Screeing Of Functional Oleaginous Fungi And The Optimization Of Fermention Conditions

Nutritional studies have shown that the intake of long-chain polyunsaturated fatty acids in diet has a potential benefit to the human health.Omega-3 series of fatty acids,including EPA and DHA,may have a significant effect on the prevention of coronary heart disease.For example GLA has been long recommended for the treatment of various diseases,because these fatty acids are not been synthesized by body,but can only be obtained through the diet.The traditional method of obtaining these essential fatty acids is relatively high cost and yield is very low production of polyunsaturated fatty acids by microbial fermentation,is a promising new industry.But in order to achieve industrial production it is necessary to find high-yield strains,the improvement of fermentation process and the extraction of polyunsaturated fatty acids.This study was based on the screening and identification of fungi in soil which could product polyunsaturated fatty acids.By screening multiple soil samples to preliminary isolate the strain that has good ability of synthesizing polyunsaturated fatty acids,then genotype identification was carried out.Study on the system of metabolic regulation by the optimization of the culture conditions to explore the condition of large-scale industrial production,in order for providing the foundation for small-scale industrial production of microbial production of polyunsaturated fatty acids.（1）The collected soil samples were separated and purified by gradient dilution and plate scribbling,and a total of 147 fungal strains were obtained.The 39 strains were obtained to ferment in flask after plate-streaking screening and the mycelium being picked for Sudan staining.The results showed that the oils of WHJ-13,HNH-14,WHJ-20,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHJ-18,WHN-M and WHZ-15 in mycelia were stained darker than other strains.（2）After finished initial shake flask screening,12 strains of oil-producing strains were got by the second shake flask fermentation.After 7 days’ culture,the cells and the biomass was detected.Then the crude fat was extracted and the fat yield was calculated.oil content of Strain FP of was highest（up to 35.58%）,may contain functional unsaturated fatty acid composition,with the value of further study.（3）The crude oils of 12 oil-producing strains were extracted by Soxhlet extraction method and were methylated,then the fatty acid methyl esters were analyzed by gas chromatography.The results showed that the 12 oil-producing strains produced few C14 fatty acid,as well as C4^C12 and the content of C16: 0,C18: 1 and C18:2 were higher than 10%,the composition of fatty acids of these fungi oil were similar with plant oil.According to the biomass,oil content and yield,total PUFAs andgamma-linolenic acid content of 12 strains,WHJ-13 had higher functional potential.（4）The FP was inoculated on PDA medium,inverted culture for three days,at25℃there were single colonies clearly visible,whose diameter about being 1cm,after cultured for four days,the aerial mycelium was white and woolly,the center of colony was thin and loose,while the outer of colony was full,dense and indicating dry.There were two types of conidia in the microscope: small conidia were ellipse or oval.FP was observed lipid droplets in hyphae by electron microscope.The ITS fragment was amplified and sequenced,and strain FP was identified as Fusarium sp.joint with the Wuhan University of China Typical Culture Collection（CCTCC）.（5）With standard linoleic acid（LA）and gamma linolenic acid（GLA）concentration（A）as abscissa.The standard area of quantitative fatty acid methyl ester peak area（P）was established as ordinate.The standard curve for Y = 1659.50 X + 201.10R² = 0.9996;the standard curve equation for γ-linolenic acid is Y = 823.55 X + 8.87 R² =0.9996.（6）In the experiment of fermentation condition optimization,the effects of various fermentation conditions on the fat production of FP were discussed.The fermentation conditions were as follows: inoculation amount 6%（v/v）,rotation speed150r/min,initial pH 6.The fermentation temperature was 28℃,the inoculation time of seed liquid was 48 h,and the final fermentation time was 7d.After optimization,the fat yield of FP was 2.47g/L,which was 69.72% higher than before yield.The linoleic acid（LA）and γ-linolenic acid（GLA）production increased by nearly 1times and 2 times,respectly.（7）The effects of various nutrition factors on the fat production of strain FP were discussed.The optimum conditions were as follows: 4.45% glucose,0.95% yeast extract,1.5% colleseed oil,KH₂PO₄,CaCl₂,MgSO₄ were 3g/L,500 mg/L,50 mg/L,respectively.（8）Based on the single factor experiment,nine factors influencing the fermentation of oil were selected by Planckett-Burman factor design experiment,and then further optimized by Box-Benhnken center composite design.The final oil yield reached7.29 g/L,increased by about 5 times higher than before optimization（1.49g/L）.