CN103509723B - Aspergillus flavus producing no aflatoxin and application thereof - Google Patents

Aspergillus flavus producing no aflatoxin and application thereof Download PDF

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CN103509723B
CN103509723B CN201310445854.6A CN201310445854A CN103509723B CN 103509723 B CN103509723 B CN 103509723B CN 201310445854 A CN201310445854 A CN 201310445854A CN 103509723 B CN103509723 B CN 103509723B
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flavus
aflatoxin
aspergillus flavus
poison
producing
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CN103509723A (en
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刘阳
魏丹丹
周露
张初署
邢福国
赵月菊
王龑
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Institute of Food Science and Technology of CAAS
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Abstract

The invention discloses an aspergillus flavus strain producing no aflatoxin. The aspergillus flavus strain producing no aflatoxin, provided by the invention, is specifically aspergillus flavus GZ-17; the preservation number of the aspergillus flavus strain in the general microbiology center of the China Committee for Culture Collection of Microorganisms is CGMCC No.8050. Experimental results show that the aspergillus flavus strain GZ-17 provided by the invention plays a role in restraining the poison production of the aspergillus flavus strain producing the aflatoxin; when the concentration ratio of the strain to a toxic producing fungus spore is 105: 105, the poison production restrained ratio of bacterium producing no poison to the bacterium producing poison is nearly 96%. The strain has important significance for restraining the infection of the aspergillus flavus producing poison to agricultural products and reducing the aflatoxin pollution in the agricultural products.

Description

Aspergillus flavus and the application thereof of aflatoxin are not produced in one strain
Technical field
The invention belongs to agricultural product security field, relate to the aspergillus flavus strain that aflatoxin is not produced in a strain, and this bacterial strain suppress flavus produce malicious in application.
Background technology
Aflatoxin (Aflatoxin, AFT) one class, primarily of mycetogenetic secondary metabolites such as flavus (Aspergillus flavus) and Aspergillus parasiticuses (Aspergillus parasiticus), now isolates 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.In China, aflatoxin produces primarily of flavus.Aflatoxin has carcinogenic, teratogenesis, causes the effect of cell mutation, is regarded as one-level carcinogens, has a strong impact on human health, wherein AFB by the World Health Organization (WHO) 1toxicity the strongest, its toxicity is 10 times of potassium cyanide, 68 times of arsenic.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 the invention is the aspergillus flavus strain that aflatoxin is not produced in a strain, and this bacterial strain suppress flavus produce malicious in application.
Aspergillus flavus strain provided by the invention is specially flavus (Aspergillus flavus) GZ-17.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 21st, 2013 and (is called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC No.8050.
Described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 lacks 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, aflatoxin synthetic gene ver-1, aflatoxin synthetic gene verA, therefore, this bacterial strain can not synthesize aflatoxin.Wherein, these two genes of ver-1 and verA, they are the necessary genes of aflatoxin synthesis, are 4 kinds of aflatoxin (B 1, B 2, G 1, G 2) two genes of the final most critical formed.
The application that described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 is suppressing the flavus producing aflatoxin to produce in poison also belongs to protection scope of the present invention.
For the microbial inoculum suppressing the flavus producing aflatoxin to produce poison, its activeconstituents is described flavus (Aspergillus flavus) GZ-17CGMCC No.8050.
In above-mentioned application or microbial inoculum, GZ-17CGMCC No.8050 can be conidium or/and mycelium for described flavus (Aspergillus flavus).
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 water content 20%(mass percentage) peanut.
In above-mentioned application, produce in the process of poison suppressing the flavus of described product aflatoxin, the spore number of described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 and the flavus of described product aflatoxin compares for 1:10 to 10:1(is as 1:1-10:1, concrete as 1:10 or 1:1 or 10:1).
In one embodiment of the invention, suppressing the flavus of described product aflatoxin to produce in the process of poison, the spore of described flavus (Aspergillus flavus) GZ-17CGMCC No.8050, the spore of flavus of described product aflatoxin and the proportioning of described peanut are (5 × 10 3~ 5 × 10 5) individual spore: 5 × 10 4individual spore: 5g peanut.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".
