CN103937687B - Do not produce flavus hybrid bacterial strain and the application thereof of aflatoxin - Google Patents
Do not produce flavus hybrid bacterial strain and the application thereof of aflatoxin Download PDFInfo
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Abstract
The invention discloses a kind of flavus hybrid bacterial strain not producing aflatoxin.Is this hybrid bacterial strain by flavus (Aspergillus? flavus) AF-8 and flavus (Aspergillus? flavus) AF-15 mixes; Described flavus (Aspergillus? flavus) AF-8 and described flavus (Aspergillus? does is flavus) AF-15, respectively CGMCC at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center? No.8964 and CGMCC? No.8965.Experiment proves, hybrid bacterial strain provided by the present invention produces poison to the aspergillus flavus strain producing aflatoxin restraining effect, and when this two strains bacterium mixes, than good to the inhibition of toxigenic bacterium when being used alone, when not toxigenic bacterium AF-8, AF-15 is 5 × 10 with the spore concentration ratio of toxigenic bacterium GD-1
4: 5 × 10
4: 1 × 10
5time, this does not produce the suppression of malicious hybrid bacterial strain to toxigenic bacterium and produces malicious rate and reach nearly 100%.It is significant that this bacterial strain produces aflatoxin contamination in malicious Aspergillus flavus infection agricultural-food, reduction agricultural-food for suppression.
Description
Technical field
The invention belongs to agricultural product security field, relate to a kind of flavus hybrid bacterial strain not producing aflatoxin, and this bacterial strain suppress flavus produce malicious in application.
Background technology
Aflatoxin (Aflatoxins, AFT) belong to mycotoxins, be by flavus (Aspergillus.flavus), Aspergillus parasiticus (A.parasiticus), collection honeybee aspergillus (A.nonius) and Aspergillus tamarii (A.tamarii) produce there is teratogenesis, carinogenicity, mutagenic secondary metabolite.Usually be present in soil, animals and plants, various nut particularly in peanut and walnut.Now isolate AFB
1, AFB
2, AFG
1, AFG
2, AFM
1, AFM
2deng the aflatoxin of 18 kinds of different structures, wherein the most important thing is AFB
1, AFB
2, AFG
1, AFG
2.Wherein AFB
1toxicity the strongest, its toxicity is 10 times of potassium cyanide, 68 times of arsenic.There is strong carinogenicity, and can the disease of domestic animals be caused, thus produce huge financial loss.Within 1993, aflatoxin delimited as first kind carcinogens by the Agency for Research on Cancer of the World Health Organization (WorldHealthOrganization, WHO).Aflatoxin mainly pollutes peanut and other crops, has a strong impact on the outlet of China's peanut and goods thereof, brings massive losses to the export trade of China.Therefore, effective prevention and control aflatoxin contamination, for ensureing China's food safety and safeguarding that national economic interest is significant.
Wherein, utilize and do not produce malicious flavus or Aspergillus parasiticus to suppress toxigenic bacterium Microflora and to produce the important means that poison amount is prevention and control aflatoxin contamination.Environmental Defense have registered the atoxigenic aspergillus flavus strain of two strains, for preventing and treating cotton and aflatoxin pollution of peanuts, and extensively tries out in the experimental plot in the multiple state of the U.S..But in suppression toxigenic bacterium, have certain scope of application from the not toxigenic bacterium strain of different areas, the not toxigenic bacterium that such as Africa screening obtains can suppress local flavus to produce poison preferably, and the U.S. screen bacterial strain that obtains the malicious flavus of suppression Africa product produce malicious in just there is no good effect.
Summary of the invention
The object of this invention is to provide a kind of flavus hybrid bacterial strain not producing aflatoxin, and this bacterial strain suppress flavus produce malicious in application.
The flavus hybrid bacterial strain not producing aflatoxin provided by the present invention, is specifically mixed by flavus (Aspergillusflavus) AF-8 and flavus (Aspergillusflavus) AF-15;
Described flavus (Aspergillusflavus) AF-8 is CGMCCNo.8964 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Described flavus (Aspergillusflavus) AF-15 is CGMCCNo.8965 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
In described hybrid bacterial strain, the spore count proportioning of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 and described flavus (Aspergillusflavus) AF-15CGMCCNo.8965 specifically can be 1:1.
In addition, described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 also belongs to protection scope of the present invention.
Described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 has large fragment gene to lack, disappearance aflatoxin synthetic gene aflT, aflatoxin synthetic gene hypE, aflatoxin synthetic gene nor-1, aflatoxin synthetic gene hypD, aflatoxin synthetic gene fas-2, aflatoxin synthetic gene aflR, aflatoxin synthetic gene norA, aflatoxin synthetic gene ver-1, aflatoxin synthetic gene hypB, aflatoxin synthetic gene omtB, aflatoxin synthetic gene omtA, aflatoxin synthetic gene vbs, aflatoxin synthetic gene hypA.Therefore, this bacterial strain can not synthesize aflatoxin.
Described flavus (Aspergillusflavus) AF-15CGMCCNo.8965 has large fragment gene to lack, disappearance aflatoxin synthetic gene norB, aflatoxin synthetic gene cypA, aflatoxin synthetic gene aflT, aflatoxin synthetic gene pksA, aflatoxin synthetic gene nor-1, aflatoxin synthetic gene fas-2, aflatoxin synthetic gene fas-1, aflatoxin synthetic gene aflR, aflatoxin synthetic gene aflJ, aflatoxin synthetic gene adhA, aflatoxin synthetic gene estA, aflatoxin synthetic gene norA.Therefore, this bacterial strain can not synthesize aflatoxin.
