| Food security is more concerned with improvement of living level and health protection recongnition.Toxicology and analytic technology confirmed that many synthesized pigments should be limited to use in many fields because they are harmful to people's health.Many researchers had attention to natural pigments because of high security and health protection functions.Researches indicated that some natural pigment can be produced by microbiology.Based on this,a red-pigment strain was studied in this paper.The results as follows.(1) PDA medium was the best medium with SDAY medium,czapek's medium, yeast-sucrose medium and amylum medium screening and growth rate comparing.(2) There were 15 strains for secondary screen by the value of OD and dry weight of strains.The value of OD outside cell of RCEF4029 was 0.39,inside cell of its was 0.63, the weight of dry mycelium was 3.11g by comparing outside and inside cell OD value and the weight of dry mycelium,which was significantly higher than other strains.So RCEF4029 was the target strain.(3) According to the shape of strain,culture features,morphological characteristics of hyphae,conidiophores,conidiogenous cells,conidia etc.,RCEF4029,the red pigment-producing fungus was identified as Fusarium.Then ITS-rDNA sequence analysis was used to confirm it.The sequence was submitted to National Center for Biotechnology Information(NCBI),compared with DNA sequence in GeneBank by Blaster,and was confirmed that it was a Fusarium momiliforme.(4) The mycelium of the red pigment was used to research technics of extraction in this paper.In the experiment,three means of extraction,ultrasonic,microwave and soaking were compared in extracting effect,and means of soaking with organic solvents was determined to be the optimal method,which was described as follows:the optimal organic solvents was dichloromethane:acetone=2:1(V/V),the optimal temperature was 40℃,the optimal time was 180min,the optimal material/liquid was 1:30(m/v) and the optimal extracting grade was third grade.(5) The nature of the red pigment was discussed in detail:the red pigment was fat-soluble,it could be dissolved in methanol,ethanol,ethyl acetate,n-hexane,toluene, petroleum ether,and other organic solvents,it also could be dissolved in chloroform,2-chloromethane,acetone;the maximum absorption value of it was 435nm;it had good heat resistance;it was relatively stable to darkness,indoor and outdoor light,but the UV-ray irradiation had certain impact on its stability;it was relatively stable to most metal ions oxidants,reducing agent,vitamins,acidity regulator,commonly used food additives and preservatives.Citric acid,sodium hydroxide,VE,gelatin and sodium benzoate had enriched the color;According to DPPH antioxidant screening model law the red pigment had certain antioxidant activity while according to CHO Cytotoxicity Assay RCEF4029 model it had non-toxic whhich has a natural pigment of high security with the good development in utilization value in dyeing,printing and foodstuff.(6) Based on the results of an orthogonal experiment for RCEF4029 fermentation conditions with the content of color values and secretion rate as an estimate,effects of nutritional and non-nutritional factors on the color values of RCEF4029 were studied.The results showed that the optimal composition of medium was:soluble starch 5%,potato 20%,pupar powder 0.25%and KH2PO4 0.075%.The optimal culture condition was as follows:initial pH at 7.5,inocula amount at 10%,culture temperature at 28℃,the agitation rate at 150 rpm,culture duration for 10 d.(7) The pigment was isolated with HPLC,LC-MS,and obtained the pure pigment.It molecular formular was C14H14O6 and molecular weight was 278.261.The compound of the molecule C14H14O6 was deduced with aid of natural product database,the results indicated that there was a known compound producing red pigment in fungus whose color, UV and molecular formula were the same as the tested one,it can be concluded that the one in the test which produces red pigment may be the compound. |
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