| Malania oleifera Chun et S.Lee,a national second-class protected plant,is a characteristic forest resource in Guangxi and Yunnan.There is rich nervonic acid in Malania oleifera oil,which is super-long chain monounsaturated fatty acid.It has a variety of physiological and pharmacological activities and high economic value.It is widely used in food,medicine and other fields.Based on the qualitative and quantitative analysis of five kinds of mixed fatty acids,the production of microbial oil rich in nervoic acid from nervonic acid crystallization mother liquid by endophytic fungi isolated from Malania oleifera branches,leaves and fruits was studied.The results are as follows:1.The main fatty acids in nervonic acid crystallization mother liquid are stearic acid,oleic acid,arachidic acid,erucic acid and nervonic acid.In order to monitor the changes of these five fatty acids in microbial oils produced by endophytic fungi from Malania oleifera,a gas chromatography method for simultaneous quantitative determination of these five fatty acids was established.For the following conditions were Rtx-Wax@ column(30 m×0.25 mm,0.25?m),the temperature programmed was the initial temperature of 180?,retention 1 min,5 ?/min to 240 0C,retention 9 min.Through the precision,recovery and sample test,the results showed that the method was accurate and reliable,and it can be used for process monitoring of nervonic acid crystallization mother liquid fermentation and subsequent process optimization.2.Endophytic fungi in healthy branches,leaves,pulp,shells and kernels of Malania oleifera were isolated and purified by tissue patch and grinding coating plate culture.Results 58 strains of endophytic fungi were isolated.The mycelia of 58 strains were stained with Sudan Black-B and observed by microscopy.19 strains with high oil content were screened.The primary strains were cultured in shaking flask,and the contents of fatty acids such as nervonic acid in microbial oils were quantitatively analyzed by gas chromatography.Two strains STGR-6 and STGK-3 were screened to produce rich nervonic acid by fermentation.Endophytic fungus STGR-6 was identified as Penicillium olsonii and STGK-3 as Aspergillus flavus.3.The fermentation conditions of Penicillium olsonii STGR-6 to produce microbial oil rich in nervonic acid were investigated,and the effects of medium formulation on the fermentation were studied.The results showed that the optimum fermentation conditions were as follows:improved Martin medium,40 mL/250 mL triangular flask,3.75 g/L concentration of nervonic acid crystallization mother liquid,pH7.5,in shaking bed at 30 0C for 5 days,and 200 r/min shaking speed.Under these conditions,the growth of the fungi was 28.5 g/L,and the content of nervonic acid in the fungi was 55.29 mg/g.When oleic acid or erucic acid were used as substrates,no nervonic acid was detected in the fungi after fermentation.When oleic acid and nervonic acid mixed solution were used as substrates,the amount of nervonic acid in the oil did not increase after calculating fermentation.When ethanol solution of erucic acid and nervonic acid was used as substrates,the growth of bacteria with erucic acid content greater than 50%was inhibited,and less than 50%of bacteria grew well,but nervonic acid was consumed as nutrient.4.Enlarged culture of endophytic fungi STGR-6 and STGK-3 was carried out under suitable conditions,and microbial oil was obtained by Soxhlet extraction.In the fermentation enlargement experiment,the biomass of STGK-3 was 8.31 g/L and the oil content was 44.85%,which was 23.43%higher than that of the initial screening,the content of nervonic acid in the oil was 20.34%and the yield of nervonic acid was 83.43%.The highest biomass of Penicillium olsonii STGR-6 was 13.69 g/L and the oil content was 23.97%,which was 6.72%higher than that of the first screening,the content of nervonic acid in the oil was 26.67%and the yield of nervonic acid was 87.95%.Two kinds of microbial oils were analyzed by GC-MS.The main components of the oils were oleic acid,erucic acid and nervonic acid. |
PDF Full Text Request