Purification And Identification Of Effective Components Extracted From Antiaflatoxigenic Hitwh-B05Strain

Aflatoxins were a type of toxic compounds produced by fungus. They werefound in soil, peanuts, corn and other crops, not only causing pollution to theenvironment, but also causing the body disease of human or animal and even death.Therefore, it was a pressing problem about how to fast and effective inhibitaflatoxin pollution on crops, persons, livestock and the environment, as well asresearching hotspot of scholars at home and abroad. In this study, theantiaflatoxigenic effective components in microbial natural products were used toeffect on agricultural products, improving the ability of agricultural productsagainst mold and aflatoxin, which effectively prevented fungal infection andreduced the productin of aflatoxins, and played an important role in order to reducechemical preservative on the environment and human health.The Bacillus subtilis Hitwh-B05strain used in this study was isolated in thislaboratory and was a new Bacillus subtilis strain with independent intellectualproperty rights. The fermentation conditions of antiaflatoxigenic effctivecomponents were opmized. The separation methods were established from variousangles for the antiaflatoxigenic effective components. The antiaflatoxigeniceffective components separated were identified. The inhibitive mechanism wasdicussed. The tip culture method used in this study was the first time in China todetect the antiaflatoxigenic abilities of effective components produced byHitwh-B05. Its supernatant could inhibit the growth of Aspergillus parasiticus andthe production of Norsolorinic Acid (NA)—the first stable intermediate product ofaflatoxin, and then inhibited the production of aflatoxins. The inhibition had broadspectrum to aflatoxins. The merit of this method was direct-viewing, easy operating,short experiment period, could be controlled by quantificationally analyzing and theresults were more accurate and reliable. The Aspergillus parasiticus mutant strainused in this study was nonhazardous and reducing the harm of experiment personneland environmental pollution compared with the aflatoxigenic strain.The activities of antiaflatoxigenic effective components were obviouslyincreased through opmizing fermention conditions. The felicity conditions ofantiaflatoxigenic effective components produced were as follows:35℃,6d,pH6.00,MnSO40.04g/L, yeast powder10g/L, beef extract12g/L, glucose40g/L, soyapeptone10g/L, MgSO44g/L and K2HPO44g/L. Comparing the effects of optimalmedium and GY under the same condition, we found that the inhibitory rate of NAaccumulation using optimal medium was2.98-fold to using GY medium, and5.21-fold to the inhibitory rate of mycelia growth. The lipopeptides were2.16-fold to GY liquid medium.Macroporous resin enrichment method was the best method of separated theantiaflatoxigenic effective components from Bacillus subtilis Hitwh-B05in actualproduction application. Macroporous resins DM130could absorb amount ofantiaflatoxigenic effective components, but was not that easy to absorb pigmentimpurities. The recovery rate of peptide could reach69.43%, while the averageremove rate of pigment could reach above96%.By the detection of molecular biological techniques, lipopeptides bacillomycin,bacilysin, ericin, fengycin, mersacidin, mycosubntilin, iturin A, surfactin andsublancin were found in the fermentation broth of Hitwh-B05, which indicated thatthe lipopeptides produced by Hitwh-B05were with wide ranges. Analized byMALDI/TOF/MS showed that the type of antiaflatoxigenic effective componentswere lipopeptides: iturin A, bacillomycin D, surfactin and fengycin A. The types oflipopeptides were more than other majority Bacillus subtilis had been reported.There were fewer reports at home and abroad about four different types oflipopipties detected in one strain, and most of them were only detected one to threetypes of lipopeptides.The mould proof and inhibitory ability of active substances separated fromHitwh-B05on the inhibition of Aspergillus parasiticus infected peanuts was muchhigher than the commonly used chemical synthetic preservatives sodium diacetate,the substances had great applictional potential, applying theoretical and techinicalsupports for the developing of the new biological pesticides with China’sindependent intellectual property rights and inhibitory ability to aflatoxin synthesis,making the use of agricultural products in China more reliable and superior. TheAspergillus parasiticus spores germination time was delayed under the treatment ofantiaflatoxigenic effective components. The mycelia were dry, surface was rough,fluid loss seriously and some had broken into pieces. This demonstrated that underthe treatment of antiaflatoxigenic effective components, the plasma membrane ofthe Aspergillus parasiticus cells form pores and the struture of plasma membranewas damaged, leading to the disability of absorbing the nutrient substances normally,and influenced the synthesis of aflatoxin.