| We have studied Hitwh-B05 strain, which can inhibit a stable intermediate product Norsolorinic acid (NA) in aflatoxin biosynthesis pathway and is one of the owner of Chinese own intellectual property rights biocontrol bacterium, about 16S rDNA molecular identification, morphological and physiological characteristics and heredity stability of produce antiaflatoxigenic active substances, suitable temperature for fermentation, antiaflatoxigenic active substances effective storage period study, and we have established the extraction and purification methods of its antiaflatoxigenic active substances by fermentation which make a base for further purification and identification the antiaflatoxigenic active substances,identify its mechanism of inhibit NA thus inhibit aflatoxin form.By Hitwh-B05 strain of morphological features and 16S rDNA identification, clear as Bacillus of Bacillus subtilis, the strain has good ability of genetic instability to produce antiaflatoxigenic active substances (at least 10 genetic stability generations), 10 subcultures supernatant of the NA formation inhibition ratio by Aspergillus parasiticus NA mutant was 100%, mycelium growth inhibition ratio is 75%~98.7%. The strain suitable fermentation temperature is 35℃, fermentation supernatant of the mycelium inhibition ratio is 76.32% over and the inhibition of the NA formation can reach 100%. And the fermentation supernatant keeping time in less than a year will not affect the stability of antiaflatoxigenic active substances, preserve 0 day, preserve half of the year and preserve one year of the fermentation supernatant on NA formation inhibition ratio are 100%, mycelium growth inhibition ratio are 84.78%,98.04% and 92.96%. The difference was not significant with CK by t-test; the supernatant that can inhibit the NA formation also can completely inhibit the aflatoxin formation by the research of Aspergillus parasiticus aflatoxin formation strain.The research of different polarity organic solvent on Hitwh-B05 fermentation supernatant of antiaflatoxigenic active substances extraction and purification shows that apart from chloroform extract has only inhibitive effect on mycelium growth by Aspergillus parasiticus NA mutant, benzene, EtOAc and n-butyl alcohol extracts have better inhibitive effect on mycelium growth and NA formation, the IC50 of mycelium growth are benzene 1.218mg/ml, ethyl acetate 0.075mg/ml, n-butyl alcohol 0.530mg/ml, the IC50 of NA formation are benzene 0.481mg/ml, Ethyl 0.029mg/ml, n-butanol 0.318mg/ml. These show that Hitwh-B05 strain fermentation supernatant containing different types of antiaflatoxigenic active substances, and we can initial judgment that the batter inhibitive effect of the antiaflatoxigenic active substances are of the polarity higher.Of ethyl acetate and n-butyl alcohol extracts by TLC further purification, respectively got 14 and 13 zones, of these, the better inhibition zones'totals are 5 and 7. The zones of ethyl acetate extract which has the inhibitive effect, zone 9 concentrations in 0.3mg/ml can inhibit NA formation completely, and zone 11, 12, 13 and 14 concentrations in 0.075mg/ml can inhibit NA formation completely, and have no significant effect on mycelium growth, even their concentrations in 0.3mg/ml also have no significant effect on mycelium growth. N-butyl alcohol separated zones which inhibited best is zone 1, the result of tip culture indicates that in a concentration of 0.05mg/ml, it can inhibit the NA formation completely, and the inhibition ratio of mycelium growth is 32.30%. The result of microwell plate experiment indicates that in a concentration of 1.0mg/ml, it can completely inhibit the germination of spores.The result of the research indicated that Hitwh-B05 strain is a good and new biocontrol bacterium strain, the further purification and structure identification of the antiaflatoxigenic active substances from the supernatant of it will provide lead compound and new biopesticide for the biocontrol of aflatoxin contamination.
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