Described hybrid bacterial strain, or described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, also belong to protection scope of the present invention in the application suppressing the flavus producing aflatoxin to produce in poison.
Present invention also offers a kind of microbial inoculum for suppressing the flavus producing aflatoxin to produce poison.
Microbial inoculum for suppressing the flavus producing aflatoxin to produce poison provided by the present invention, its activeconstituents specifically can be described hybrid bacterial strain, or described flavus (Aspergillusflavus) AF-8CGMCCNo.8964.
In above-mentioned application or microbial inoculum, each flavus (Aspergillusflavus) all can be conidium or/and mycelium.
In above-mentioned application or microbial inoculum, describedly suppress the flavus producing aflatoxin to produce poison to be embodied in: the toxin producing amount of the flavus of described product aflatoxin is reduced, specifically as made the described product poison of flavus in agricultural-food producing aflatoxin measure reduction.
Described agricultural-food can be cereal, oil crops, nut, spice, feedstuff raw material, herbal medicine and fruit etc.In one embodiment of the invention, described agricultural-food are peanut, are specially the peanut of water content 25% (mass percentage).In another embodiment of the present invention, described agricultural-food are corn, are specially the corn of water content 25% (mass percentage).
Suppress the flavus of described product aflatoxin to produce in the process of poison at described hybrid bacterial strain, the spore number of the flavus of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, described flavus (Aspergillusflavus) AF-15CGMCCNo.8965 and described product aflatoxin is than being 1:1:2.
Concrete, in the present invention, suppressing the flavus of described product aflatoxin to produce in the process of poison, the spore of the spore of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, described flavus (Aspergillusflavus) AF-15CGMCCNo.8965, the spore of flavus of described product aflatoxin and the proportioning of described peanut are 5 × 10
4individual spore: 5 × 10
4individual spore: 1 × 10
5individual spore: 10g peanut (or corn).In addition, suppressing the flavus of described product aflatoxin to produce in the process of poison, culture condition is 30 DEG C, cultivates 14 days.
Suppress the flavus of described product aflatoxin to produce in the process of poison at described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, the spore number of the flavus of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 and described product aflatoxin is than being 1:1.
Concrete, in the present invention, suppressing the flavus of described product aflatoxin to produce in the process of poison, the spore of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, the spore of flavus of described product aflatoxin and the proportioning of described peanut are 1 × 10
5individual spore: 1 × 10
5individual spore: 10g peanut (or corn).In addition, suppressing the flavus of described product aflatoxin to produce in the process of poison, culture condition is 30 DEG C, cultivates 14 days.
In above-mentioned application or microbial inoculum, the flavus of described product aflatoxin specifically can be flavus (Aspergillusflavus) GD-1.
In above-mentioned application or microbial inoculum, the described aflatoxin that do not produce is specially and does not at least produce AFB
1, B
2, G
1and G
2.
" poison " in described product poison all above all refers to " aflatoxin ", is " AFB further
1" and/or " AFB
2".
Described hybrid bacterial strain, or described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, the application in the described microbial inoculum of preparation also belongs to protection scope of the present invention.
Described hybrid bacterial strain, or described flavus (Aspergillusflavus) AF-8CGMCCNo.8964, in production conidium or/and the application in mycelium also belongs to protection scope of the present invention.
Described hybrid bacterial strain provided by the present invention, or the colonial morphology of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 is similar to common flavus, but do not synthesize aflatoxin; Its not toxogenic mechanism is: AF-8 lacks the gene on 13 aflatoxin Biosynthetic pathways, and AF-15 lacks the gene on 12 aflatoxin Biosynthetic pathways.
Experiment proves, the hybrid bacterial strain be made up of described flavus (Aspergillusflavus) AF-8CGMCCNo.8964 and described flavus (Aspergillusflavus) AF-15CGMCCNo.8965 provided by the present invention produces poison to the aspergillus flavus strain producing aflatoxin restraining effect, and when this two strains bacterium mixes, than good to the inhibition of toxigenic bacterium when being used alone, when not toxigenic bacterium AF-8, AF-15 is 5 × 10 with the spore concentration ratio of toxigenic bacterium GD-1
4: 5 × 10
4: 1 × 10
5time, this does not produce the suppression of malicious hybrid bacterial strain to toxigenic bacterium and produces malicious rate and reach nearly 100%.It is significant that this bacterial strain produces aflatoxin contamination in malicious Aspergillus flavus infection agricultural-food, reduction agricultural-food for suppression.
Preservation explanation
Strain name: flavus
Latin name: (Aspergillusflavus)
Strain number: AF-8
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on March 25th, 2014
Register on the books numbering: CGMCCNo.8964 at preservation center
Strain name: flavus
Latin name: (Aspergillusflavus)
Strain number: AF-15
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on March 25th, 2014
Register on the books numbering: CGMCCNo.8965 at preservation center
Strain name: flavus
Latin name: (Aspergillusflavus)
Strain number: GZ-17
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on August 21st, 2013
Register on the books numbering: CGMCCNo.8050 at preservation center
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of aflatoxin hybrid standard product.Wherein, 1 is AFG in aflatoxin standard substance
2, retention time is 8.914min; 2 is AFG in aflatoxin standard substance
1, retention time is 10.092min; 3 is AFB in aflatoxin standard substance
2, retention time is 11.879min; 4 is AFB in aflatoxin standard substance
1, retention time is 13.713min.
Fig. 2 is the HPLC collection of illustrative plates of strains A F-8.
Fig. 3 is the HPLC collection of illustrative plates of strains A F-15.
