Production Of Carotenoids By Neurospora Crassa On Agro-industrial Residues

In this paper, high-value added carotenoid was produced by Neurospora crassaon low-cost agro-industrial residues in solid-state fermentation. Neurospora crassa,which could produce high amount of cellulase and carotenoids, was isolated andmutated from the traditional fermented food in Jiangxi province. The optimalconditions of the solid-state fermentation were optimized to improve the yield ofcarotenoids. Meanwhile carotenoids were separated by high-performance liquidchromatography and the stability of carotenoids from the spores and acetone extractof Neurospora crassa were studied. Finally in vitro fermentation test and five kindsof in vitro antioxidant methods were used to evaluate the functional properties ofcarotenoids from Neurospora crassa, providing theoretical foundation and scientificbasis for the further application of carotenoids. The main results are as follows:1. The carotenoid location experiment proved that carotenoids existed in theform of pigment particle which mainly located around the cell wall of mycelium andspore wall of spores. Besides the amount of pigment particles in the spores is morethan that in the mycelium.2．Plackett-Burman Design and Response Surface Methodology (RSM) wereused to optimize the concentration of most significant factors and their interaction,consequently, the carotenoids production increased dramatically from266.53to966.21μg/gds when the fermentation was carried out in the optimized mediumcomposed of0.51%ammonium sulphate,20%brewer’s grain,34.25%sweet potatoresidues and45.75%rice bran (calculated in dry weight) with the initial moisturecontent of72.15%at pH6.5for120h.3．Through the optimization of HPLC method, the optimal condition were：methanol: acetonitrile: isopropyl alcohol(54:44:2）; detection wavelength:460nm;injection mode: auto-injection; sample size:20μL. Fifteen kinds of carotenoidswere separated with this method, as follows: Apo-4’-lycopenoic acid，-apo-4’-carotenal， neurosporaxanthin， γ-carotene，3,4-didehydrolycopene，all-trans-lycopene，15or15-cis-lycopene， neurosporene， all-trans-torulene， ζ-carotene，all-trans-carotene，13or13-cis--carotene，three kinds of cis-torulene.With external standard method, the content of all-trans lycopene2.51%and all-trans-carotene were18.56%respectively.4．The stability test of carotenoid showed that with the protection of the sporewall, the carotenoid can resist high temperature, heavily acidic conditions, andoxidization. Moreover, these extreme conditions can improve the efficiency ofcarotenoid extraction. On the contrary, high temperature, oxidant, sunlight andacidic conditions can speed up the degradation of the carotenoid in the acetoneextract;low temperature, dark condition, and alkaline conditions have little effecton the carotenoid stability; some antioxidant such as BHT can prevent degradationof carotenoid, While VC unexpectedly promote oxidation of carotenoid. Of all thecommon metal ions, Fe3+can greatly speed up the degradation of the carotenoid,while Al3+just has less influence on the carotenoids, the other metal ions had littleeffect on the stability of the carotenoid. These researches can provide some measuresfor reducing the degradation of carotenoid in the laboratory and the industrialproduction, moreover, under these conditions, inactivating spores can be taken intoconsideration as the microencapsulation of carotenoids.5． The results of antioxidant experiments indicated that: compared withcommon antioxidant, lycopene and-carotene, the scavenging capability ofcarotenoids produced by Neurospora crassa towards DPPH， superoxide andhydroxyl radical was stronger in the experimental concentration. In addition,carotenoids produced by Neurospora crassa exhibited high total antioxidantcapability and good chelating ability of metals.6．The functional properties of carotenoids produced by Neurospora crassa wereevaluated by in vitro fermentation. The results found out that carotenoids fromNeurospora crassa had good fermentation characteristics. The pH of parts ofcarotenoids groups were obviously lower than those of the control group5.03，andwhen the concentration of carotenoids reached100mg/L, it has the lowest pH was4.95. With the increasing of the mass concentration of carotenoids, the ammonia Ncontent was declining, and the lowest content of ammonia N was recorded when thecarotenoids group was at the concentration of100mg/L, which was41.5mg/L. The carotenoids group of100mg/L produced the highest content of acetic acid reaching113.28μmol/mL. In addition it also produced the highest amount of the total shortchain fatty acid, which was225.36μmol/mL.