Study On Interact Of Bacillus Atrophaeus To Aspergillus Flavus

Fungus Aspergillus flavus and aflatoxin is serious threat to the health of people and animals,cause the contamination to cereal products,avariety of food and feed,nontoxic and antiseptic storage of them is a global concern,among which the biological control of Aspergillus flavus and aflatoxin have became a hot area of research,cause it’s friendly to the environment and effect is obvious,the biological control become more and more important. Separating and screening bacteria which have abvious antagonistic effect on Aspergillus flavus,extracting and purifing the active ingredients inhibited the growth of Aspergillus flavus and aflatoxin by biochemical method,clarifing the mechanism of action of antagonistic bacteria,have both theoretical value and practical significance.This thesis aimed at further research about Bacillus atrophaeus,the bacteria screened from the soil which have abvious antagonistic effect on Aspergillus flavus. Study the mechanism about it from four parts,including nutrition competition,direct parasitic,inducible resistance and antibiotic effect.The mechanism of nutrition competition researched from two parts, Bacillus atrophaeus inhibited the growth of Aspergillus flavus and the germination of spore.The results showed that, all the treatments of Bacillus atrophaeus could play a role of inhibition,especially the preparated fermentation broth and bacteria suspension,this means Bacillus atrophaeus had obvious nutrition competition with Aspergillus flavus; the cell-free suspension fluids also showed inhibitory effect to a certain extent.The mechanism of direct parasitic researched from two parts, Bacillus atrophaeus inhibited the growth of hyphae of Aspergillus flavus and the changes of the mycelial morphplogy.The results showed that,the preparated fermentation broth of Bacillus atrophaeus had the best effect,and then was the bacteria suspension,after the two treatments,the mycelial morphplogy were mutation,became distortion,scattered,moniliform stigmas,part of the cell contents out of the cavity and partial melting,while the cell-free suspension fluids and fermentation broth of Bacillus atrophaeus with121℃sterilization treatments not showed significant effect.The mechanism of inducible resistance researched by vaccinating Bacillus atrophaeus been different treatments and Aspergillus flavus on peanuts,then detected the activity of SOD,CAT,PAL,POD and PPO. The results showed that, Bacillus atrophaeus could improved the activities of SOD and POD,reduce the activity of CAT,while the effects to PAL and PPO were not so obviously.Through the study of nutrition competition and inducible resistance found that,there may has one or more secondary metabolites in the cell-free suspension fluids of Bacillus atrophaeus had the antagonism function to Aspergillus flavus,this showed that antibiotic effect is one of the mechanisms.Optimization of fermentation conditions by orthogonal experiment,determined the best conditions for fermentation was:incubation temperature35℃,time48h, inoculum size1.5%, agitation speed of rotary shaker180r/min, initial pH of medium7.0and liquid medium volume30mL.Choosing the optimal coarse extract method from ammonium sulfate precipitation,hydrochloric acid precipitation,ethanol precipitation and extraction by organic solvent,and the best one was80%ammonium sulfate precipitation.Using Sephadex G-25and DEAE Sepharose Fast Flow ion-exchange column chromatography to isolating and purificating antimicrobial substance,then detecting it’s molecular weight by SDS-PAGE and Tricine-SDS-PAGE,and found that SDS-PAGE showed no protein bands,Tricine-SDS-PAGE showed one clear protein band,and the molecular weight was about5.8KDa.Using LC-MS to comparing the amino acid sequence of antimicrobial substance with other protein,found that the similarity was not enough to judged they are the same material,the highest similarity is just17.61%,so speculated it could be a kind of new material which had not been reported.The main ingredient of antimicrobial substance is protein detected by infrared wavelength scanning,and also found that the main groups of the protein were:N-H、C-H、-C=C=C-、C-N.The biological characteristics of the antimicrobial substance studied from several aspects.The results showed that,the antimicrobial substance had good stability about temperature、pH and organic solvent,while the antimicrobial substance could be degradated by papain partly,it still had some antimicrobial activity.Both the ninhydrin reaction and biuret reaction results showed positive.In addition,the antimicrobial substance has inhibitory effect on many kinds of pathogenic fungi;the MIC is20μg/mL;the antifungal material contained in1mL fermentingliquor as same as1.5mg potassium sorbate.The result of inhibition of Aspergillus flavus on peanuts showed that,different contentrations of antimicrobial active substances could inhibited the growth of Aspergillus flavus obviously,along with the increase in protein content, the growth of Aspergillus flavus reduced gradually,when the contentration of protein reached80μg/mL,almost can’t see the growth of Aspergillus flavus on peanuts by eyes.