CN107245453B - Aflatoxin-producing aspergillus flavus and application thereof in degrading aflatoxin - Google Patents
Aflatoxin-producing aspergillus flavus and application thereof in degrading aflatoxin Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and particularly discloses aspergillus flavus JZ2 (with the preservation number of CGMCC No.12865) without producing toxin and application thereof in degrading aflatoxin. The invention provides the aspergillus flavus strain capable of degrading the aflatoxin for the first time, and also develops the new application of the aspergillus flavus strain JZ2 without producing the toxin in the aspect of degrading the aflatoxin for the first time. The aflatoxin-free aspergillus flavus strain JZ2 can efficiently degrade aflatoxin, can be used as a biological material for degrading aflatoxin, and has good application prospect in the aspects of developing new biodegradable microbial agents and biodegradable sterile preparations.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to aflatoxin-free aspergillus flavus and application thereof in degrading aflatoxin.
Background
Aflatoxins (AFT) are secondary metabolites produced by fungi such as Aspergillus flavus (a. flavus), Aspergillus parasiticus (Aspergillus parasiticus) and Aspergillus oryzae (Aspergillus nomius), have carcinogenic, teratogenic and cytogenic effects, and are commonly referred to as AFB1、AFB2、AFG1、AFG2And AFM1Etc. wherein AFB1The toxicity is strongest. The main action target of aflatoxin is liver, and a large number of epidemiological regulationsResearch studies have shown that 28% of hepatocellular carcinoma (HCC; a major type of liver cancer) worldwide is caused by aflatoxins (Liu et al, 2010; Liu et al, 2012; Wu, 2014). In addition, aflatoxins can also cause acute lesions of the kidneys and adrenal glands (Poirier et al, 2000; Kensler et al, 2011).
The basic structures of aflatoxins are the difuran ring, which is the basic toxic structure, and the oxanaphthaleno-ketones (coumarins), which are the major carcinogenic structures. Aflatoxin mainly exists in foods such as peanuts, corns, dried fruits and the like, and seriously threatens the food safety and the body health of people in China; in addition, feed is also often contaminated with aflatoxins. Therefore, a highly efficient detoxification method for removing such toxins is urgently needed. The traditional aflatoxin detoxification method comprises physical and chemical methods, including an ammoniation method, an alkaline method, a high-temperature method, a ray irradiation method, an ultrafiltration-osmosis method, an adsorption method and the like, and the methods have the defects of large nutrient loss, complex degradation products, unclear toxicity of the degradation products, unstable effect and the like. The biological detoxification method is a method for degrading aflatoxin by utilizing microorganisms and metabolic products such as enzymes secreted by the microorganisms, has the advantages of strong specificity, high efficiency, no pollution to food, feed and environment and the like, is a main development direction of aflatoxin detoxification, and has wide application prospect. At present, the following aflatoxin-degrading microorganisms and enzymes thereof are mainly reported: pseudomonas aeruginosa, Nocardia DSM12676, stenotrophomonas maltophilia, Armillaria pseudomeliloti, Mycobacterium DSM 44556T, Aspergillus niger, Rhodococcus erythropolis, Pleurotus ostreatus, etc. and their extracted degrading enzymes.
No study and report about the aspergillus flavus capable of degrading the aflatoxin is found.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a strain of aspergillus flavus without producing toxin and application thereof in degrading aflatoxin.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
the invention firstly provides a strain of Aspergillus flavus (Aspergillus flavus) JZ2 without producing toxin, which is preserved in China general microbiological culture Collection center (CGMCC for short, address: No. 3 of West Lu 1 of Beijing Inward region, Microbiol research institute of Chinese academy of sciences, postal code: 100101), the preservation date is 2016, 9 and 7 days, and the preservation number is CGMCC No. 12865.
The aspergillus flavus colony which does not produce toxin is green, generates a large amount of conidia, grows faster on a PDA culture medium, and can cover a flat plate with the diameter of 9cm after being cultured in the dark at the temperature of 30 ℃ for 5 days. The strain is deleted with an aflatoxin synthesis gene, wherein the aflatoxin synthesis gene of the aspergillus flavus strain is deleted with an aflatoxin synthesis gene aflT, an aflatoxin synthesis gene pkSA, an aflatoxin synthesis gene nor-1, an aflatoxin synthesis gene fas-2, an aflatoxin synthesis gene fas-1, an aflatoxin synthesis gene aflR, an aflatoxin synthesis gene aflJ, an aflatoxin synthesis gene adhA, an aflatoxin synthesis gene estA, an aflatoxin synthesis gene norA, an aflatoxin synthesis gene ver-1 and an aflatoxin synthesis gene ver A, so the strain cannot synthesize aflatoxin; the circular arc azoic acid synthesis gene maoA is deleted, so that the strain cannot synthesize circular arc azoic acid; therefore, the strain does not produce aflatoxin and azocycloate.
