Optimization Of Fermentation Conditions And Study On The Antioxidant Activity Of Lycopene Produced From Neurospora Crassa By Solid Fermentation

Lycopene is an oxygen-free carotenoid,which is isoprene compounds with 11 conjugated double bonds and two non-conjugated double bonds,exists in natural plants and has a strong antioxidant effsct.In this paper,Neurospora crassa,which was simple in nutrition,rich in growth and safety,was used as the starting strain to optimize and determine the fermentation parameters.And effects of the addition of fermentation promoters on lycopene production and the antioxidant activity were studied.Finally,the yield of lycopene produced by the method of solid fermentation and liquid fermentation was compared.The fermentation conditions of lycopene production by solid culture were studied,and using N.crassa 3.1607 as the starting strain,through collecting spores to research.According to the single-factor experiments and response surface methodology,the three factors which had obvious influence on the yield of fermentation products were optimized.The pigment was extracted with ethyl acetate acetone?1:1,V/V?mixed organic solvent and analyzed by HPLC,and the UV detection wavelength was 472 nm,the proportion of mixed mobile phase acetonitrile dichloromethane?V/V?were 60:40,under this condition,lycopene could be well separated.The optimal fermentation conditions were as follows: the PH of initial culture was 5.5,the fermentation temperature was 30?,the inoculation amount was 0.95%,the fermentation time was 95 h,and the whole process of illumination,under that condition,the yield of lycopene was 2.48mg/g.The production of lycopene from N.crassa in solid culture and liquid culture were compared,solid culture was more simple and convenient,short cycle and easy collection of spores in the operation;the more spores and lycopene in yield.The solid culture of N.crassa that adding additives was used to achieve the purpose of accumulating lycopene.A single factor experiment was carried out with the addition of various additives as independent variables,and the productivity of lycopene as dependent variable,then from citric acid,geraniol,BHT,ketoconazole and terbinafine hydrochloride,2-isopropyl imidazole screened four substances,and analyzed and response surface experimental designed by Box-Behnken principle,and amount of geraniol,BHT,terbinafine hydrochloride and 2-isopropyl imidazole which were four significant influence additives were optimized.Finally,the optimal amount of composite additives were:BHT 0.29g/L,geraniol 24.78mg/L,terbinafine hydrochloride 0.95mg/L and 2-isopropyl imidazole 571.21mg/L,under that condition,the lycopene yield reached 5.10mg/g,which was 77.7% higher than that of the blank group 2.87mg/g.It was proved that the addition of the promoters had a certain effect in promoting on the lycopene productivity.The antioxidant activity of lycopene from N.crassa by solid state fermentation was studied.Mainly through the scavenging rate of DPPH radical,hydroxyl radical,and superoxide anion radical were determinated,at the same time,compared with some common antioxidants ?-carotene,BHA,VE,BHT and VC.The results showed that the lycopene of N.crassa by solid-state fermentation had a certain scavenging effect on DPPH radical?hydroxyl radical and superoxide anion radical,and the obvious dose effect relationship.In a certain concentration range,the order of DPPH radical scavenging ability was: BHA> lycopene > VE > ?-carotene;the order of hydroxyl radical scavenging ability was: lycopene > VC > BHT;the order of superoxide anion radical scavenging ability of lycopene > VC > VE.The EC50 of DPPH·,·OH and O2-· on lycopene were 0.039mg/m L?0.008mg/m L and 0.054mg/m L.It could be seen that the lycopene obtained from solid-state fermentation of N.crassa had strong free radical scavenging and antioxidant capacity.Meanwhile,lycopene and VE could inhibit the oxidative modification of LDL by Cu²⁺ inducing,and lycopene was stronger than VE.It had been shown that VE could slow down the oxidation process of As,and provided new clues for the early treatment of As,so lycopene may also have the effect of inhibiting As.Then,this provided a broader space for the study of the biological activity of lycopene.The spore yield and the lycopene yield of solid-state fermentation and liquid fermentation were compared from four aspects: medium,fermentation temperature,inoculation amount and fermentation time.The results showed that the sporulation of solid fermentation was 1.58 times higher than that of liquid fermentation under the conditions that effective components and dosage of medium were the same,so the sporulation of solid fermentation was high;the temperature of solid fermentation and liquid fermentation wass basically the same,probably because the same species were used;the inoculation of solid fermentation was less than liquid fermentation,and the former of lycopene production was 1.56 times higher than that the latter under the same conditions of effective components and dosage of medium,due to the mycelial growth took a long time,sporulation slower in liquid fermentation,while the solid fermentationdirectly produced spores and less time-consuming.In addition,through the study of antioxidant activity in vitro and the inhibition of LDL induced by Cu²⁺,the lycopene obtained by solid fermentation had the same effect as that obtained by liquid fermentation.Therefore,the use of solid fermentation to obtain lycopene had the advantages of low cost,short cycle,high yield of sporulation and high yield of lycopene.