Simultaneous Detection And Trichoderma's Biocontrol On Aspergillus Flavus And Aflatoxins In Grains And Oil

Aflatoxin is the most toxic group among mycotoxins,which can cause acute or chronic damage to livers,and has serious carcinogenic,teratogenetic and mutagenic effects on humans and animals.Aflatoxin is produced mainly by Aspergillus flavus and A.parasiticus,which contaminates on agro-products such as peanuts,maize,wheat,rice,cotton and nuts in worldwide areas,especially on peanuts and maize.A.flavus contamination is widespread in China,whereas A.parasiticus contamination is rare.The contamination of A.flavus and aflatoxin can not only pose a serious threat to the health of humans and animals,but also seriously reduce the nutritional and economic values of agro-products.Undoubtedly,it has been becoming a huge restriction of agro-products quality and safety.In this study,the rapid and simultaneous detection methods and efficient biocontrol technologies based on Trichoderma were developed,which were important for the earlier detection and scientific supervision of the contaminants to ensure the quality and safety of grains and oil.Detection technology is the“eye”for timely detection and effective control of contaminations.Currently,a number of techniques for aflatoxin detection have been developed,such as high performance liquid chromatography,enzyme-linked immunosorbent assay,immunosensor and immunochromatographic assays.However,simultaneous analysis methods especially the simultaneous detection for small molecular contaminants and food-borne microorganisms were scare.Because it is an environmentally friendly,high efficient and safe method,the biological control has been popular in biocontrol of pathogens and toxins in recent years.However,the biological control of A.flavus and aflatoxin is still facing many challenges,such as the lack of efficient agents,the unknown mechanisms of the action and the lack of practical applications.To solve the above problems,some research works were developed.The main contents and conclusions are as follows:1.The RT-PCR method was established for the simultaneous detection of total aflatoxins and zearalenone in grains and oil products.Two phages V_2-5 and V_8#,displaying the anti-idiotypic nanobodies that bind aflatoxin-or zearalenone-specific monoclonal antibodies?1C11 or 2D3?,were used as competitors for corresponding analytes.Firstly,specific DNA sequences encoding anti-idiotypic nanobodies were used to design the primers for PCR amplification.Secondly,through the combination of immunoreaction and real-time PCR,the simultaneous detection of aflatoxins and zearalenone were realized.The results showed that detection limits for total aflatoxins and zearalenone were 0.03 and 0.09ng/mL,respectively.Recoveries of spiked aflatoxins and zearalenone were 80–118%and 76.7–111%,respectively.Finally,the method was successfully used in the simultaneous detection of aflatoxins and zearalenone in maize,rice,wheat and feed.The results were in good agreement with the gold-standard high performance liquid chromatography method.These results indicated that the detection technology of the RT-PCR based on nanobody-phages could provide new methods for the simultaneous detection of contaminants in grains and oil products.2.The RT-PCR method was developed for the simultaneous detection of total aflatoxins and its main fungi in maize.Aflatoxins are a group of highly toxic and carcinogenic small moleculars,which are produced mainly by Aspergillus flavus and A.parasiticus from the section Flavi of Aspergillus.The detection of aflatoxins and its main fungi was carried out depending on the nanobody-phage V_2-5specially recognizing monoclonal antibody 1C11 and aflatoxin-synthesis related gene nor-1?=aflD?,respectively.Based on the taqman principles in RT-PCR,two specific pairs of primers were designed according to the specific gene sequences of V_2-5 and nor-1.Finally,the simultaneous detection of aflatoxins and its main fungi was realized by RT-PCR.The detection limits for aflatoxins and A.flavus were 0.02 ng/mL and 8×10² spores/g,respectively.Naturally contaminated maize samples?n=25?were analyzed for a further validation.The results were in good agreements between the new developed method and the referential methods?high performance liquid chromatography and the conventional plating counts?.The results indicated that the simultaneous detection technology created with aflatoxin and A.flavus as an example,for the first time,has broken through the bottleneck problem for the simultaneous determination of small molecular contaminants and food-borne microorganisms in grains and oil products.3.This study screened Trichoderma strains T60 and T44 with the highest biological efficiency against the growth of and the aflatoxin production by A.flavus,and explored mechanisms of the actions.Through dual-culture assay and metabolic assays,twenty Trichoderma isolates were investigated for their antagonistic potentials and metabolic activities against the growth of and the aflatoxin production by A.flavus.It was found that T.atroviride T32 and T50,T.harzianum ITEM908 and T61,T.polysporum T60 and T.viride T62 grew very rapidly,and inhibited A.flavus growth completely by mycoparasitism and by the capacity to compete with nutritions and living spaces.Metabolites produced by 15 of 20 Trichoderma isolates exhibited metabolic abilities to significantly inhibit A.flavus growth in Czapex Dox Agar in vitro after 3–5 days'growth,while metabolites of T60 could completely inhibit A.flavus growth by 100%.Metabolites produced by 14 of 20 Trichoderma isolates could significantly inhibit aflatoxin B₁ production in peanut-agar in vitro after 3–5 days'growth,while metabolites produced by T44?T.harzianum?are capable of reducing AFB₁ production at rates as high as 85±6%.The results indicated that the Trichoderma could inhibit A.flavus through antagonistic and metabolic activities,while some had very high efficiency on the inhibition which could be potentially used as biological agents.4.This study screened the most efficient strain of T.reesei CGMCC3.5218 on aflatoxins degradation,explored mechanisms and studied practical applications in grains and oil products.Firstly,we compare the capacity of 65 Trichoderma isolates covering 23 species on degradation of 50 ppb AFB₁in liquid culture.T.reesei CGMCC3.5218 could degrade 50 ppb?500 ppb?10 ppm and 15 ppm AFB₁ by100%?95%?88%and 66%,respectively,after 5 days'growth in liquid culture,while it also could degrade 500 ppb AFB₂,AFG₁ and AFG₂ by 86%,88%and 87%,respectively,after 7 days'growth in liquid culture.The assays revealed that the AFB₁ degradation was the effect of extracellular metabolites.The degradation was enzymatic actions and the enzymes were thermostable and non-metal-binding proteins.The culture conditions of CGMCC3.5218 were optimized,the results indicating that the optimum temperatures were 28–37?,and it could grew well and degraded AFB₁ efficiently in pH values of 4.5–8.3 and in various carbon-and nitrogen-containing media.Importantly,the strain can efficiently degrade aflatoxins in peanut cake,maize and feed,with degradation of AFB₁ from 1657?g/kg to a safe level?20?g/kg?in naturally contaminated maize.Hence,T.reesei CGMCC3.5218 could be used as biological agents to degrade aflatoxins in grains and oil products.In summary,this study provided new methods for the rapid detection and efficient control of A.flavus and aflatoxins in grains and oil products.The study has important theoretical significance and practical values for the prevention and control of A.flavus and aflatoxins,which is beneficial for the protection of consumption safety.