Further, the invention provides application of the non-toxigenic aspergillus flavus JZ2 in degrading aflatoxin.
More specifically, the application is to degrade aflatoxin by using aspergillus flavus JZ2 which does not produce toxin, or bacterial suspension thereof, or fermentation liquor thereof, or crude extract thereof.
The invention further provides application of the non-toxigenic aspergillus flavus (A. flavus) JZ2 in preparation of products for degrading aflatoxin.
Further, the product for degrading aflatoxin can contain non-toxigenic aspergillus flavus JZ2, or a bacterial suspension thereof, or a fermentation liquor thereof, or a crude extract thereof.
Based on the technical scheme, the inventor also provides a method for degrading aflatoxin, namely, aspergillus flavus JZ2 which does not produce toxin, or bacterial suspension thereof, or fermentation liquor thereof, or crude extract thereof is used for degrading aflatoxin.
The method specifically comprises the following steps: mixing the aflatoxin-free JZ2, or a bacterial suspension thereof, or a fermentation broth thereof, or a crude extract thereof with a sample containing aflatoxin.
The sample can be an agricultural product processing raw material, food, feed, an environmental sample and the like containing aflatoxin.
The invention further provides a reagent for degrading flavomycotoxin, which contains the non-toxigenic aspergillus flavus JZ2 or a bacterial suspension thereof, or a fermentation liquor thereof, or a crude extract thereof.
The aflatoxin in the technical scheme of the invention is a B-family aflatoxin and/or a G-family aflatoxin. The B group aflatoxin is aflatoxin B1And/or aflatoxin B2(ii) a The G-family aflatoxin is aflatoxin G1And/or aflatoxin G2。
The raw materials or reagents involved in the invention are all common commercial products, and the operations involved are all routine operations in the field unless otherwise specified.
The invention has the beneficial effects that:
the invention provides the aspergillus flavus strain capable of degrading the aflatoxin for the first time, and also develops the new application of the aspergillus flavus strain JZ2 without producing the toxin in the aspect of degrading the aflatoxin for the first time.
The aflatoxin-free aspergillus flavus strain JZ2 can efficiently degrade aflatoxin, can be used as a biological material for degrading aflatoxin, and has good application prospect in the aspects of developing new biodegradable microbial inoculum and biodegradable sterile preparations.
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FIG. 1 is a schematic diagram showing deletion of a toxin synthesis gene of a. flavus non-toxigenic strain JZ2 according to the present invention.
FIG. 2 is the aflatoxin detection in the Aspergillus flavus JZ2 culture solution; (A) aflatoxin B1A standard solution liquid chromatogram; (B) and (3) culturing the aspergillus flavus JZ2 on a YES culture medium for 7 days to obtain the liquid chromatogram of the aflatoxin extracting solution.
FIG. 3 is detection of circular arc azoic acid (CPA) in Aspergillus flavus JZ2 culture solution according to the present invention; (A) CPA standard solution liquid chromatogram map; (B) a liquid chromatogram of CPA extract was obtained by culturing Aspergillus flavus JZ2 on YES medium for 7 days.
FIG. 4 shows the treatment of aflatoxin B by Aspergillus flavus JZ2 in accordance with the present invention1Mass spectrum of the degradation product of (1).
FIG. 5 is a derivative of the aflatoxin degradation pathway.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantification experiments in the following examples were repeated three times and the results were averaged.
DG18 solid medium: casein peptone 5.0g/L, anhydrous glucose 10.0g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate (MgSO)4·H2O)0.5g/L, niclosamide 0.002g/L, anhydrous glycerin 200g/L, agar 15g/L, chloramphenicol 0.1g/L, and distilled water dissolved to 1L.
PDB liquid medium: glucose 20g, rhizoma Solani Tuber osi 200g (boiling in water for 20-30min, filtering with 2 layers of gauze while it is hot, collecting filtrate), and adding distilled water to 1L.
PDA solid medium: 15g/L of agar was added to the PDB liquid medium to obtain a PDA solid medium.
YES liquid medium: yeast extract 20g, sucrose 150g, magnesium sulfate (MgSO)4·7H2O), dissolving to 1L by distilled water.
