Mechanisim Of The Difference Of Aflatoxin B₁ Production In Different Feed Ingredients By Aspergillus Flavus

There was a different AFB₁ contamination in various feed ingredients.The aim of the study was to better understand the potential factors affecting aflatoxin B1?AFB₁?accumulation varies in different feed ingredients.The nutrient composition and contents of feed ingredients were detected,additionally,according to the nutrient content of the feed ingredients,the effects of nutrients on AFB₁ production;mycelial growth and AFB₁ relative m RNA/proteins expression were examined to identify the nutrients responsible for the different AFB₁ contamination in feed ingredients.And these results will provide new concepts for antifungal methods to protect feed from AFB₁ contamination.The main research contents and results are as follows: 1.Effects of oils on A.flavus growth,AFB₁ production and its possible mechanismFirst,the experiment was conducted to study the AFB₁ accumulation in different feed ingredients and effects of different oils in various materials on AFB₁ production by adding A.flavus spores in different feed ingredients and adding four oils?corn oil,soybean oil,peanut oil and wheat germ oil?into different defatted materials and inoculated with A.flavus spore solution.Next to further study the effects of four oils on A.flavus growth and AFB₁ production,and to find its possible mechanism by using Real time-PCR and i TRAQ technology.1)The results showed that the defatted feed ingredients were slightly contaminated by A.flavus and had low levels of AFB₁ accumulation.However,AFB₁ accumulation sharply increased with the addition of 10 % corn oil.The concentrations of AFB₁ in full-fat substrates were also higher than in the defatted substrates.Peanut oil significantly promoted AFB₁ production by A.flavus,while wheat germ oil had relatively weak effect.However,when adding the same oil in different materials,the contents of AFB₁ were various.These results suggested that the oil content might be one of the main causes of aflatoxin B1?AFB₁?accumulation varies in different feed ingredients.In addition,although lipids are one of the most important factors supporting AFB₁ syntheses,other nutrients in substrate also play key roles.2)With the increase of the amount of oils,AFB₁ production also increased,when the amount of oil was more than 1.0%,peanut oil significantly affected?P<0.05?AFB₁ production and increased 5.5-91.3 times,followed by corn oil > soybean oil> wheat germ oil.Oils also could promote the growth of A.flavus colonies.With the increase of the amount of oils,A.flavus colony diameter increased significantly?P<0.05?,and peanut oil had strongest effect,but there was no obvious difference between the four oils.Four kinds of oils had a certain extent effect on A.flavus spore,and the inhibition of wheat germ oil and soybean oil was the most obvious.Peanut oil promoted A.flavus spores germination of A.flavus most obviously?P<0.05?,but soybean oil had less influence?P?0.05?.3)A total of 14 key genes?afl R,afl S,afl C,afl D,afl E,afl F,afl J,afl K,afl L,afl M,afl O,afl P,afl Q and afl U?associated with the AF synthesis were detected in the study.Four oils could significantly increase the expression of afl R and afl S,and the effect of peanut oil was the most significant,followed by wheat germ oil> corn oil> soybean oil.At the same time,the four oils had a great influence on the front genes of AF synthesis pathway,and afl C and afl D significantly increased in four groups?P<0.05?.In corn oil and peanut oil groups,gene afl O,afl Q and afl U significantly up-regulated?P <0.05?,while no significant differences in the other two groups.The application of i TRAQ technology found that compared to the control group?CON?,the amount of different proteins in peanut oil group?P?,corn oil group?C?,soybean oil group?S?and wheat germ oil group?W?were 298,236,231 and 168,respectively.And 179,88,123 and 86 proteins were up-regulated,respectively;proteins 119,148,108 and 82 proteins were down-regulated,respectively.Peanut oil had he most obviously effect on related proteins of AF synthesis,a total of seven proteins,including 6 proteins?Afl G,Afl K,Afl L,Afl O,Afl P and Afl V?significantly increased?P<0.05?,but protein Afl B significantly down-regulated?P<0.05?,while the effect of soybean oil was minimal.Proteins Afl G,Afl O and Afl P in four groups were significantly up-regulated.2.Effects of unsaturated fatty acids on A.flavus growth and AFB₁ productionFirstly,this trial was conducted to study the effects of four kinds of unsaturated fatty acids?oleic acid,linoleic acid,linolenic acid and arachidonic acid?on AFB₁ production and A.flavus growth in medium,and the content of unsaturated fatty acids was 0.005 %-6.0 %.Secondly,according