CN109913375A - A kind of Aspergillus flavus of not toxin producing, the composition containing it and its application - Google Patents
A kind of Aspergillus flavus of not toxin producing, the composition containing it and its application Download PDFInfo
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Abstract
The present invention relates to a kind of not Aspergillus flavus of toxin producing, the composition containing it and its applications.The Aspergillus flavus of the not toxin producing is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC No.17189.
Description
Technical field
The present invention relates to a kind of Aspergillus flavus of not toxin producing, especially containing its composition and its application.
Background technique
Aflatoxin is that had by one kind of the generations such as Aspergillus flavus, aspergillus parasiticus bacterium, black-koji mould and aspergillus tamarii bacterium
Carcinogenic, teratogenesis, mutagenesis secondary metabolite, mainly pollute peanut, corn, cotton, nut etc..Aflatoxin
Basic structure is two furan nucleus and cumarin, is the similar compound of one group of structure, the aflatoxin having now been found that has 20
Kind or so, it is common and very harmful in food to have aflatoxin B1、B2、G1、G24 kinds.Aflatoxin B1Toxicity is most strong,
It is 10 times of potassium cyanide, 68 times of arsenic.1993 international cancer tissue (IARC) that aflatoxin is determined as level-one is carcinogenic
Substance.Various countries are different to the limit standard of aflatoxin, and China is for AFB in corn, peanut and its productlLimit standard
For no more than 20 μ g/kg.
Aflatoxin is not only very big to human health risk, also brings about great losses to national economy, it is therefore desirable to fast
Fast efficient aflatoxin preventing control method.Aflatoxin to the pollution of grain before harvest, harvest during, storage and add
There is generation in each stage such as work.Therefore, there is the anti-prosecutor of different aflatoxin to each stage of harvest in plantation
Method can be irrigated by beginning sowing in good time in planting process, crop rotation, late season, during harvest in due course harvest, rapid draing, divide
Choosing removes mildew grain, controls the methods of temperature, humidity, ventilation condition in the storage after harvest and reduces aflatoxin
Pollution, but the usual efficiency of these methods it is lower and due to different regions condition limitation be difficult to carry out.
It and mainly include physics, chemistry and biological method, physics side for the minimizing technology of aflatoxin contamination grain
Method mainly includes irradiation method etc., and chemical method mainly includes ammoniation process, alkaline process, oxidizing process etc., and biological wayss are to utilize micro- life
The secondary metabolite of object or the enzyme of secretion decompose and destroy toxin.Wherein, the method for physics and chemistry that there are effects is unstable,
The disadvantages of nutrient component damages are larger and are difficult to large-scale production.And the research of biological method is then lacked to degradation at present
The further investigation of mechanism, and lack the enzyme preparation or bacteria preparation of efficient stable aflatoxin degradation.
It is prevention aflatoxin contamination using the biological prevention and control method that the Aspergillus flavus of not toxin producing carries out antenatal prevention and control
Effective measures are also the current most successful method of prevention and control aflatoxin, this method by introduce the not Aspergillus flavus of toxin producing or
Aspergillus parasiticus controls the microbial flora in soil, makes the bacterial strain of the not toxin producing preferential substitution soil in plant growing process
The bacterial strain of original toxin producing in earth inhibits the growth of the bacterial strain of toxin producing using the bacterial strain of not toxin producing, reduces aspergillus flavus poison
A possibility that element generates, and then reduce aflatoxin contamination；Or it is yellow bent using the degradation of interbiotic adhesive attraction, removal
Mould toxin.
However, the Aspergillus flavus of toxin producing be not colonized and aflatoxin yield is inhibited to directly influence biological prevention and control effect
Fruit.
Summary of the invention
One of present invention provides a kind of Aspergillus flavus (Aspergillus flavus) of not toxin producing, is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.17189.
The two of the present invention provide a kind of composition comprising Aspergillus flavus as described in one of present invention and acceptable
Carrier.
In a specific embodiment, the acceptable carrier includes peanut meal.In addition, may be used also in the composition
Think or including at least one of Soybean Meal, rice and barley etc..
In a specific embodiment, the content of the Aspergillus flavus in the composition is 107To 108cfu/kg。
In a specific embodiment, the water content of the composition is 25wt% to 35wt%.For example, the combination
The water content of object is 30wt%.
In a specific embodiment, the grain diameter of the peanut meal is less than or equal to 1mm.