Fig. 4 is aspergillus flavus strain AF-8 and AF-15 toxin synthetic gene disappearance schematic diagram.The gene representation disappearance that open circles is corresponding; Gene representation corresponding to filled circles does not lack.
Fig. 5 is the HPLC collection of illustrative plates of Aspergillus flavus GD-1 on peanut substratum producing separately aflatoxin.Wherein, elution peak for the purpose of 1 and 2.
Fig. 6 is the HPLC collection of illustrative plates of Aspergillus flavus GD-1 in corn culture medium producing separately aflatoxin.Wherein, elution peak for the purpose of 1 and 2.
Fig. 7 is AF-8:AF-15:GD-1 (5 × 10
4: 5 × 10
4: 1 × 10
5) HPLC collection of illustrative plates on peanut substratum.Wherein, elution peak for the purpose of 1.
Fig. 8 is AF-8:AF-15:GD-1 (5 × 10
4: 5 × 10
4: 1 × 10
5) HPLC collection of illustrative plates in corn culture medium.Without object elution peak.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Produce the Aspergillus flavus GD-1 of aflatoxin: be recorded in " administration at the beginning of. Aspergillus flavus distribution in China's four ecotope peanut soil, produce malicious feature and genetic diversity Journal of Sex Research [D]. the Chinese Academy of Agricultural Sciences, 2013. " literary composition.
AFB
1(A6636), B
2(A9887), G
1(A0138), G
2(A0263) Sigma company is purchased from.
The collection of embodiment 1, aspergillus flavus strain AF-8 with AF-15, be separated, qualification
One, from peanut cultivation soil, aspergillus flavus strain AF-8 and AF-15 is separated
AF-8 with AF-15 is separated aspergillus flavus strain with DG18 substratum respectively from Hubei Province and Guangdong Province's peanut cultivation soil.Concrete operations are as follows:
1, the preparation of pedotheque bacteria suspension
Get 10g soil sample, add 90mL0.1% peptone sterilized water (w/v), at room temperature concussion 30min, make 10
-1bacteria suspension; Get 0.5mL10 again
-1bacteria suspension adds 4.5mL0.1% peptone sterilized water, prepares 10
-2dilution bacteria suspension; Prepare 10 as stated above
-3dilution bacteria suspension.
2, the isolation and purification of bacterial strain
Each extent of dilution gets 0.1mL bacterium liquid, be coated on DG18 substratum (formula: casein peptone 5.0g, dextrose anhydrous 10.0g, potassium primary phosphate 1.0g, magnesium sulfate 0.5g, dicloran 0.002g, agar 15.0g, paraxin 0.1g, dissolve with distilled water 1000mL, add the glycerine of 200.0g again, mixing, 121 DEG C of sterilizings.) on, 30 DEG C of dark culturing 5d, each extent of dilution repeats 3 times.Picking is long has the bacterial strain of yellow spore on DG18 substratum, to carry out secondary line separation, until obtain single bacterium colony.The single bacterium colony of picking is in MEA slant tube substratum (formula: containing Fructus Hordei Germinatus leaching powder 30.0g in often liter of substratum, soy peptone 3.0g, agar 15.0g; PH5.8), on, be stored in 4 DEG C after cultivating 3d in 30 DEG C.Wherein will be designated as AF-8 and AF-15 by two strain bacterium.
Two, the qualification of strains A F-8 and AF-15
1, Morphological Identification
Picking is stored in strains A F-8 on MEA substratum and AF-15 in AFPA substratum (formula: peptone 10.0g/L, yeast leaching powder 20.0g/L, ferric ammonium citrate 0.5g/L, chlorine ammonium nitrate 0.002g/L, paraxin 0.1g/L, agar 15.0g/L, pH are 6.3) on, cultivate 3-5d for 30 DEG C, the visible AFPA substratum back side is bright orange.
2, Molecular Identification
Gene order is adjusted to carry out Molecular Identification (Rodrigues to strains A F-8 and AF-15 by fungi calcium, P., Santos, C., Venancio, A., Lima, N., 2011.SpeciesidentificationofAspergillussectionFlaviisola tesfromPortuguesealmondsusingphenotypic, includingMALDI-TOFICMS, andmolecularapproaches.JApplMicrobiol111,877-892).Flavus genome calmodulin pcr amplification primer used is CL1 and CL2A (sequence is as follows).Pcr amplification reaction program is: 94 DEG C of denaturation 5min, 1 circulation; 94 DEG C of sex change 30s, 54 DEG C of annealing 30s, 72 DEG C extend 90s, totally 30 circulations; 72 DEG C finally extend 7min.After amplification, product is stored in 4 DEG C.Product delivers to the order-checking of Shanghai Sheng Gong biotechnology company limited.And on BLASTresearches comparison sequencing result (http://www.ncbi.nlm.nih.gov/).
CL1：5’-GARTWCAAGGAGGCCTTCTC-3’；
CL2A：5’-TTTTTGCATCATGAGTTGGAC-3’。
The sequencing result of the pcr amplification product of strains A F-8 and AF-15 is respectively as shown in sequence in sequence table 1 and sequence 2.Submitted to by the sequencing result of the Calmodulin gene of strains A F-8 and AF-15 on upper NCBI and compare, find, sequence 1 and sequence 2 are 99% with the homology of Aspergillus flavus NRRL3357 and NRRL21882.
Through above Morphological Identification and Molecular Identification, known strains A F-8 and AF-15 is flavus (Aspergillusflavus).