The application of described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 in the described microbial inoculum of preparation also belongs to protection scope of the present invention.
Described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 is producing conidium or/and the application in mycelium also belongs to protection scope of the present invention.
The colonial morphology of aspergillus flavus strain GZ-17 of the present invention is similar to common flavus, but does not synthesize aflatoxin; Its not toxogenic mechanism is: disappearance appears in the toxin synthetic gene of this bacterial strain from gene aflT to synthetic gene verA, the gene between verA gene to last toxin synthetic gene hypA is complete.
Experiment proves, aspergillus flavus strain GZ-17 of the present invention produces poison to the aspergillus flavus strain producing aflatoxin restraining effect, is 10 at this bacterial strain and the spore concentration ratio of toxigenic bacterium 5: 10 5time, this not the suppression of toxigenic bacterium to toxigenic bacterium produce malicious rate and reach nearly 96%.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: (Aspergillus flavus)
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 in preservation center: CGMCC No.8050
Accompanying drawing explanation
Fig. 1 is AFB 1, B 2, G 1, G 2the UPLC collection of illustrative plates of hybrid standard product (LB96951).Wherein, 1 is AFG in aflatoxin standard substance 2, retention time is 1.273min; 2 is AFG in aflatoxin standard substance 1, retention time is 1.483min; 3 is AFB in aflatoxin standard substance 2, retention time is 1.720min; 4 is AFB in aflatoxin standard substance 1, retention time is 2.057min.
Fig. 2 is the UPLC collection of illustrative plates of bacterial strain GZ-17.
Fig. 3 is aspergillus flavus strain GZ-17 toxin synthetic gene disappearance schematic diagram.
Fig. 4 is the flat-plate bacterial colony form of aspergillus flavus strain GZ-17.
Fig. 5 is the competition bacterium colony figure of aspergillus flavus strain GZ-17 to the Aspergillus flavus GD-1 of product aflatoxin.Wherein, A is GZ-17:GD-1 (10 4: 10 5); B is GZ-17:GD-1 (10 5: 10 5); C is GZ-17:GD-1 (10 6: 10 5).
Fig. 6 is the UPLC collection of illustrative plates of aflatoxin B1 standard substance.Wherein, 1 AFB is 1standard substance.
Fig. 7 is the UPLC collection of illustrative plates of the Aspergillus flavus GD-1 producing separately aflatoxin.Wherein, elution peak for the purpose of 1.
Fig. 8 is GZ-17:GD-1 (10 6: 10 5) UPLC collection of illustrative plates.Wherein, elution peak for the purpose of 1.
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 " Chu-Shu Zhang; Fu-Guo Xing, JonathanNimal Selvaraj et al.The effectiveness of ISSR profiling for studying genetic diversity ofAspergillus flvcus from peanut-cropped soils in China.Biochemical Systematics andEcology50 (213): 147-153 " civilian.
AFB 1, B 2, G 1, G 2hybrid standard product (LB96951): be purchased from SUPELCO company.
AFB 1standard substance (LB88848): be purchased from SUPELCO company.
AFB 1elisa euzymelinked immunosorbent assay (ELISA) test kit (HEM0396/HEM0348): be purchased from Beijing Huaan Magnech Bio-Tech Co., Ltd..Test kit carries AFB 1enzyme mark thing, AFB 1the related reagents such as anti-reagent, substrate solution A liquid, substrate solution B liquid.
Embodiment 1, the collection of aspergillus flavus strain GZ-17, separation, qualification
One, from peanut cultivation soil, aspergillus flavus strain GZ-17 is separated
Aspergillus flavus strain is separated with DG18 substratum from Guangdong peanut planting 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 GZ-17 by a strain bacterium.