In a specific embodiment, amount of application of the composition in crop field is 0.01kg/m2To 0.03kg/m2。
In a specific embodiment, amount of application of the composition in crop field is 0.03kg/m2。
The three of the present invention provide a kind of method for preparing the composition as described in the two of the present invention comprising following step
It is rapid: the Aspergillus flavus is mixed with the acceptable carrier.
In a specific embodiment, the Aspergillus flavus is mixed with peanut meal.
In a specific embodiment, after the seed fermentation liquid of the Aspergillus flavus being mixed with the peanut meal, training
It supports 5 to 10 days.
In a specific embodiment, after the seed fermentation liquid of the Aspergillus flavus being mixed with the peanut meal,
It is cultivated 6 to 8 days at 20 to 30 DEG C.
In a specific embodiment, after the seed fermentation liquid of the Aspergillus flavus being mixed with the peanut meal,
It is cultivated 7 days at 25 DEG C.
In a specific embodiment, the Aspergillus flavus is with amount ratio of the peanut meal both when just mixed
103Cfu/kg to 104cfu/kg。
In a specific embodiment, the mass ratio of the peanut meal and the fermentation liquid be 70:1 (≈ 99 × 70%:
1) to 63:10 (90 × 70%:10).
In a specific embodiment, the fermentation liquid ferments in the following way is made:
(1) Aspergillus flavus is inoculated on the solid medium suitable for Aspergillus flavus growth, cultivates 2 at 28 to 32 DEG C
Activation bacterium was obtained to 7 days；
(2) the activation bacterium is inoculated into the seed culture medium suitable for Aspergillus flavus growth shaken cultivation 8 to 24 hours,
Obtain the seed fermentation liquid of the Aspergillus flavus.
In a specific embodiment, the fermentation liquid ferments in the following way is made:
(1) Aspergillus flavus is inoculated on the solid medium, cultivates 3 to 5 days and is activated at 28 to 32 DEG C
Bacterium；
(2) the activation bacterium is inoculated into the seed culture medium shaken cultivation 10 to 15 hours, is obtained described yellow bent
The seed fermentation liquid of mould.
In a specific embodiment, the fermentation liquid ferments in the following way is made:
(1) Aspergillus flavus is inoculated on the solid medium, is cultivated at 30 DEG C and obtains within 4 days activation bacterium；
(2) the activation bacterium is inoculated into shaken cultivation 12 hours in the seed culture medium, obtains the Aspergillus flavus
Seed fermentation liquid；Wherein, seed culture based formulas is as follows: sucrose 50g/L, peptone 10g/L, potassium dihydrogen phosphate 0.2g/L,
Bitter salt 0.2g/L, polysorbate60 15g/L, pH 6.0.Wherein, seed culture medium needs just to can be used after sterilizing.
In a specific embodiment, before mixing the Aspergillus flavus with peanut meal, first to the peanut meal
Middle addition water.
In a specific embodiment, the mass ratio of the preferably described peanut meal and water is 3:7 to 9:1.
In a specific embodiment, the mass ratio of the preferably described peanut meal and water is 3:2 (6:4) to 4:1 (8:2).
In a specific embodiment, the mass ratio of the peanut meal and water is 7:3, and the peanut meal and the water are
Peanut meal and water after sterilizing.
The four of the present invention provide one of according to the present invention described in Aspergillus flavus, the present invention two described in composition or
The composition that method described in any one of three of the present invention is prepared is in the Aspergillus for inhibiting production aflatoxin
(Aspergillus) it is colonized and/or inhibits the application in aflatoxin.
In a specific embodiment, the aspergillus includes Aspergillus flavus (Aspergillus flavus), parasitic song
Mould (Aspergillus parasiticus), black-koji mould (Aspergillus niger), aspergillus tamarii bacterium
(Aspergillus tamarii), aspergillus oryzae (Aspergillus oryzae) and soy sauce koji mould (Aspergillus
At least one of sojae).
In the present invention, term " not toxin producing " refers to that microbial strains itself cannot synthesize aflatoxin, wherein
Aflatoxin include it is existing it has been found that type, have aflatoxin for example including common in food and very harmful
B1、B2、G1、G24 kinds.
In the present invention, term " toxin producing " refers to that microbial strains itself can synthesize aflatoxin, therein
Aflatoxin includes the existing type having found, has aflatoxin for example including common in food and very harmful
B1、B2、G1、G24 kinds.