3, utilize high performance liquid chromatography to detect strains A F-8 and AF-15 and whether produce aflatoxin
(1) testing sample preparation
(formula: 20g yeast extract/L, 150g sucrose/L, 15g agar/L) is cultivated 7 days under 30 DEG C of dark conditions aspergillus flavus strain AF-8 and AF-15 to be inoculated into respectively YES substratum.From cultured flat board, picking 3 blocks of agar put into the centrifuge tube of 4ml, add the methyl alcohol of 1ml, shake 60min at a high speed, then filter with sterilized filter paper, after filtrate being diluted, then filter with microfibre filter paper with distillation water with high vibrator; Get 10mL filtrate to join in immune affinity column (Huaan wheat section, HCM0125) (aflatoxin is suspended on pillar, thus effectively removes impurity), liquid is flowed out with the speed of 2-3ml/min; After drain, with distilled water or deionized water wash 2 times, each 10mL, flow velocity 3-4ml/min; After drain, loading 1mL methyl alcohol (chromatographic grade), with sample bottle graft elutriant, flow velocity 1ml/min; After wash-out, liquid is by the membrane filtration with 0.45 μm, and filtrate utilizes HPLC to measure the content of aflatoxin.
(2) high performance liquid chromatography detects testing sample
Get the AFB of Sigma company
1(A6636), B
2(A9887), G
1(A0138), G
2(A0263), prepare flavus hybrid standard product voluntarily, be mixed with solution with hplc grade methanol, make wherein AFB
1final concentration be 10ng/ μ l, AFB
2final concentration be 5ng/ μ l, AFG
1final concentration be 10ng/ μ l, AFG
2final concentration be 5ng/ μ l.Get testing sample prepared by step (1) again, carry out high performance liquid chromatography detection according to following condition respectively.See whether the color atlas of testing sample has chromatographic peak to occur at the retention time place identical with aflatoxin standard substance.
Wherein, high-efficient liquid phase chromatogram condition is: C18 chromatographic column (4.6mm × 150mm, 5 μm); Fluorimetric detector 2475 type, excitation wavelength 360nm, emission wavelength: 440nm; Column temperature: 30 DEG C; Moving phase: with methyl alcohol: water (1:1, V:V) is moving phase; Sample size: 20 μ L; Flow velocity: 1.0mL/min.
The HPLC collection of illustrative plates of aflatoxin hybrid standard product as shown in Figure 1, as can be seen from Figure 1, AFB in aflatoxin standard substance
1retention time be 13.713min, AFB
2retention time be 11.879min, AFG
1retention time be 10.092min, AFG
2retention time be 8.914min.
The HPLC collection of illustrative plates of strains A F-8 as shown in Figure 2, as can be seen from Figure 2, does not all have chromatographic peak to produce at above four retention time places corresponding with aflatoxin standard substance.As can be seen here, strains A F-8 does not produce aflatoxin.
The HPLC collection of illustrative plates of strains A F-15 as shown in Figure 3, as can be seen from Figure 3, does not all have chromatographic peak to produce at above four retention time places corresponding with aflatoxin standard substance.As can be seen here, strains A F-15 does not produce aflatoxin.
By above qualification result, the strains A F-8 of determining step two gained and AF-15 is the flavus (Aspergillusflavus) not producing aflatoxin.And respectively preservation has been carried out to strains A F-8 and AF-15, depositary institution: (address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms's common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101), preservation date is: 2014 3 years 25 days, the preserving number of strains A F-8: CGMCCNO.8964; The preserving number of strains A F-15: CGMCCNO.8965.
Aflatoxin synthetic gene disappearance qualification in embodiment 2, flavus (Aspergillusflavus) AF-8 and AF-15
The present inventor take Genbank accession number as the sequence of the toxin synthetic gene of AY510451 flavus is reference, devise toxin synthesis related gene hypB, hypC, hypD and hypE tetra-pairs of primers, the primer of other genes is with reference to Perng-KuangChang (Chang, P.K., Horn, B.W.andDorner, J.W. (2005) Sequencebreakpointsintheaflatoxinbiosynthesisgenecluster andflankingregionsinnonaflatoxigenicAspergillusflavusiso lates.FungalGenetBio42, 914 – 923.).29 genes to be measured and the primer sequence of correspondence as shown in table 1.
The primer sequence of the different aflatoxin synthetic gene of table 1
Extract the genomic dna of flavus (Aspergillusflavus) AF-8CGMCCNO.8964 and flavus (Aspergillusflavus) AF-15CGMCCNO.8965 respectively, with it for template, carry out Standard PCR reaction with each primer pair in table 1 respectively, each reaction all arranges genomic DNA control and the negative control (using sterilized water as template) of the Aspergillus flavus GD-1 producing aflatoxin.
PCR reaction system: 1 μ LDNA template, upstream and downstream primer (10 μMs) each 1 μ L, Gotag (Gotagcolorlessmastermix, Promega company, M7133) 10 μ L, sterilized water complements to 20 μ L.
Reaction conditions: 95 DEG C of denaturation 2min, 94 DEG C of sex change 30s, each group primer 55 DEG C annealing 30s, 72 DEG C extend 1min30s, carry out 30 circulations, and last 72 DEG C extend 7min.
The PCR primer sepharose of 1.0% after electrophoresis, is taken pictures with gel imaging system in 1 × TAE damping fluid.
Result shows, verified by the repeatedly PCR of group primer each in table 1, flavus (Aspergillusflavus) AF-8CGMCCNO.8964 of embodiment 1 gained lacks aflatoxin synthetic gene aflT, aflatoxin synthetic gene hypE, aflatoxin synthetic gene nor-1, aflatoxin synthetic gene hypD, aflatoxin synthetic gene fas-2, aflatoxin synthetic gene aflR, aflatoxin synthetic gene norA, aflatoxin synthetic gene ver-1, aflatoxin synthetic gene hypB, aflatoxin synthetic gene omtB, aflatoxin synthetic gene omtA, aflatoxin synthetic gene vbs, aflatoxin synthetic gene hypA.Therefore, this bacterial strain can not synthesize aflatoxin.
