Fermentation Optimization And Preliminary Study On Metabolic Mechanism Of Penicillium Citrinum Asc2-4 From Pyrosomella Verticilliata For Oil-producing

Penicillium citrinum Asc2-4(KX462901)is an oil-producing fungus isolated from Pyrosomella verticilliata in Leizhou.The oil products has the advantages of high content of polyunsaturated fatty acids(PUFAs)and simple component.Polyunsaturated fatty acids(PUFAs)have functions of anti-inflammatory and anti-cancer effects that applicable for food additives,health products as well as feed.As the demand of unsaturated oil has been increasing and microbial oil has the advantages of low cost and high efficiency,studies on the development of microbial oil received more concern.However,the oil-producing microorganisms isolated and screened have low biomass and oil content.In order to be used in industrial production,it is necessary to optimize the fermentation conditions and improve the strain to improve the biomass and oil content.In this paper,the medium composition and fermentation conditions of Penicillium citrinum Asc2-4 were optimized in order to increase the biomass,oil concentration and oil content.The mechanism of oil synthesis was discussed by key enzymes activity and the fatty acid composition of oil was analyzed,which provided an important basis for the further application of this strain.The main conclusions are as follows:(1)Optimization of fermentation medium composition of Penicillium citrinum Asc2-4The optimal medium consists of a carbon source that was glucose with the concentration of 100 g/L,a nitrogen source with peptone and yeast extract,a C/N of 100.On the basis of single factor experiment of eight kinds of inorganic salts,the orthogonal test was used to optimize three kinds of inorganic salts.It was determined that the type and concentration of inorganic salts were KH₂PO₄4 g/L,Mg SO₄ 150 mg/L and Fe Cl₃30.0?g/L,respectively.In this complex,oil concentration reached 4.63 g/L.The study on its growing pattern and the law of sugar-oil transformation showed that the oil conversion efficiency was the best on the 4th day,the optimum fermentation time was the 6th day,and the 4th day was the best feeding point.The diameter of mycelium pellet of 0.1?0.2 cm was conductive to the accumulation of oil.(2)Optimization of fermentation conditions of Penicillium citrinum Asc2-4Through the single factor experiment,the optimal fermentation conditions of oil production were determined that the temperature was 29?,initial p H was 5.5,inoculation volume was 3%,loading volume was 100m L in 250m L conical flask,rotation speed was 220rpm.Under this condition,the biomass,oil concentration and oil content reached 18.73 g/L,8.83 g/L and 47.24%,respectively.The optimal inoculation volume,loading volume and rotation speed were analyzed by Box-Behnken response-surface analysis.The fermentation conditions were further optimized as follows:inoculum volume of 3.26%,loading volume of118.5 m L/250 m L,rotation speed of 226 rpm.Under this condition,oil concentration was up to8.92 g/L.(3)A preliminary study on the mechanism of oil synthesis pathway of Penicillium citrinum Asc2-4Under the condition of C/N of 40 and C/N of 100(carbon source in both medium was100g/L,nitrogen source was 2.5 g/L and 1.0 g/L,respectively),the key enzyme activity of metabolize cycle and metabolic pathway of oil accumulation was analyzed.The results showed that citrate synthase(CS),ATP-citrate lyase(ACL)and malic enzyme(ME)played key roles in the oil synthesis and metabolism of this strain.C/N of 100 was conductive to stimulate pentose phosphate pathway and tricarboxylic acid cycle to accumulate oil.Therefore,nitrogen-limited strategy was applicable to oil accumulation of this strain.The fatty acid composition of the oil produced by different glucose concentrations(50 g/L,100 g/L and 150 g/L)was analyzed,in which the contents of unsaturated fatty acid were more than 53%.In glucose concentration100g/L group,the unsaturated fatty acid content was 65.19%,among which oleic acid(C18:1)and linoleic acid(C18:2)contents were 41.46%and 21.62%,respectively.So Penicillium citrinum Asc2-4 was worth of research and development,and the fatty acid compositions were slight differences in different glucose concentrations media.