Beneficial effects of the present invention:
The aspergillus flavus strain of not toxin producing of the invention is strong in field field planting power, can effectively prevent Aflatoxin in Peanut byHigh
Pollution.
Detailed description of the invention
Fig. 1 shows the morphological feature of Aspergillus flavus.
Fig. 2 shows the morphological feature of aspergillus parasiticus bacterium.
Fig. 3 shows the NheI digestion result of aflR promoter pcr amplification product.The result shows that the pcr amplification product does not have
There is NheI restriction enzyme site.
Fig. 4 shows the HincII digestion result of aflR structural gene pcr amplification product.
Fig. 5 shows the PvuII digestion result of aflR structural gene pcr amplification product.
Fig. 6 shows the meoh eluate and aflatoxin hybrid standard product of the aspergillus flavus strain ASAG40 of not toxin producing
HPLC chromatogram.Wherein, four peaks in figure are followed successively by G from left to right2、G1、B2And B14 kinds of aflatoxin are formed by
Peak.
Culture presevation
The fungal strain that the present invention screens is named as ASAG40, which is preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, deposit number is CGMCC No.17189, the deposit date is on March 18th, 2019, preservation
Location are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, postcode: 100101.Its system point
Class is Aspergillus flavus (Aspergillus flavus).
Specific embodiment
Below with reference to embodiment, the invention will be further described, but the exemplary only explanation of the embodiment of the present invention, should
No matter embodiment does not constitute a limitation of the invention under any circumstance.
Unless otherwise instructed, reagent used in the following embodiment is commercially available.
18% glycerin agar basal medium of botran (DG18 culture medium): casein peptone 5g/L, DEXTROSE ANHYDROUS 10g/
L, potassium dihydrogen phosphate 1g/L, chloramphenicol 0.1g/L, magnesium sulfate 0.5g/L, agar powder 15g/L, botran 0.002g/L, glycerol
200g/L, pH value 5.6 ± 0.2.115 DEG C of sterilizing 20min.
Aspergillin agar medium (AFPA culture medium): peptone 10g/L, yeast extract 20g/L, ferric citrate
0.5g/L, chlorine ammonium nitrate 0.002g/L, chloramphenicol 0.1g/L, agar 15g/L, pH value 6.3 ± 0.2.115 DEG C of sterilizing 20min.
Czapek's medium: dipotassium hydrogen phosphate 1g/L, sodium nitrate 3g/L, ferrous sulfate 0.01g/L, magnesium sulfate 0.5g/L, chlorine
Change potassium 0.5g/L, sucrose 30g/L.115 DEG C of sterilizing 20min.
Potato dextrose agar (PDA culture medium): potato 200g/L, glucose 20g/L, agar 20g/L,
PH value 6.5.115 DEG C of sterilizing 20min.
Seed culture medium: sucrose 50g/L, peptone 10g/L, potassium dihydrogen phosphate 0.2g/L, bitter salt 0.2g/L,
Polysorbate60 15g/L, pH 6.0.115 DEG C of sterilizing 20min.
Embodiment 1
The not high frequency zone of the aspergillus flavus strain ASAG40 of toxin producing
(1) separation of bacterial strain: the big Tanaka of peanut from Wuyang city, Henan Province, Zhenyang city, Henan Province and Xiangyang City, Hubei Province adopts
Collect soil and peanut.It prepares soil dilution liquid: 10g pedotheque is added to the 0.1wt% sterile peptone solution that 90mL is housed
500mL conical flask in, 30 DEG C, 220r/min vibrate 30min, take 1mL in equipped with 9mL 0.1wt% peptone sterile water be centrifuged
Guan Zhong, abundant whirlpool mix, and the sample diluting liquid of 1:10 is made, until 10-3, each dilution takes 100 μ L in DG18 culture medium
On, after coating uniformly, inversion is incubated in 30 DEG C of incubators, and each dilution does three parallel, each flat-plate bacterial colonies of close observation
Growing state, peanut sample then directly place it at DG18 culture medium center, 30 DEG C of stationary cultures.It observes bacterium colony and grows feelings
Condition.Wherein, Aspergillus flavus and aspergillus parasiticus bacterium can initially generate the mycelia of white on DG18 culture medium, extend around, until
Third day starts to generate spore, and spore color is in bright yellow, and as time goes by, spore color is gradually dimmed.