AFPA medium: 10.0g/L of peptone, oh 20.0.0 g/L of yeast picking, 0.5g/L of ferric ammonium citrate, 0.002g/L of nitramine chloride, 0.1g/L of chloramphenicol and 15.0g/L of agar, wherein the pH value is 6.3.
Example 1 Collection, isolation and identification of non-toxigenic Aspergillus flavus
Method for separating aspergillus flavus strain JZ2 from peanut planting soil
Aspergillus flavus strains were isolated from peanut planting soil in Guangdong using DG18 medium. The specific operation is as follows:
1. preparation of soil sample bacterial suspension
10g of soil sample was added with 90mL of 0.1% peptone sterile water (w/v), and shaken at room temperature for 30min to prepare 10-1Bacterial suspension; taking 0.5mL of 10-1Adding 4.5ml of 0.1% peptone sterile water into the bacterial suspension to prepare 10-2Diluting the bacterial suspension; preparation of 10 by the above method-3And (5) diluting the bacterial suspension.
2. Isolation and purification of the strains
0.1mL of bacterial liquid is taken for each dilution, the bacterial liquid is coated on a DG18 culture medium, dark culture is carried out for 5d at the temperature of 30 ℃, each dilution is repeated for 3 times, and strains with yellow spores are selected to carry out secondary streak separation on a DG18 culture medium until a single colony is obtained. Single colonies were picked up on PDA slant tube medium, cultured at 30 ℃ for 3 days and stored at 4 ℃. One of the strains was designated as JZ 2.
II, identification of a strain JZ 2:
1. morphological identification:
selecting strain JZ2 stored in PDA culture medium and AFPA culture medium, and culturing at 30 deg.C for 3-5 days to obtain AFPA culture medium with bright orange back.
2. And (3) molecular identification:
strain JZ2 was molecularly characterized by the fungal calmodulin sequence (Rodrigues, P., Santos, C., Venancio, A., Lima, N., 2011. specificity identification of Aspergillus section from porous microorganisms using phenotypical, included MALDI-ICMS, and molecular profiling. J. applied Microbiol 111877. cost 892). Primers used for the Aspergillus flavus genomic calmodulin PCR amplification were CL1 and CL2A (sequences are as follows). The PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5min for 1 cycle; denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, and extension at 72 ℃ for 90s for 30 cycles; final extension at 72 ℃ for 7 min. After amplification, the product was stored at 4 ℃. The product is sent to Shanghai Bioengineering Co., Ltd for sequencing. And the sequencing results were aligned on BLASTREARCHEs (http:// www.Ncbi.nlm.nih.gov /).
CL1：5’-GARTWCAAGGAGGCCTTCTC-3’；
CL2A：5’-TTTTTGCATCATGAGTTGGAC-3’。
The sequencing result of the calmodulin gene of the strain JZ2 is submitted to NCBI for comparison, and the homology of the strain with Aspergillus flavus NRRL3357 and NRRL21882 is 99%.
Through the morphological identification and the molecular identification, the strain JZ2 is Aspergillus flavus.
3. Detection of aflatoxin and circular arc azoic acid (CPA) synthetic gene integrity in Aspergillus flavus (Aspergillus flavus) JZ2
Six pairs of primers for toxin synthesis-related genes, damT, maoA, hypB, hypC, hypD and hypE, were designed with reference to the Sequence of the toxin synthesis gene of the Aspergillus flavus having Genbank accession No. AY510451, and the primers for the other genes were referred to as long-Kuang Chang (Chang, p.k., Horn, b.w. and Dorner, J.W. (2005) Sequence break points inter-graft antibiotic gene cluster and fluorng regions in biochemical gene 42, 941. 923).
TABLE 1 primer information for identifying deletion of toxin-synthesizing genes
Extracting genome DNA of Aspergillus flavus JZ2(CGMCC No.12865), and performing conventional PCR reaction with the primer pairs in Table 1 by using the genome DNA as a template, wherein each reaction is provided with genome DNA control and negative control (sterile water is used as a template) of Aspergillus flavus GD-1 for producing aflatoxin.
And (3) PCR reaction system: mu.L of the DNA template, 1. mu.L of each of the upstream and downstream primers (10. mu.M), 10. mu.L of Gotaq (Gotag colorlessmaster mix, Promega, M7133), and sterile water to make up to 20. mu.L.
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 2min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min30s, performing 30 cycles, and final extension at 72 ℃ for 7 min.