Flavus (Aspergillusflavus) AF-15CGMCCNO.8965 lacks aflatoxin synthetic gene norB, aflatoxin synthetic gene cypA, aflatoxin synthetic gene aflT, aflatoxin synthetic gene pksA, aflatoxin synthetic gene nor-1, aflatoxin synthetic gene fas-2, aflatoxin synthetic gene fas-1, aflatoxin synthetic gene aflR, aflatoxin synthetic gene aflJ, aflatoxin synthetic gene adhA, aflatoxin synthetic gene estA, aflatoxin synthetic gene norA.Therefore, this bacterial strain can not synthesize aflatoxin.
Aspergillus flavus strain AF-8 and AF-15 toxin synthetic gene disappearance schematic diagram are as shown in Figure 4.
Embodiment 3, flavus (Aspergillusflavus) hybrid bacterial strain " AF-8+AF-15 " in peanut to the restraining effect of Aspergillus flavus of producing aflatoxin
One, experimental technique
(1) preparation of peanut
Select the complete peanut pellets not having breakage, then take 10g peanut of uniform size respectively, carry out surface sterilization 2min with 75% alcohol, put in the triangular flask of sterilized 150ml after blotting with aseptic filter paper; Moisture content in sample is adjusted to 25% (mass percentage), bottle is biological to be cultivated 7 days at 30 DEG C under dark condition in climatic chamber with safety film sealing, observes the effect of surface sterilization.Result shows, and peanut surface, without any the growth of miscellaneous bacteria, will be used for the surface sterilization of sample in follow-up test by this method.
(2) preparation of bacteria suspension
Malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964 will not be produced, do not produce malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, and the bacterial classification producing malicious flavus GD-1 is inoculated on MEA slant tube substratum respectively, cultivate 5 days for 28 DEG C, then spore on substratum is dipped in sterilized water with cotton swab, turbula shaker is utilized to shake evenly, then adjust spore concentration with blood counting chamber, be formulated as follows five kinds of bacteria suspensions:
Bacteria suspension 1: containing 5 × 10 in every mL bacteria suspension
4the individual spore, 5 × 10 not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964
4the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 2: containing 1 × 10 in every mL bacteria suspension
5the individual spore not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 3: containing 1 × 10 in every mL bacteria suspension
5the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 4: only containing 1 × 10 in every mL bacteria suspension
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 5: containing 5 × 10 in every mL bacteria suspension
4the individual spore, 5 × 10 not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964
4the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965.
(3) competition is cultivated
Select the complete peanut pellets not having breakage, then 10g peanut of uniform size is taken respectively, carry out putting in the triangular flask of sterilized 150ml after surface sterilization 2min aseptic filter paper blots with 75% alcohol, in triangular flask, add not toxigenic bacterium and toxigenic bacterium (10 that 1ml now prepares
5: 10
5) pityrosporion ovale suspension as experimental group, obtain 3 experimental group altogether, respectively corresponding bacteria suspension 1, bacteria suspension 2 and bacteria suspension 3.Experiment arranges the toxigenic bacterium (10 adding 1ml and now prepare in triangular flask simultaneously
5) pityrosporion ovale suspension (corresponding bacteria suspension 4) as positive controls; And in triangular flask, add the not toxigenic bacterium (10 that 1ml now prepares
5) pityrosporion ovale suspension (corresponding bacteria suspension 5) as negative control group.After incubation, the moisture content of peanut sample is 25%.Each process do three parallel, 30 DEG C, under dark condition cultivate 14 days.
(4) aflatoxin content measures
Cultured peanut sample to be put in high-pressure sterilizing pot 121 DEG C, under 30min, carry out sterilizing (making flavus inactivation); The peanut of bacterium of having gone out is put into high speed Universalpulverizer smash to pieces, and in triangular flask, add the methanol solution (solvent is water) of 50ml80% (volume fraction) then, 30min is shaken at a high speed with vibrator, then filter with sterilized filter paper, after filtrate being diluted with distillation water, then filter with microfibre filter paper; Get 10mL filtrate to join in immune affinity column (Huaan wheat section, HCM0125) (aflatoxin is suspended on pillar, thus effectively removes impurity), liquid is flowed out with the speed of 2-3ml/min; After drain, with distilled water or deionized water wash 2 times, each 10mL, flow velocity 3-4ml/min; After drain, loading 1mL methyl alcohol (chromatographic grade), with sample bottle graft elutriant, flow velocity 1ml/min; After wash-out, liquid is by the membrane filtration with 0.45 μm, and filtrate utilizes HPLC to measure the content of aflatoxin.
Wherein, high-efficient liquid phase chromatogram condition is with described in embodiment 1 step 23 (2).
Get the AFB of Sigma company
1(A6636), B
2(A9887), G
1(A0138), G
2(A0263), prepare flavus hybrid standard product voluntarily, be mixed with hplc grade methanol the serial solution that each flavus poison all has different concentration known.High performance liquid chromatography detection is carried out according to as above condition.Carry out regression Calculation with the sample size (X, ng/ μ l) of the peak area of the aflatoxin of particular types (Y) to the aflatoxin of corresponding kind, obtain typical curve equation.
The peak area of chromatographic peak identical with the aflatoxin standard substance retention time of particular types in each treatment group is substituted in corresponding typical curve equation, calculates the content of the aflatoxin of corresponding kind in testing sample.