(2) picking single colonie: from step (1) resulting plate, sterile water is dipped with oese, by doubtful single colonie
Spore is carefully transferred on DG18 culture medium, is placed in 30 DEG C of inversion culture 4d.Then by the list of resulting doubtful Aspergillus flavus
Bacterium colony, scraping spore are transferred in the glycerine water solution of 20wt%, -20 DEG C of preservations.
(3) Morphological Identification: the single colonie of doubtful Aspergillus flavus or aspergillus parasiticus is inoculated on AFPA culture medium, 30
At DEG C be inverted culture 5 days, observe culture medium back color, in crocus be Aspergillus flavus (such as Fig. 1) or aspergillus parasiticus bacterium (such as
Fig. 2).By the Conidia preservation of the doubtful Aspergillus flavus filtered out or aspergillus parasiticus bacterium in the glycerine water solution of 20wt%, -20 DEG C
Short-term preservation is spare.Wherein one plant of bacterium number is ASAG40.
(4) ASAG40 is transferred on DG18 culture medium, 30 DEG C are cultivated 4 days.After it grows spore, spore is transferred to
In Czapek's medium, 220r/min shaken cultivation 2.5 days, mycelia is obtained using Suction filtration device, then extracted with fungal genomic DNA
Kit (Beijing DingGuo ChangSheng Biology Technology Co., Ltd) extracts genomic DNA.
(5) PCR amplification calcium tune gene order carries out Species estimation to bacterial strain.Wherein, PCR amplification upstream primer CL1 is (such as
Shown in SEQ ID No.1), downstream primer CL2A (as shown in SEQ ID No.2).PCR reaction system is 1 μ L DNA profiling (step
Suddenly (4) genomic DNA, about 4ng), each 1 μ L of 10 μM of upstream and downstream primers,HS (Premix) (TAKARA company)
12.5 μ L add water to 25 μ L.Reaction condition is that 98 DEG C of initial denaturation 5min, 98 DEG C of denaturation 30s, 52 DEG C of annealing 20s, 72 DEG C extend
1min carries out 30 circulations, finally extends 10min.PCR product is sent to Hua Da gene Co., Ltd and is sequenced.(such as by sequencing result
Shown in SEQ ID No.3) it is committed on NCBI and is compared, find the calcium tune gene of ASAG40 bacterial strain and the calcium tune of Aspergillus flavus
Gene identity is 99%.
(6) special for key controlling gene aflR promoter sequence (515bp) design during aspergillus flavus toxin producing
Property primer, upstream primer aflRPF (as shown in SEQ ID No.4) and downstream primer aflRPR (as shown in SEQ ID No.5)；
Specific primer, upstream primer aflRF (as shown in SEQ ID No.6) and downstream are designed for aflR structural gene (798bp)
Primer aflRR (as shown in SEQ ID No.7) carries out PCR, PCR condition using step (4) extracted genomic DNA as template
With reference to step (5), its size of electrophoresis detection.
(7) two kinds of PCR products resulting to step (6) carry out digestion, are carried out using NheI enzyme to aflR promoter sequence
Digestion carries out single endonuclease digestion to aflR structural gene using HincII and PvuII respectively.The aflR promoter PCR of ASAG40 bacterial strain
The NheI digestion result of amplified production is shown in Fig. 3.The HincII digestion knot of the aflR structural gene pcr amplification product of ASAG40 bacterial strain
Fruit sees Fig. 4.The PvuII digestion result of the aflR structural gene pcr amplification product of ASAG40 bacterial strain is shown in Fig. 5.According to Fig. 3 to Fig. 5
Result can determine that ASAG40 is the aspergillus flavus strain of not toxin producing.
(8) the aspergillus flavus strain ASAG40 of the not toxin producing screened is inoculated into PDA culture medium, 30 DEG C are protected from light training
It supports, observes mycelial growth rate and sporiparous ability, ASAG40 speed of growth in PDA culture medium is very fast, and mycelia is after 72h to connect
Kind point is the center of circle, covers with the plate that diameter is 90mm, and bacterium colony back is in yellow-white, wherein after cultivating 48h, plate front is begun to show
The spore of yellow green, until covering with entire plate after culture 84h, with the growth of time, spore density increases, and color is dimmed.
It places it in the glycerol liquor of 20wt%, -20 DEG C of preservations.