to the unsaturated fatty acids proportion of the oils,mixed fatty acids were prepared to study the influence of fatty acid ratio on AFB₁ production and A.flavus growth.1)With the increase amount of oleic acid,linoleic acid and arachidonic acid,AFB₁ production also increased,but linolenic acid significantly inhibited the synthesis of AFB₁.Four fatty acids also promoted the growth of A.flavus colonies,and the effect of linolenic acid was the most obvious.Linolenic acid can promote sporulation when the amount was less than 0.5 %,while the other three fatty acids had little influence on spores production.Linoleic and linolenic acids promoted A.flavus spore germination significantly?P<0.05?,but arachidonic acid obviously inhibited the germination of spores?P<0.05?.0.05% and 0.2% oleic acid promoted the spores germination,when the amount of oleic acid further increased,the spores germination were inhibited significantly?P <0.05?.2)0.5% and 2.0% mixture as peanut oil and corn oil treatments can promote AFB₁ synthesis,while 2.0% and 8.0% mixture as soybean oil treatment inhibited the AFB₁ production.Wheat germ oil ratio treatment significantly inhibited AFB₁ synthesis and promoted A.flavus growth,and peanut oil ratio treatment had no significant effect on the growth of A.flavus colonies.Four mixed fatty acids significantly inhibited A.flavus spore production?P<0.05?,when the amount reaches 8.0%.3.Effects of four kinds of nutrients on A.flavus growth,AFB₁ synthesis and its possible mechanismThe content of starch,soluble sugars,amino acids and trace elements in different feed ingredients were detected in the study.The effects of these nutrients on AFB₁ production,A.flavus growth and key genes expression?afl R,afl S,afl D,afl M and afl P?in AF synthesis were studied based on the components of the nutrients in feed ingredients.1)Effects of starch,soluble sugars.The results showed that starch can not promote AFB₁ production.Low concentrations of soluble sugars could not promote AFB₁ biosynthesis;however,when the soluble sugar concentration reached 3.0% and 6.0%,the soluble sugars promoted AFB₁ production sharply,especially glucose,sucrose,stachyose and maltose?P<0.05?.Raffinose exhibited the weakest enhancement.The dose relationship between the mycelial growth and soluble sugars revealed that higher concentrations of soluble sugars resulted in improved mycelial growth.The growth promoting effect of stachyose was the most obvious.Compared to 1.0 % sucrose,glucose,maltose and stachyose,3.0% soluble sugars significantly promoted afl S,afl D,afl M and afl P gene expression,and 3.0 % glucose?P<0.01?and stachyose?P<0.001?promoted afl R expression.2)Effects of amino acids.As the amount of amino acids increased,AFB₁ synthesis decreased.However,0.5 % glutamic acid,aspartic acid,and glycine significantly stimulated AFB₁ production?P<0.05?.Glutamic acid,aspartic acid,arginine,and alanine stimulated mycelial growth as their concentrations increased.Aspartic acid,glutamic acid and arginine were varying degrees to promote these five key genes expression.3)Effects of trace elements.Only zinc significantly promoted AFB₁ biosynthesis;the other three trace elements significantly inhibited AFB₁ production?P<0.05?.All trace elements,except copper,can promote A.flavus mycelial growth to varying degrees.In 3 days,zinc can significantly increase the expression of the 5 key genes?P<0.001?and on 5 days,copper inhibited the five key genes expression significantly.In conclusion,1)oil is an important factor affecting AFB₁ production,and also affects A.flavus growth.And peanut oil promoted AFB₁ production and A.flavus growth significantly.Oils stimulated AFB₁ synthesis by promoting A.flavus growth and increased the key genes and proteins expression of AF biosynthetic pathway.2)Unsaturated fatty acids had a certain impact on AFB₁ production and A.flavus growth.There was a great relationship between the kinds and proportions of unsaturated fatty acids in oils and the promotion of oils on AFB₁ production and A.flavus growth.3)Aspartic acid,glutamic acid,arginine,sucrose,glucose,maltose,stachyose and zinc can significantly stimulate AFB₁ production;while copper,iron and manganese significantly inhibited AFB₁ synthesis.Amino acids,soluble sugar and trace elements?iron,zinc,and manganese?could promote A.flavus growth,and the effect of stachyose was the most significant.These nutrients promote the synthesis of AFB₁ possibly by up-regulating genes expression.4)AFB₁ contamination various in different feed ingredients was the result of combine effects of various components.And oils,soluble sugars,amino acids,trace elements affected AFB₁ production by A.flavus may through regulating A.flavus growth and the expression of key genes and proteins.