Study On The Production Of Polyunsaturated Fatty Acids By Filamentous Fungi

Taking Mortierella isabellina AS3.3410 as the starting strain,a high-yielding strain of polyunsaturated fatty acids was bred by mutagenesis,and corn sugar was used as the carbon source.The fermentation process conditions for the production of polyunsaturated fatty acids by mutant strains were optimized,and the purification of oils and fats was studied,including:1.In order to improve the oil yield,Mortierella isabellina AS3.3410 was used as the starting strain,and ultraviolet(UV)and lithium chloride(Li Cl)were used for compound mutagenesis.After primary screening with acetylsalicylic acid and Sudan black B staining,Shake flask re-screening,screen high-yielding polyunsaturated fatty acid mutant strains,and analyze their genetic stability and oil composition.The results showed that the concentration of lithium chloride was 0.6 g/L,the UV mutagenesis time was 20 s,and the concentration of acetylsalicylic acid in the initial screening was 0.6 g/L,and a genetically stable mutant SLZH-20 was screened out by mutagenesis.and oil content were 22.76 g/L and 52.30%,respectively,which were 34.60%and 24.30%higher than the starting strain,respectively;the unsaturated fatty acids in the oil produced by this strain included palmitonic acid,oleic acid,linoleic acid,?-linolenic acid,?-linolenic acid,the content is81.41%,which is 5.72%higher than that of the original strain AS3.3410.The polyunsaturated fatty acid content was 16.24%,which was 2.18%higher than that of the starting strain AS3.3410.2.Using biomass and oil yield as evaluation indicators,the effects of nitrogen source type and concentration,carbon source concentration,magnesium sulfate concentration,inoculum amount,p H,temperature and time on the fermentation effect of the strain were studied.The fermentation conditions were determined by single factor and orthogonal experiment optimization.The results showed that the selected nitrogen source was yeast powder,the ratio of carbon and nitrogen sources was 30:1,and the optimal fermentation conditions were carbon source mass concentration of 80.0 g/L and nitrogen source of 2.7g/L.,magnesium sulfate 0.7 g/L,sodium citrate 2.0 g/L,potassium dihydrogen phosphate2.0 g/L,inoculum size 10%,p H 6,temperature 26?,time 6 d.The verification experiment was carried out according to the optimization results of the orthogonal experiment of biomass and oil yield.The results showed that the bacterial biomass and oil yield reached26.12 g/L and 14.63 g/L,respectively.After gas chromatography analysis,the fatty acid composition of the oil of Mortierella aflavus mutant SLZH-20 was myristic acid,palmitic acid,palmitonic acid,stearic acid,oleic acid,linoleic acid,arachidic acid,?-linolenic acid,?-linolenic acid,of which the unsaturated fatty acid content is 81.45%,and the polyunsaturated fatty acid content is 16.28%.3.In order to increase the content of PUFAs in fats and oils after fermentation,studies on methyl esterification and urea inclusion are required.Taking the fatty acid oil yield as the evaluation index,the effects of oleyl alcohol ratio(oil:methanol),potassium hydroxide concentration,reflux temperature and reflux time on the methyl esterification of fatty acids were investigated.The esterification conditions of fatty acid were determined by single factor experiment.The results showed that the optimal esterification conditions were oleyl alcohol ratio of 1:5,potassium hydroxide concentration of 1.3%,reflux temperature of60?,and reflux time of 30 min.Taking the yield of fatty acid oil and the content of polyunsaturated fatty acid as evaluation indexes,the effects of the ratio of fatty acid oil and urea,the ratio of urea and ethanol,the inclusion temperature and the inclusion time on the urea inclusion process were studied.The urea inclusion process was optimized through single factor and orthogonal experiments.The results showed that the optimal conditions were that the ratio of fatty acid oil and urea was 1:2.5(g:g),the ratio of urea and ethanol was 1:4(g:m L),The inclusion temperature was-10°C and the inclusion time was 18 h.The verification experiment was carried out according to the orthogonal experimental optimization results of the urea inclusion yield and urea inclusion content.The results showed that the yield of fatty acid oil and the content of polyunsaturated fatty acids reached 58.7%and 54.6%.After gas chromatography analysis,the polyunsaturated fatty acids obtained by urea inclusion and separation of fatty acid oils are mainly composed of C₁₈unsaturated fatty acid oils,namely oleic acid,linoleic acid and?-linolenic acid,of which linoleic acid is 34.4%,gamma-linolenic acid was 19.1%.