Two, the qualification of bacterial strain GZ-17
1, Morphological Identification
The bacterial strain GZ-17 that picking is stored on MEA substratum (fills a prescription: peptone 10.0g/L in AFPA substratum, 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 bacterial strain GZ-17 by fungi calcium, P., Santos, C., Venancio, A., Lima, N., 2011.Species identification of Aspergillus section Flavi isolatesfrom Portuguese almonds using phenotypic, including MALDI-TOF ICMS, and molecularapproaches.J Appl Microbiol111877-892).Flavus genome calmodulin pcr amplification primer used is that 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 bacterial strain GZ-17 is as shown in sequence in sequence table 1.Submitted to by the sequencing result of the Calmodulin gene of bacterial strain GZ-17 on upper NCBI and compare, find, the homology of itself and Aspergillus flavus NRRL3357 and NRRL21882 is 99%.
Through above Morphological Identification and Molecular Identification, known bacterial strain GZ-17 is flavus (Aspergillus flavus).
3, utilize ultra-performance liquid chromatography to detect bacterial strain GZ-17 and whether produce aflatoxin
(1) testing sample preparation
Bacterial strain GZ-17 is inoculated on MEA slant tube substratum, cultivates 5 days, 5ml stroke-physiological saline solution is added MEA slant tube substratum for 28 DEG C, rinse, obtain bacteria suspension.Bacteria suspension is added in the test tube of 50ml, then adds the nutrient solution (150g sucrose/L, 20g yeast extract/L, 10g soy peptone/L, pH5.9) of 10ml, cultivate 5 days for 30 DEG C.By liquid fermentation liquid filter paper filtering, get 1ml filtrate and join in immune affinity column (VICAM, G1010) and (make aflatoxin be suspended on pillar, thus effectively remove impurity), after drain, with distilled water or deionized water wash 2 times, each 10mL, flow velocity 2 ~ 3d/s; After drain, loading 1mL methyl alcohol, with sample bottle graft elutriant, after wash-out, liquid is testing sample, detects for UPLC.
(2) ultra-performance liquid chromatography detects testing sample
Get AFB 1, B 2, G 1, G 2hybrid standard product (LB96951), are mixed with solution with methyl alcohol, then get testing sample prepared by step (1), carry out Ultra Performance Liquid Chromatography detection respectively according to following condition.See whether the color atlas of testing sample has chromatographic peak to occur at the retention time place identical with aflatoxin standard substance.
Wherein, the condition of Ultra Performance Liquid Chromatography is as follows: chromatographic column: C18 chromatographic column (50mm × 2.1mm, 1.7 μm); Detector: fluorimetric detector ACQUITY type, excitation wavelength 365nm, emission wavelength: 440nm; Column temperature: 40 DEG C; Moving phase: methyl alcohol: water (45:55, volume ratio), is aided with methyl alcohol (the pure methyl alcohol of chromatographic grade) for cleaning; Sample size: 10 μ L; Flow velocity: 0.2mL/min.
AFB 1, B 2, G 1, G 2the UPLC collection of illustrative plates of hybrid standard product (LB96951) as shown in Figure 1, as can be seen from Figure 1, AFB in aflatoxin standard substance 1retention time be 2.057min, AFB 2retention time be 1.720min, AFG 1retention time be 1.483min, AFG 2retention time be 1.273min.The UPLC collection of illustrative plates of bacterial strain GZ-17 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, bacterial strain GZ-17 does not produce aflatoxin.
By above qualification result, the bacterial strain GZ-17 of determining step two gained is the flavus (Aspergillus flavus) not producing aflatoxin, and preservation has been carried out to it, 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: 2013 8 years 21 days, preserving number: CGMCC NO.8050.
Aflatoxin synthetic gene disappearance qualification in embodiment 2, flavus (Aspergillus flavus) GZ-17CGMCC No.8050
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-Kuang Chang(Chang, P.K., Horn, B.W.and Dorner, J.W. (2005) Sequencebreakpoints in the aflatoxin biosynthesis gene cluster and flanking regions innonaflatoxigenic Aspergillus flavus isolates.Fungal Genet Bio42, 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 (Aspergillus flavus) GZ-17CGMCC No.8050, 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 L DNA profiling, each 1 μ L, the Gotag(Gotag colorlessmaster mix of upstream and downstream primer (10 μMs), 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, there is disappearance (wherein three putative protein genes hypC, hypD, hypE exist) in flavus (Aspergillus flavus) the GZ-17CGMCC No.8050 of embodiment 1 gained, the gene between avnA gene to last toxin synthetic gene hypA is complete (as shown in Figure 3) from gene aflT to synthetic gene verA.Specifically, described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 lacks 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, aflatoxin synthetic gene ver-1, aflatoxin synthetic gene verA, therefore, this bacterial strain can not synthesize aflatoxin.Wherein, these two genes of ver-1 and verA, they are the necessary genes of aflatoxin synthesis, are 4 kinds of aflatoxin (B 1, B 2, G 1, G 2) two genes of the final most critical formed.