(9) the further verifying of ASAG40 bacterial strain not toxin producing characteristic: ASAG40 is inoculated on DG18 culture medium, 30 DEG C
It is inverted culture 4 days, to grow spore on plate, takes spore into 1mL sterile water with oese.Soybean is pulverized, is taken
75g soy meal is added into each conical flask, while preparing ultrapure water for 250mL conical flask, 121 DEG C of sterilizing 20min.After sterilizing,
It puts to room temperature, is added 25mL sterile water into each triangular flask in superclean bench, the mass ratio of soy meal and sterile water is
3:1.After mixing evenly with glass bar, the spore suspension of 1mL is added.It seals with sealing film, is placed in 30 DEG C of stationary culture 30d.
By 121 DEG C of tunning, 20min sterilizes twice, it is ensured that aspergillus flavus strain spore inactivation.Tunning in conical flask is whole
It takes out, is crushed using pulverizer.It takes 5g to crush sample, 1g sodium chloride is added and 100mL extracting solution (70% methanol-water solution) is mixed
It is even.High speed homogenization 3min is filtered with fast qualitative filter paper, collects filtrate.It takes 10mL filtrate that the dilution of 20mL water is added, then uses fento
Tie up filter paper filtering.Take 15ml filtrate that immune affinity column (Beijing Huaan Magnech Bio-Tech Co., Ltd.) is added, to drain
Afterwards, it is washed with deionized 2 times, each 10ml, is eluted with 1ml methanol.Eluent is detected with HPLC.ASAG40 meoh eluate
HPLC map and aflatoxin hybrid standard product (i.e. G1、G2、B1And B2The mixed mark of 4 kinds of aflatoxin) HPLC map
Superposition compares, and as a result sees Fig. 6.The result shows that the extraction sample of ASAG40 does not have G1、G2、B1And B24 kinds of aflatoxin
Chromatographic peak generate.Therefore ASAG40 does not produce aflatoxin.
Embodiment 2
The preparation of ASAG40 microbial inoculum
(1) it activates for the first time: the aspergillus flavus strain ASAG40 of not toxin producing is transferred on DG18 culture medium, 30 DEG C of inversions
Culture 4 days to growing a certain amount of spore.
(2) it activates for second: by the spore inoculating of the Aspergillus flavus of first time activation into seed culture medium, in 220r/
Min shaken cultivation 12h obtains primary seed solution (i.e. seed fermentation liquid), and measuring its concentration by colony counting method is 105cfu/
kg。
(3) peanut oil expression by-product peanut meal is crushed with high speed Universal pulverizer, crosses 1mm sub-sieve, 121 DEG C go out
Then nothing is added with the ratio that the mass ratio of dry peanut meal and water is respectively 3:7,6:4,7:3,8:2 and 9:1 in bacterium 20min
Bacterium water is configured to the peanut meal culture medium of five kinds of water contents.The seed fermentation liquid of the 10g not Aspergillus flavus of toxin producing is turned respectively
It is connected in the peanut meal culture medium of five kinds of water contents of 90g, 28 DEG C of stationary cultures observe the production of spore.The result shows that flower
Spore can be generated when the mass ratio of the raw dregs of rice and water is 3:7 and 9:1 after culture 7 days respectively, the mass ratio of peanut meal and water is 6:4
With when 8:2 respectively culture 6 days after can generate spore；When the mass ratio of peanut meal and water is 7:3, spore can be generated after 4 days.
It should be pointed out that not heat production or quantity of heat production substantially is little since the culture amount in the stage is smaller, therefore, setting is stood
The temperature of culture can be in optimum growth temperature the floating up and down by a small margin of the bacterium.
(4) 1g, 5g, 10g seed fermentation liquid are accessed into peanut meal culture medium respectively (mass ratio of peanut meal and water is 7:3)
In, make final gross mass 100g, therefore the initial concentration of peanut meal culture medium and seed liquor after mixing is respectively 103、5
×103With 104cfu/kg.32 DEG C of stationary cultures, observe the production of spore.The result shows that inoculum concentration be 5g and 1g when according to
It is secondary to generate spore after culture 6 days and 7 days；When inoculum concentration is 10g, strain growth is fastest, can generate spore after 4 days
Son.Similarly, since the culture amount in the stage is smaller, not heat production or quantity of heat production substantially is little, therefore, stationary culture is arranged
Temperature can be in optimum growth temperature the floating up and down by a small margin of the bacterium.
Embodiment 3
The aspergillus flavus strain ASAG40 of toxin producing does not prevent the application of aflatoxin contamination in peanut cultivation
(1) it activates for the first time: the aspergillus flavus strain ASAG40 of not toxin producing is transferred on DG18 culture medium, 30 DEG C of inversions
Culture 4 days to growing a certain amount of spore.