Two, experimental result
1, the HPLC collection of illustrative plates of each treatment group
The Aspergillus flavus GD-1 of the independent product aflatoxin HPLC collection of illustrative plates on peanut substratum as shown in Figure 5.Visible, compared with the HPLC collection of illustrative plates (Fig. 1) of aflatoxin hybrid standard product, toxigenic bacterium poison that GD-1 produces is mainly AFB
1(in Fig. 5 peak 1) and AFB
2(in Fig. 5 peak 2), does not produce AFG substantially
1and G
2.
AF-8:AF-15:GD-1 (5 × 10
4: 5 × 10
4: 1 × 10
5) HPLC collection of illustrative plates on peanut substratum as shown in Figure 7.Compared with the HPLC collection of illustrative plates of the independent Aspergillus flavus GD-1 shown in Fig. 5, only there is AFB
1elution peak (in Fig. 7 peak 1), without the elution peak of other three kinds of aflatoxin; And AFB
1the peak height of elution peak and peak area much smaller than the peak 1 in Fig. 5.
In addition, the HPLC collection of illustrative plates display of negative control group (not producing malicious mixed bacterium " AF-8+AF-15 ", without toxigenic bacterium GD-1), without aflatoxin chromatographic peak.
2, each treatment group produces the calculating of malicious inhibiting rate
The peak area at the peak 1 in HPLC collection of illustrative plates each in step 1 is substituted into AFB
1typical curve equation, calculate AFB in testing sample
1content; By the peak area at the peak 2 in HPLC collection of illustrative plates each in step 1 for people's AFB
2typical curve equation, calculate AFB in testing sample
2content.The total content adding and be namely considered as aflatoxin in testing sample of two kinds of aflatoxin contents.And then calculate product poison amount, the suppression product poison amount of each treatment group and suppress the malicious rate of product.
Result shows, and under P<0.05 level, does not produce malicious mixed bacterium " AF-8+AF-15 " to the inhibition of toxigenic bacterium GD-1 apparently higher than the inhibition of when toxigenic bacterium is not used alone, toxigenic bacterium GD-1 being produced to poison.Compare for (5 × 10 when not producing the spore concentration of malicious mixed bacterium " AF-8+AF-15 " with toxigenic bacterium GD-1
4: 5 × 10
4: 1 × 10
5) time, this does not produce malicious mixed bacterium " AF-8+AF-15 " suppression to toxigenic bacterium GD-1 and produces malicious rate and reach 99.99%, concrete outcome (negative control group does not produce poison) as shown in table 2.And when toxigenic bacterium does not suppress toxigenic bacterium GD-1 separately in two strains, when toxigenic bacterium AF-8 and toxigenic bacterium GD-1 spore concentration ratio are not 10
5: 10
5time, this not the suppression of toxigenic bacterium AF-8 to toxigenic bacterium GD-1 produce malicious rate nearly 97.76%; When toxigenic bacterium AF-15 and toxigenic bacterium GD-1 spore concentration ratio are not 10
5: 10
5time, malicious rate is not produced in the suppression of toxigenic bacterium AF-15 to toxigenic bacterium GD-1 is 78.60% for this.Do not produce malicious mixed bacterium " AF-8+AF-15 " as can be seen from Table 2 than product poison inhibition good (P<0.05) of single bacterium to toxigenic bacterium GD-1, almost completely inhibit the product poison of toxigenic bacterium GD-1.And this does not produce the inhibition of malicious mixed bacterium " AF-8+AF-15 " to toxigenic bacterium GD-1 product poison also produces poison to toxigenic bacterium GD-1 inhibition apparently higher than GZ-17 bacterial strain in patent applied for 201310445854.6.In sum, this does not produce malicious mixed bacterium " AF-8+AF-15 " and produce the malicious inhibition had clearly to toxigenic bacterium GD-1 on peanut substratum.
Table 2 does not produce malicious mixed bacterium " AF-8+AF-15 " and produce the rejection ratio of poison comparatively to toxigenic bacterium GD-1 in peanut
Note: " μ g/g " expression " aflatoxin μ g/g peanut "." suppress to produce malicious rate " in row, lowercases different after data represents difference significant difference in P<0.05 level each other.
Embodiment 4, flavus (Aspergillusflavus) hybrid bacterial strain " AF-8+AF-15 " in corn to the restraining effect of Aspergillus flavus of producing aflatoxin
One, experimental technique
(1) preparation of corn
Selecting complete does not have damaged peanut pellets, then takes 10g corn of uniform size respectively, carries out surface sterilization 2min with 75% alcohol, with aseptic water washing three times, puts in the triangular flask of sterilized 150ml after then blotting with aseptic filter paper; Moisture content in sample is adjusted to 25% (mass percentage), bottle is biological to be cultivated 7 days at 30 DEG C under dark condition in climatic chamber with safety film sealing, observes the effect of surface sterilization.Result shows, and corn surface, without any the growth of miscellaneous bacteria, will be used for the surface sterilization of sample in follow-up test by this method.
(2) preparation of bacteria suspension
Malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964 will not be produced, do not produce malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, do not produce malicious flavus (Aspergillusflavus) GZ-17CGMCCNO.8050 (patent applied for 201310445854.6), and the bacterial classification producing malicious flavus GD-1 is inoculated on MEA slant tube substratum respectively, cultivate 5 days for 28 DEG C, then spore on substratum is dipped in sterilized water with cotton swab, turbula shaker is utilized to shake evenly, then spore concentration is adjusted with blood counting chamber, be formulated as follows six kinds of bacteria suspensions:
Bacteria suspension 1: containing 5 × 10 in every mL bacteria suspension
4the individual spore, 5 × 10 not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964
4the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 2: containing 1 × 10 in every mL bacteria suspension
5the individual spore not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 3: containing 1 × 10 in every mL bacteria suspension
5the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 4: only containing 1 × 10 in every mL bacteria suspension
5the spore of the malicious flavus GD-1 of individual product.