Embodiment 3, flavus (Aspergillus flavus) GZ-17CGMCC No.8050 on PDA substratum to the restraining effect of Aspergillus flavus of producing aflatoxin
One, experimental technique
Preparing spore concentration is respectively 10 4, 10 5, 10 6individual/ml flavus (Aspergillus flavus) GZ-17CGMCCNo.8050 and 10 5individual/ml produces the Aspergillus flavus GD-1 pityrosporion ovale suspension of aflatoxin, be that 1:1 mixes with volume ratio by both, PDA substratum (the formula: potato powder 6.0g/L that 30 μ l pityrosporion ovale suspensions join 6cm is got after mixing, glucose 20.0g/L, agar 20.0g/L) in plate, cultivate 10 days at 30 DEG C, observe colonial morphology, and measure the content of wherein aflatoxin with ultra-performance liquid chromatography.
Wherein, with ultra-performance liquid chromatography measure wherein aflatoxin (with AFB 1for example) the method for content as follows:
(1) preparation of testing sample
Cultivate at 30 DEG C after 10 days, PDA substratum is put into high speed Universalpulverizer smash to pieces, substratum after smashing to pieces is put into triangular flask, and in triangular flask, add the methyl alcohol of 20ml, 30min is shaken at a high speed with vibrator, then filter with sterilized filter paper, filtrate measures the content of wherein aflatoxin with ultra-performance liquid chromatography, get 1ml filtrate and join immune affinity column (VICAM, G1010) (aflatoxin is made to be suspended on pillar in, thus effectively remove impurity), after drain, with distilled water or deionized water wash 2 times, each 10mL, flow velocity 2 ~ 3d/s, after drain, loading 1mL methyl alcohol, with sample bottle graft elutriant, after wash-out, liquid is testing sample, for UPLC.
(2) standard curve making
Get AFB 1standard substance (LB88848), with the solution of methyl alcohol preparation series concentration.By the AFB of series concentration 1standard solution carries out Ultra Performance Liquid Chromatography detection according to following condition.With AFB 1the concentration of standard solution is X-coordinate, take peak area as ordinate zou, production standard curve.
Wherein, Ultra Performance Liquid Chromatography condition is as follows: chromatographic column: C18 chromatographic column (50mm × 2.1mm, 1.7 μm); Detector: fluorimetric detector ACQUITY type, excitation wavelength 365nm, emission wavelength: 440nm; Column temperature: 40 DEG C; Moving phase: methyl alcohol: water (45:55, volume ratio), is aided with methyl alcohol (the pure methyl alcohol of chromatographic grade) for cleaning; Sample size: 10 μ L; Flow velocity: 0.2mL/min.
(3) AFB in testing sample 1assay
Testing sample step (1) prepared also carries out Ultra Performance Liquid Chromatography detection according to as above condition, will with AFB 1the peak area of the chromatographic peak that standard substance retention time is consistent substitutes into step (2) gained typical curve, thus calculates the AFB in testing sample 1content, and then AFB in the PDA substratum obtaining cultivating at 30 DEG C unit weight after 10 days 1content.Calculate the not suppression of toxigenic bacterium GZ-17 to toxigenic bacterium GD-1 more further and produce malicious rate.
In experiment, the initial spore concentration 10 of independent GZ-17(is set for each process 5individual/ml) as negative control, the initial spore concentration 10 of independent GD-1(is set for each process 5individual/ml) as positive control, each process do four parallel.
Two, experimental result
1, respectively strain morphology is processed
Aspergillus flavus strain GZ-17 and GD-1, and the colonial morphology that the competition of two bacterial strains is cultivated all significantly is distinguished specifically as shown in Figure 4 and Figure 5.
