Optimization Of EPA Fermentation Process Of Mortierella Alpina And Its Application In Hen's Feed

Eicosapentaenoic acid(EPA)is a kind of omega-3 series long-chain polyunsaturated fatty acid,which has special physiological functions such as anti-inflammatory,anti-cancer,anticoagulation,lipid-lowering and anti-cardiovascular disease.However,EPA is mainly derived from deep-sea fish oil.Due to the limited fish resources,the pollution of the marine environment and the complexity of the extraction process,finding a sustainable alternative source of EPA has become a research hotspot.Among them,microbial oils have received extensive attention and research.` Mortierella alpina(M.alpina),with high production of oil and rich in polyunsaturated fatty acids,is an FDA-safely certified industrial strain for the production of arachidonic acid.In the early stage,we obtained a recombinant strain of M.alpina CCFM698 by expressing the omega-3 desaturase PPD17,which achieved a breakthrough in converting ARA to EPA at room temperature.In order to further improve the productivity of EPA by this strain,a series of fermentation process optimizations were carried out,and twostage pH control combined with oxygen-enriched air strategies with fed-batch fermentation for the highly efficient production of EPA in M.alpina CCFM698 were proposed.Finally,M.alpina was used as a PUFAs feed additive in laying hens,and its effects on the components of PUFAs in eggs and the organization of laying hens were investigated.The main findings are as follows:1.On the basis of shake flask fermentation,the optimal carbon and nitrogen source was added with 50 g/L glucose and 20 g/L soybean meal under batch fermentation conditions.In the 7.5 L fermentor experiment,this study investigated the factors affecting fermentation dissolved oxygen,such as fermenter speed,oxygen-enriched medium and oxygen-enriched air.The results show that the fermenter rotation speed of 400 r/min can prevent the excessive shear stress from causing damage to the M.alpina.However,under this condition,the high dissolved oxygen demand in the process of cell growth and lipid accumulation cannot be satisfied.Therefore,the dissolved oxygen supply strategies were studied.Studies had shown that the addition of 4% n-hexane as an oxygen-enriched medium can improve the dissolved oxygen in the fermentation system,but it is not conducive to the growth of cell and the accumulation of EPA.The reason may be that hexane is toxic to the M.alpina.The use of oxygen-enriched air strategy to maintain the relative oxygen solubility of the fermentation system at about 30% can effectively meet the dissolved oxygen demand and significantly increase the EPA production,which is 1.8 times higher than the control group.Therefore,the oxygen-enriched air strategy was chosen as the dissolved oxygen regulation in the fermentation process.2.Due to the sharp increase of glucose consumption rate under the oxygen-enriched air strategy,the lack of glucose in the late fermentation stage hindered the synthesis and accumulation of lipids.Therefore,the fed-batch fermentation was studied.The results of the fed-batch fermentation showed that the initial glucose content was controlled at 30 g/L,and the residual glucose concentration was maintained at 10~30 g/L by intermittent feeding,which increased the yield of EPA by 79.1%.The results of different pH control strategies show that the use of ammonia to regulate pH can effectively increase the level of biomass and lipid production.M.alpina has different pH requirements during the growth phase and lipid synthesis period.pH 6.0 is beneficial to the growth of M.alpina,while pH control at 6.5 is beneficial to lipid synthesis.Finally,a fed-batch fermentation strategy based on dissolved oxygen and twostage pH controled to achieve the high-yield EPA of M.alpina in a 7.5 L fermenter was developed.The final biomass reached 41.2 g/L,and the total lipid accounted for 31.6% of the biomass,and EPA yield is 3.5 g/L,which is the highest yield in the current.3.Through the laying hen experiment,the effects of M.alpina as a feed additive for PUFAs in laying hens on the components of PUFAs in eggs and organization of laying hens were investigated.The results showed that with the increase of the proportion of M.alpina,the content of ARA,DPA and DHA in eggs increased gradually,and the proportion of ?-6/?-3 PUFAs gradually decreased.The content of ARA,DPA and DHA in the eggs increased by 21.1%,67.9% and 68.9%,and the ?-6/?-3 PUFAs decreased by 28.6%,respectively,when 2% of the M.alpina were added.In addition,the addition of M.alpina feed additivies can significantly reduce the ratio of ?-6 and ?-3 PUFAs in laying hens.The proportion of omega-6/?-3 PUFAs in chicken liver,chicken blood and chicken breast decreased by 46.5%,61.4% and 73.7%,respectively,when 2% of the M.alpina were added at the end of the supplementary period.