Study On The Effect Of Oleic Acid On The Lycopene Production In Neurospora Crassa And Its Promoting Action

Lycopene,one kind of carotenoid,is a straight-chain hydrocarbon without oxygen atoms.Due to its high degree of conjugation,lycopene shows superior antioxidant properties.Additionally,dietary supplementation with lycopene has many benefits for human health,such as improving immunity,preventing and treating cardiovascular,cancer and other types of diseases.Hence,it is increasing industrially in functional food,medicine and more fields.In recent years,microbial fermentation technology applied to produce lycopene has attracted extensive attention.Among them,it has been verified that Neurospora crassa could synthesis lycopene through the mevalonate(MVA)pathway,using acetyl-Co A as the starting substrate.Meanwhile,there are no toxic metabolites have been detected up to now.Thus,N.crassa could be considered as a promising microorganism for producing lycopene.To further improve the lycopene yield,the effect of unsaturated fatty acids on microbial growth and lycopene production in N.crassa 3.1608 was studied.Moreover,the optimal culture conditions and potential promoting mechanism of lycopene production were also explored in this paper.Besides,the optimal multi-enzymatic extraction conditions of lycopene in N.crassa and the stability of lycopene were investigated.The main research results were listed as follows:1.The effects of three unsaturated fatty acids on the growth of N.crassa and lycopene production were studied,and the results shown that the promoting effect of oleic acid was more significant than that of linoleic acid and linolenic acid.Further,the optimal culture conditions obtained through the single factor experiment and response surface methodology were as follows:oleic acid concentration of 0.9 g/L,shaking rate of 100 r/min,shake culture temperature of 34?.Under this condition,the lycopene yield in N.crassa was 4.21±0.11 mg/L,which was 2.77-fold higher than that of the control group.2.The metabolic changes between the control and oleic acid-treated groups were determined to explore the promoting mechanism of oleic acid on lycopene production in N.crassa.In light of our results,when compared with the control group,the levels of acetyl-Co A and NADPH in the oleic acid-treated group were increased by 37.64%and 24.63%,respectively.Thus,it could supply more available carbon flux and cofactors for N.crassa.Furthermore,the key factors associated with lycopene biosynthesis in the MVA pathway were all increased significantly after adding oleic acid to the medium.The levels of HMGS,HMG-Co A,HMGR and GGPPS were raised from 142.40,115.57,94.83 and 88.81 ng/L in the control group,respectively to 200.71,165.25,134.84 and 151.28 ng/L in the oleic acid-treated group,respectively.Therefore,the above up-regulated key factors indicated that the carbon flux was effectively channelized toward the MVA pathway,promoting the synthesis of lycopene.On the other hand,intracellular triacylglycerol(TAG)content and its fatty acids composition could reflect the storage capacity for lipophilic metabolites.With the addition of oleic acid,the fatty acid synthetase(FAS)level was significantly increased to 10.83 nmol/L from 8.69 nmol/L(the control group),indicating that a certain amount of acetyl-Co A could enter the de novo process for synthesizing more fatty acids.Besides,the prominent enhancement of oleic acid proportion in the total lipids and TAG implied that exogenous oleic acid could be directly incorporated into the intracellular lipids through the ex novo process.Ultimately,the TAG content was ascended from 0.91mmol/gprot to 1.24 mmol/gprot through the above two processes,as well as its unsaturated fatty acids percentage was up to 81.10%from 74.23%.As a result,such changes in TAG were contributed to improving the storage capacity for accumulating more lipophilic lycopene via lipid bodies(LBs).3.The Viscozyme L and cellulase were utilized to hydrolyze the N.crassa cell wall for improving the extraction efficiency of lycopene,and the multi-enzymatic hydrolysis conditions were further optimized.With the 20 mg of Viscozyme L and cellulase(1:1,m:m),the single factor experiment and response surface methodology were applied to determine the optimal conditions.Results shown that the extraction yield of lycopene was risen to 0.66±0.03 mg/g under the conditions of hydrolysis temperature of 45?,hydrolysis time of 78 min,and hydrolysis pH of 4.5,which was59.98%higher than that of the control group without enzymes.4.The effects of light,temperature,pH and metal ions on the stability of lycopene were studied in this paper.According to our results,both the ultraviolet light(UV)and natural light had a strong destructive influence on lycopene,which was decreased to1.40%and 11.45%after illuminating for 60 min,respectively.However,it had no obvious variation for lycopene when exposed to incandescent light.Similarly,a minor destructive effect was also observed when lycopene stored at 25-70?for 6 h,and the relative amounts of lycopene were 99.20%,99.18%,93.10%,92.43%and 91.22%,respectively.Besides,lycopene was sensitive to the alkaline condition,while the opposite results were found under the acidic and neutral conditions.The relative amounts of lycopene were decreased to 22.75%,20.08%,24.74%and 20.74%when treated with Na⁺,K⁺,Mg²⁺and NH⁺for 6 h,respectively.Additionally,the phenomenon of flocculent precipitate was observed when lycopene coexisted with Fe²⁺.Thus,lycopene is sensitive to the above metal ions.