2, UPLC detected result
AFB 1the UPLC collection of illustrative plates of standard substance as shown in Figure 6, as can be seen from the figure AFB 1the retention time of standard substance is 2.025min.The UPLC collection of illustrative plates of independent GD-1 as shown in Figure 7, as can be seen from the figure, with AFB 1the retention time place that standard substance are identical, Fig. 7 has obvious chromatographic peak.GZ-17:GD-1 (10 6: 10 5) HPLC collection of illustrative plates as shown in Figure 8, compared with chromatographic peak peak area identical with flavus standard substance retention time in figure, Fig. 7 reduces greatly.
On the substratum of different treatment, the content detection result of aflatoxin specifically (independent GZ-17 does not produce poison) as shown in table 2, when GZ-17 and GD-1 spore concentration ratio is 10 6: 10 5time, this not the suppression of toxigenic bacterium GZ-17 to toxigenic bacterium GD-1 produce malicious rate and reach more than 96%.
Table 2 bacterial strain GZ-17 produces the suppression of poison to GD-1
Strain name Produce poison amount (μ g/kg) Suppress to produce poison amount (μ g/kg) Suppress to produce malicious rate %
GD-1（1×10 5） 949.6±152.1 - -
GZ-17:GD-1(10 4:10 5) 528.4±133.0 421.2±133.0 44.36±14.01
GZ-17:GD-1(10 5:10 5) 334.6±57.8 615.0±57.8 64.76±6.09
GZ-17:GD-1(10 6:10 5) 34.3±22.4 915.3±22.4 96.39±2.36
Note: " μ g/kg " expression " aflatoxin μ g/kg PDA substratum "
Embodiment 4, flavus (Aspergillus flavus) GZ-17CGMCC No.8050 in peanut to the restraining effect of Aspergillus flavus of producing aflatoxin
One, experimental technique
Take 5g epidermis and there is no damaged peanut pellets of uniform size (the biodiversity percentage composition of peanut pellets is 9.25%), after carrying out surface sterilization 2min with 75% alcohol, put in the triangular flask of 50ml; Preparing spore concentration is respectively 10 4, 10 5, 10 6individual/ml flavus (Aspergillus flavus) GZ-17CGMCC No.8050 and 10 5individual/ml produces the Aspergillus flavus GD-1 pityrosporion ovale suspension of aflatoxin, be that 1:1 mixes with volume ratio by both, get 1ml pityrosporion ovale suspension after mixing to join in triangular flask and (the biodiversity percentage composition of peanut pellets is adjusted to about 20%), cultivate 14 days at 30 DEG C.Observe colonial morphology, and use AFB 1eLISA euzymelinked immunosorbent assay (ELISA) test kit (HEM0396/HEM0348) measure wherein AFB 1content.Calculate the not suppression of toxigenic bacterium GZ-17 to toxigenic bacterium GD-1 more further and produce malicious rate.
Wherein, wherein AFB is measured by ELISA kit 1the method of content as follows:
(1) preparation of testing sample
After being ground by cultured peanut with high-speed tissue mashing machine, the methyl alcohol adding 20ml mixes, and shake 30min at a high speed with turbula shaker, then filter with sterilized filter paper, filtrate is testing sample, measures for ELISA kit.
(2) mensuration of testing sample
Use AFB 1eLISA euzymelinked immunosorbent assay (ELISA) test kit (HEM0396/HEM0348) testing sample is measured, operate and carry out see test kit specification sheets, specific as follows:
A) AFB 1eLISA euzymelinked immunosorbent assay (ELISA) measure:
1. standard substance/sample is added: add testing sample 50 μ l prepared by aflatoxin B1 standard substance or step (1) in the micropore of correspondence, add AFB 1enzyme mark thing, 50 μ l/ holes, then add AFB 1anti-reagent, 50 μ l/ holes, shake mixing gently, react 30min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
2. wash plate: carefully open cover plate film, dried by liquid in hole, with wash operating solution (providing in test kit) 300 μ l/ hole, fully washing 5 times, every minor tick 30s, pats dry with thieving paper.