(2) it activates for second: the aspergillus flavus bacterium colony of the first activation being seeded in seed culture medium, is vibrated in 220r/min
12h is cultivated, primary seed solution (i.e. seed fermentation liquid) is obtained, measuring its concentration by colony counting method is 105cfu/kg。
(3) peanut oil expression by-product peanut meal is crushed with high speed Universal pulverizer, crosses 1mm sub-sieve, 121 DEG C go out
Bacterium 20min, then with the mass ratio of dry peanut meal and water be 7:3 amount be added sterile water, i.e., 12.6kg peanut meal and
5.4kg sterile water stirs evenly, and obtains peanut meal culture medium.By final concentration 104The inoculum concentration of cfu/kg is by 2kg not toxin producing
The seed fermentation liquid of Aspergillus flavus is transferred to 25 DEG C stationary culture 7 days in the peanut meal culture medium that 18kg is prepared.It may be noted that
, since the culture amount in the stage is larger, fermentation process is also easy to produce a large amount of heat, thus the initial incubation temperature being arranged compared with
It is low, with the growth of incubation time, in the case where heat production is more than 32 DEG C it should be noted that radiating and cooling.It is measured and is sent out by oven drying method
The water content in product after ferment is 30wt%, and measuring Aspergillus flavus in content wherein by colony counting method is 107cfu/
kg。
(4) plot plantation peanut is chosen respectively in Xiangyang City, Hubei Province.Divide before peanut cultivation into two blocks of peanut soil
Do not apply the peanut meal bacterial manure for spilling the Aspergillus flavus for the not toxin producing that 1kg and 3kg are prepared according to above-mentioned steps (3) uniformly, then into
Row a surname soil and sowing, carry out according to traditional peanut cultivation mode later, while the blank control group that setting is not processed.Test
Plot area is 100m2, three repetitions.
(5) when distinguishing before planting with peanut maturation, according to the close of the aspergillus flavus strain in five point sampling detection soil
Degree and not the aspergillus flavus strain proportion of toxin producing, as shown in table 1.It is real before applying the not aspergillus flavus microbial inoculum of toxin producing
It tests the aspergillus flavus strain quantity in group and blank control group and there was no significant difference for the ratio of the aspergillus flavus strain of toxin producing.
But after applying the not peanut meal bacterial manure of the Aspergillus flavus of toxin producing, the quantity of aspergillus flavus strain be increased significantly, and increase 10
Times or so.The ratio of the aspergillus flavus strain of the not toxin producing of high dose group and low dose group is all remarkably higher than blank control group, high
The ratio of the aspergillus flavus strain of the not toxin producing of dosage group is more up to 99.5%.Illustrate that such microbial inoculum applying mode realizes not
The field planting of the Aspergillus flavus of toxin producing in the soil.
The field planting of the not aspergillus flavus strain of toxin producing of table 1
Yield is calculated to the peanut after harvest, 2 is the results are shown in Table, shows that applying bacterial manure of the invention not influences the production of peanut
Amount.
After peanut after harvest is stored 3 months and 6 months, HPLC method measures the content of Aflatoxin in Peanut byHigh, such as
Shown in table 2, show that the aspergillus flavus strain of not toxin producing of the invention can effectively inhibit aflatoxin in peanut cultivation to generate, and
And having apparent inhibitory effect in storage 6 months, the inhibitory effect of high dose group becomes apparent from, and inhibiting rate is up to 90% or more；
The inhibiting rate of low dose group is also close to 90%.
Different phase Aflatoxin in Peanut byHigh content after table 2 harvests
It should be noted that the above list is only a few specific embodiments of the present invention.It is clear that the invention is not restricted to
Above embodiments, acceptable there are many deformations.Those skilled in the art can directly export from present disclosure
Or all deformations associated, it is considered as protection scope of the present invention.
Sequence table
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Claims (10)
1. a kind of Aspergillus flavus (Aspergillus flavus) of not toxin producing, is preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center, deposit number are CGMCC No.17189.
2. a kind of composition comprising Aspergillus flavus as described in claim 1 and acceptable carrier.
