtained, andit was also proved that AU-1had hereditary stability after passage for5times. After shake flask fermentation, the DHA production of this strain reached up to11.81g/L, improved by75.74%compared to that of the original strain (6.72g/L); the DHA content in fatty acids wasalso raised from31.93%to49.47%. After fermentation in a5-L tank, the yield of biomass, oiland DHA reached up to50.71g/L,35.65g/L,15.43g/L, respectively. In addition, theproduction intensity was up to142.87mg/L/h. All the indexes above were increasedcompared to those of the original strain, i.e.,46.78g/L,32.65g/L,13.26g/L and110.5mg/L/h, respectively. Most remarkably was that the DHA yield of this strain was increased byas high as16.37%. |
PDF Full Text Request