3. develop the color: add substrate solution A liquid 50 μ l/ hole, then add substrate solution B liquid 50 μ l/ hole, shake mixing gently, react 15min with in the rearmounted 25 DEG C of light protected environment of cover plate membrane cover plate.
4. measure: add stop buffer (providing in test kit, is 2M sulfuric acid) 50 μ l/ holes, shake mixing gently, setting microplate reader, in 450nm place, measures every hole OD value.
B) AFB 1aFB in the making of typical curve and sample to be tested 1the calculating of content
Test kit provide standard concentration 0,0.1,0.25,0.5,1.5,5ng/mL, with standard substance percentage light absorption ratio (equaling the absorbance of percentage absorbance divided by 0ng/mL of standard substance) for ordinate zou, with AFB 1the logarithm of standard concentration (ng/mL) is X-coordinate, drawing standard curve, and gained typical curve equation is y=-16.858ln (x)+45.892, R 2=0.9986.OD value measured by above-mentioned typical curve equation and sample to be tested thereof calculates AFB in sample to be tested 1content.
In experiment, the initial spore concentration 10 of independent GZ-17(is set for each process 5individual/ml) as negative control, the initial spore concentration 10 of independent GD-1(is set for each process 5individual/ml) as positive control, each process do four parallel.
Two, experimental result
Result shows, and the colonial morphology that aspergillus flavus strain GZ-17 and GD-1 and the competition of two bacterial strains are cultivated all significantly is not distinguished, and the AFB of different treatment 1content but have obvious change, concrete outcome (independent GZ-17 does not produce poison) as shown in table 3, when GZ-17 and GD-1 spore concentration ratio is 10 5: 10 5time, this not the suppression of toxigenic bacterium GZ-17 to toxigenic bacterium GD-1 produce malicious rate nearly 96%, and when GZ-17 and GD-1 spore concentration ratio be 10 6: 10 5time, this not the suppression of toxigenic bacterium produce malicious rate and reach more than 99%.
Table 3 bacterial strain GZ-17 produces the suppression of poison to GD-1
Strain name Produce poison amount (μ g/kg) Suppress to produce poison amount (μ g/kg) Suppress to produce malicious rate %
GD-1（1×10 5） 35464.71±1797.45 - -
GZ-17:GD-1(10 4:10 5) 23603.12±5021.81 11861.59±5021.81 33.45±14.16
GZ-17:GD-1(10 5:10 5) 1449.78±991.14 34014.93±991.14 95.91±2.79
GZ-17:GD-1(10 6:10 5) 139.66±49.48 35325.05±49.48 99.61±0.14
Note: " μ g/kg " expression " aflatoxin μ g/kg peanut "
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 (10)

1. do not produce flavus (Aspergillus flavus) GZ-17 of aflatoxin, it is CGMCC No.8050 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.
2. flavus according to claim 1 (Aspergillus flavus) GZ-17CGMCC No.8050 is suppressing to produce the application in the flavus product poison of aflatoxin.
3. application according to claim 2, is characterized in that: described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 is that conidium is or/and mycelium.
4. application according to claim 2, 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. application according to claim 2, it is characterized in that: suppressing the flavus of described product aflatoxin to produce in the process of poison, described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 is 1:10 to 10:1 with the spore number ratio of the flavus of described product aflatoxin.
6. the microbial inoculum for suppressing the flavus producing aflatoxin to produce poison, its activeconstituents is flavus according to claim 1 (Aspergillus flavus) GZ-17CGMCC No.8050.
7. microbial inoculum according to claim 6, is characterized in that: described flavus (Aspergillus flavus) GZ-17CGMCC No.8050 is that conidium is or/and mycelium.
8. the microbial inoculum according to claim 6 or 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 application of flavus according to claim 1 (Aspergillus flavus) GZ-17CGMCC No.8050 in preparation claim 6-8 in arbitrary described microbial inoculum.
10. flavus described in claim 1 (Aspergillus flavus) GZ-17CGMCC No.8050 is producing conidium or/and application in mycelium.
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