3. composition according to claim 2, which is characterized in that the acceptable carrier includes peanut meal；It is preferred that institute
Stating the content of Aspergillus flavus in the composition is 107To 108cfu/kg；
It is preferred that the grain diameter of the peanut meal is less than or equal to 1mm；
It is preferred that the water content of the composition is 25wt% to 35wt%, the water content of the preferably described composition is 30wt%；
Amount of application of the more preferable composition in crop field is 0.01kg/m2To 0.03kg/m2；The most preferably described composition is big
The amount of application in field is 0.03kg/m2。
4. a kind of method for preparing composition as claimed in claim 2 or claim 3 comprising following steps: by the Aspergillus flavus
It is mixed with the acceptable carrier；It is preferred that the Aspergillus flavus is mixed with the peanut meal.
5. according to the method described in claim 4, it is characterized in that, by the seed fermentation liquid of the Aspergillus flavus and the peanut
After dregs of rice mixing, cultivate 5 to 10 days；It is preferred that being cultivated 6 to 8 days at 20 to 30 DEG C；It is cultivated 7 days more preferably at 25 DEG C.
6. method according to claim 4 or 5, which is characterized in that two when the Aspergillus flavus and the just mixed peanut meal
The amount ratio of person is 103Cfu/kg to 104cfu/kg。
7. method according to claim 5 or 6, which is characterized in that the mass ratio of the peanut meal and the fermentation liquid is
70:1 to 63:10.
8. the method according to any one of claim 5 to 7, which is characterized in that the fermentation liquid is in the following way
Fermentation is made:
(1) Aspergillus flavus is inoculated on the solid medium suitable for Aspergillus flavus growth, cultivates 2 to 7 at 28 to 32 DEG C
It obtains activation bacterium；
(2) the activation bacterium is inoculated into the seed culture medium suitable for Aspergillus flavus growth shaken cultivation 8 to 24 hours, is obtained
The seed fermentation liquid of the Aspergillus flavus；
Preferably,
(1) Aspergillus flavus is inoculated on the solid medium, is cultivated at 30 DEG C and obtains within 4 days activation bacterium；
(2) the activation bacterium is inoculated into shaken cultivation 12 hours in the seed culture medium, obtains the kind of the Aspergillus flavus
Sub- fermentation liquid；Wherein, seed culture based formulas is as follows: sucrose 50g/L, peptone 10g/L, potassium dihydrogen phosphate 0.2g/L, seven water
Close magnesium sulfate 0.2g/L, polysorbate60 15g/L, pH6.0.
9. the method according to any one of claim 4 to 8, which is characterized in that by the Aspergillus flavus and peanut
Before dregs of rice mixing, first it is added water into the peanut meal, the mass ratio of the preferably described peanut meal and water is 3:7 to 9:1；
It is preferred that the mass ratio of the peanut meal and water is 3:2 to 4:1；
It is preferred that the mass ratio of the peanut meal and water is 7:3, the peanut meal and the water are the peanut meal and water after sterilizing.
10. appointing in Aspergillus flavus according to claim 1, composition described in claim 2 or 3 or claim 4 to 9
The composition that method described in meaning one is prepared is in Aspergillus (Aspergillus) field planting for inhibiting production aflatoxin
And/or the application in inhibition aspergillus flavus toxin, the preferably described aspergillus include Aspergillus flavus (Aspergillus flavus), post
Raw Aspergillus (Aspergillus parasiticus), black-koji mould (Aspergillus niger), aspergillus tamarii bacterium
(Aspergillus tamarii), aspergillus oryzae (Aspergillus oryzae) and soy sauce koji mould (Aspergillus
At least one of sojae).
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| CN113846020B (en) * | 2021-06-24 | 2023-06-27 | 中国农业科学院油料作物研究所 | Aspergillus flavus XZCY1805 without toxicity production and application thereof |
| CN115181675A (en) * | 2022-05-06 | 2022-10-14 | 南京思农生物有机肥研究院有限公司 | Trichoderma guizhou growth promoting companion aspergillus flavus and application thereof |
| CN115181675B (en) * | 2022-05-06 | 2024-06-04 | 南京思农生物有机肥研究院有限公司 | Trichoderma Guizhou growth-promoting partner aspergillus flavus and application thereof |
| CN116064332A (en) * | 2023-02-01 | 2023-05-05 | 山东省花生研究所 | Bacterial strain for degrading aflatoxin B1 and application thereof |
| CN116064332B (en) * | 2023-02-01 | 2023-08-15 | 山东省花生研究所 | Bacterial strain for degrading aflatoxin B1 and application thereof |
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