Bacteria suspension 5: containing 5 × 10 in every mL bacteria suspension
4the individual spore, 5 × 10 not producing malicious flavus (Aspergillusflavus) AF-8CGMCCNO.8964
4the individual spore not producing malicious flavus (Aspergillusflavus) AF-15CGMCCNO.8965.
Bacteria suspension 6: containing 1 × 10 in every mL bacteria suspension
5the individual spore not producing malicious flavus (Aspergillusflavus) GZ-17CGMCCNO.8050, and 1 × 10
5the spore of the malicious flavus GD-1 of individual product.
3) competition is cultivated
Select the complete corn particle not having breakage, then 10g corn of uniform size is taken respectively, carry out putting in the triangular flask of sterilized 150ml after surface sterilization 2min aseptic filter paper blots with 75% alcohol, in triangular flask, add not toxigenic bacterium and toxigenic bacterium (10 that 1ml now prepares
5: 10
5) pityrosporion ovale suspension as experimental group, obtain 3 experimental group altogether, respectively corresponding bacteria suspension 1, bacteria suspension 2 and bacteria suspension 3.Experiment arranges the toxigenic bacterium (10 adding 1ml and now prepare in triangular flask simultaneously
5) pityrosporion ovale suspension (corresponding bacteria suspension 4) as positive controls; The not toxigenic bacterium (10 that 1ml now prepares is added in triangular flask
5) pityrosporion ovale suspension (corresponding bacteria suspension 5) as negative control group; And in triangular flask, add the not toxigenic bacterium (10 that 1ml now prepares
5) pityrosporion ovale suspension (corresponding bacteria suspension 6) as existing not toxigenic bacterium strain control group.After incubation, the moisture content of corn sample is 25%.Each process do three parallel, 30 DEG C, under dark condition cultivate 14 days.
(4) aflatoxin content measures
Cultured corn sample to be put in high-pressure sterilizing pot 121 DEG C, under 30min, carry out sterilizing (making flavus inactivation); The corn of bacterium of having gone out is put into high speed Universalpulverizer smash to pieces, and in triangular flask, add the methanol solution (solvent is water) of 50ml80% (volume fraction) then, 30min is shaken at a high speed with vibrator, then filter with sterilized filter paper, after filtrate being diluted with distillation water, then filter with microfibre filter paper; Get 10mL filtrate to join in immune affinity column (Huaan wheat section, HCM0125) (aflatoxin is suspended on pillar, thus effectively removes impurity), liquid is flowed out with the speed of 2-3ml/min; After drain, with distilled water or deionized water wash 2 times, each 10mL, flow velocity 3-4ml/min; After drain, loading 1mL methyl alcohol (chromatographic grade), with sample bottle graft elutriant, flow velocity 1ml/min; After wash-out, liquid is by the membrane filtration with 0.45 μm, and filtrate utilizes HPLC to measure the content of aflatoxin.
Wherein, high-efficient liquid phase chromatogram condition is with described in embodiment 1 step 23 (2).
Get the AFB of Sigma company
1(A6636), B
2(A9887), G
1(A0138), G
2(A0263), prepare flavus hybrid standard product voluntarily, be mixed with hplc grade methanol the serial solution that each flavus poison all has different concentration known.Be mixed with the solution of serial concentration known with hplc grade methanol, carry out high performance liquid chromatography detection according to as above condition.Carry out regression Calculation with the sample size (X, ng/ μ l) of the peak area of the aflatoxin of particular types (Y) to the aflatoxin of corresponding kind, obtain typical curve equation.
The peak area of chromatographic peak identical with the aflatoxin standard substance retention time of particular types in each treatment group is substituted in corresponding typical curve equation, calculates the content of the aflatoxin of corresponding kind in testing sample.
Two, experimental result
1, the HPLC collection of illustrative plates of each treatment group
The Aspergillus flavus GD-1 of the independent product aflatoxin HPLC collection of illustrative plates in corn culture medium as shown in Figure 6.Visible, compared with the HPLC collection of illustrative plates (Fig. 1) of aflatoxin hybrid standard product, toxigenic bacterium poison that GD-1 produces is mainly AFB
1(in Fig. 6 peak 1) and AFB
2(in Fig. 6 peak 2), does not produce AFG substantially
1and G
2.
AF-8:AF-15:GD-1 (5 × 10
4: 5 × 10
4: 1 × 10
5) HPLC collection of illustrative plates as shown in Figure 8.Without the elution peak of any one aflatoxin in figure.
In addition, the HPLC collection of illustrative plates display of negative control group (not producing malicious mixed bacterium " AF-8+AF-15 ", without toxigenic bacterium GD-1), without aflatoxin chromatographic peak.
2, each treatment group produces the calculating of malicious inhibiting rate
The peak area at the peak 1 in HPLC collection of illustrative plates each in step 1 is substituted into AFB
1typical curve equation, calculate AFB in testing sample
1content; By the peak area at the peak 2 in HPLC collection of illustrative plates each in step 1 for people's AFB
2typical curve equation, calculate AFB in testing sample
2content.The total content adding and be namely considered as aflatoxin in testing sample of two kinds of aflatoxin contents.And then calculate product poison amount, the suppression product poison amount of each treatment group and suppress the malicious rate of product.
Result shows, and under P<0.05 level, does not produce malicious mixed bacterium " AF-8+AF-15 " to the inhibition of toxigenic bacterium GD-1 apparently higher than the inhibition of when toxigenic bacterium is not used alone, toxigenic bacterium GD-1 being produced to poison.Compare for (5 × 10 when not producing the spore concentration of malicious mixed bacterium " AF-8+AF-15 " with toxigenic bacterium GD-1
4: 5 × 10
4: 1 × 10
5) time, this does not produce malicious mixed bacterium " AF-8+AF-15 " suppression to toxigenic bacterium GD-1 and produces malicious rate and reach 100%, concrete outcome (negative control does not produce poison) as shown in table 3.And when toxigenic bacterium does not suppress toxigenic bacterium GD-1 separately in two strains, the inhibition of toxigenic bacterium to toxigenic bacterium GD-1 does not reach more than 94.72% yet.When toxigenic bacterium AF-8 and toxigenic bacterium GD-1 spore concentration ratio are not 10
5: 10
5time, this not the suppression of toxigenic bacterium AF-8 to toxigenic bacterium GD-1 produce malicious rate nearly 99.62%; When toxigenic bacterium AF-15 and toxigenic bacterium GD-1 spore concentration ratio are not 10
5: 10
5time, this not the suppression of toxigenic bacterium AF-15 to toxigenic bacterium GD-1 produce malicious rate and reach 94.72%.Do not produce malicious mixed bacterium " AF-8+AF-15 " as can be seen from Table 3 than product poison inhibition good (P<0.05) of single bacterium to toxigenic bacterium, almost completely inhibit the product poison of toxigenic bacterium.And this does not produce the inhibition of malicious mixed bacterium " AF-8+AF-15 " to toxigenic bacterium GD-1 product poison also produces poison to toxigenic bacterium GD-1 inhibition apparently higher than GZ-17 in patent applied for 201310445854.6.In sum, this mixing not toxigenic bacterium in corn culture medium, poison is produced to toxigenic bacterium GD-1 and has inhibition clearly.
Table 3 does not produce malicious mixed bacterium " AF-8+AF-15 " and produce the rejection ratio of poison comparatively to GD-1 in corn
Note: " μ g/g " expression " aflatoxin μ g/g corn " " suppresses to produce malicious rate " in row, and lowercases different after data represents difference significant difference in P<0.05 level each other.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, a lot of distortion can also be had, as used in the processes such as various agricultural cultivation, results, storage, processing.More than this area all distortion also belong to protection scope of the present invention.
Claims (11)
1. do not produce a flavus hybrid bacterial strain for aflatoxin, mixed by flavus (Aspergillusflavus) AF-8 and flavus (Aspergillusflavus) AF-15;
Described flavus (Aspergillusflavus) AF-8 is CGMCCNo.8964 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center;
Described flavus (Aspergillusflavus) AF-15 is CGMCCNo.8965 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. hybrid bacterial strain according to claim 1, is characterized in that: the spore count proportioning of described flavus (Aspergillusflavus) AF-8 and described flavus (Aspergillusflavus) AF-15 is 1:1.
3. the hybrid bacterial strain described in claim 1 or 2 is suppressing to produce the application in the flavus product poison of aflatoxin.
4. application according to claim 3, is characterized in that: the described flavus product poison producing aflatoxin that suppresses is embodied in: the product poison amount of the flavus of described product aflatoxin is reduced.
5. the application according to claim 3 or 4, it is characterized in that: suppress the flavus of described product aflatoxin to produce in the process of poison at described hybrid bacterial strain, the spore number of the flavus of described flavus (Aspergillusflavus) AF-8, described flavus (Aspergillusflavus) AF-15 and described product aflatoxin is than being 1:1:2.
6. the application according to claim 3 or 4, is characterized in that: the flavus of described product aflatoxin is flavus (Aspergillusflavus) GD-1.
7. the microbial inoculum for suppressing the flavus producing aflatoxin to produce poison, its activeconstituents is the hybrid bacterial strain described in claim 1 or 2.
8. microbial inoculum according to claim 7, is characterized in that: the described flavus product poison producing aflatoxin that suppresses is embodied in: the product poison amount of the flavus of described product aflatoxin is reduced.
9. the microbial inoculum according to claim 7 or 8, is characterized in that: the flavus of described product aflatoxin is flavus (Aspergillusflavus) GD-1.
10. the application of the hybrid bacterial strain described in claim 1 or 2 in preparation claim 7-9 in arbitrary described microbial inoculum.
Hybrid bacterial strain described in 11. claims 1 or 2 is producing conidium or/and application in mycelium.
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| CN101363006A (en) * | 2008-09-08 | 2009-02-11 | 浙江大学 | Aspergillus flavus strain without producing aspergillus flavus toxin and uses thereof |
| CN103509723A (en) * | 2013-09-25 | 2014-01-15 | 中国农业科学院农产品加工研究所 | Aspergillus flavus producing no aflatoxin and application thereof |
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| CN103710456A (en) * | 2014-01-03 | 2014-04-09 | 中国农业科学院农产品加工研究所 | Primer for identifying whether bacterial strain of aspergillus flavus generates aflatoxin and application thereof |
| CN103725783A (en) * | 2014-01-03 | 2014-04-16 | 中国农业科学院农产品加工研究所 | Primer for identifying whether aspergillus flavus strain produces aflatoxin or not and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101363006A (en) * | 2008-09-08 | 2009-02-11 | 浙江大学 | Aspergillus flavus strain without producing aspergillus flavus toxin and uses thereof |
| CN103509723A (en) * | 2013-09-25 | 2014-01-15 | 中国农业科学院农产品加工研究所 | Aspergillus flavus producing no aflatoxin and application thereof |
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