The PCR product was electrophoresed in 1 XTAE buffer using 1.0% agarose gel, and photographed using a gel imaging system.
The identification result shows that the Aspergillus flavus JZ2 lacks genes aflT, pksA, nor-1, fas-2, fas-1, aflR, aflJ, adhA, estA, norA, ver-1 and verA synthesized by the aflatoxin, so the strain can not synthesize the aflatoxin; the circular arc azoic acid synthesis gene maoA is deleted, so that the strain can not synthesize circular arc azoic acid.
Third, detection of aflatoxin and CPA (circular arc azoic acid)
1. Aflatoxin detection
Activating the strain on PDA culture medium, eluting spore solution with 0.1% Tween 80, adding the spore solution into 50mL YES (20g/L yeast powder, 150g/L sucrose, 1g/L magnesium sulfate) liquid culture medium, and adjusting the final concentration of spore solution to 1 × 104cells/mL were cultured in the dark at 30 ℃ and 180rpm for 7 days. Filtering the culture solution with filter paper, adding equal volume of chloroform into 1mL, mixing with vortex oscillator, adding 1mL of chloroform into the upper water phase, extracting aflatoxin, mixing organic phases, blowing nitrogen at 60 deg.C until dried, re-dissolving the toxin in 50% methanol-water solution, and detecting aflatoxin with high performance liquid chromatography. Chromatographic conditions are as follows: mobile phase methanol: water 1:1(v/v), flow rate 1mL/min, chromatography column Agilent TC-C18(4.6 × 250mm), sample size 20 μ L; equipped with photochemical derivatization device and detected by fluorescence detector, excitation wavelength is 360nm, and emission wavelength is 440nm. The identification result shows that the aflatoxin standard product has a peak at 6.991min, while the detection result of the culture solution of the strain JZ2 shows that JZ2 has no chromatographic peak near 6.991min, which indicates that JZ2 does not produce aflatoxin.
2. CPA (circular arc azoic acid) detection
After the strain was cultured on YES medium for 7 days, 25mL of the filtrate was taken and 100mL of methanol was added: 2% sodium bicarbonate water solution (7:3, v/v), shaking for 30min at more than 200rpm, taking out 50mL of solution to a separating funnel, adding 100mL of n-hexane for extraction, shaking for layering, removing a hexane layer, then adding 50mL of 10% KCl solution, uniformly mixing, adding 2.0mL of 6.0M HCl solution for acidification, extracting CPA twice with 50mL of chloroform, collecting a chloroform layer, drying 5mL of nitrogen, and redissolving in 1mL of acetonitrile: 0.05M aqueous ammonium acetate (8:2, v/v, pH 5) was detected by HPLC. Chromatographic conditions are as follows: mobile phase acetonitrile: 0.05M aqueous ammonium acetate (8:2, v/v, pH 5), flow rate 0.6mL/min, detection wavelength 284nm, chromatography column Agilent TC-C18(4.6 × 250mm), sample size 20 μ L. The detection result shows that the standard product of the circular arc azoic acid generates a peak at 5.260min, and JZ2 does not have any chromatographic peak near 5.260min, which indicates that JZ2 does not generate the circular arc azoic acid.
Example 2 non-toxigenic Aspergillus flavus JZ2 on Aflatoxin B1Analysis of the ability to degrade
1. Aflatoxin B1Preparation of standard substance
1mg of aflatoxin B1(AFB1) The standard substance is dissolved in 2mL of chromatographic pure methanol to obtain the aflatoxin B with the concentration of 500ppm1And (3) solution.
2. Cultivation of bacterial species
Inoculating Aspergillus flavus JZ2 not producing toxin on MEA slant test tube culture medium, culturing at 28 deg.C for 5 days, dipping spores on the culture medium in sterile 0.1% Tween-80 with cotton swab, shaking with oscillator, and regulating spore concentration to 1 × 10 with blood counting plate6cfu/mL。
3. Aflatoxin B by aspergillus flavus JZ2 without producing toxin1Degradation of
Taking JZ2 spore liquid (1 × 10)6cfu/mL)1mL in sterilized 50mL PDAnd B, adding the aflatoxin subjected to filtration sterilization into the culture medium to ensure that the concentration of the aflatoxin in the culture solution is 500ppb, fully and uniformly mixing, culturing in a shaking table at 28 ℃ at 180rpm, sampling, extracting toxins and detecting on 3 days, 6 days and 9 days of the culture respectively, and marking as an experimental group. Positive control experiment: taking 1mL of sterile 0.1% Tween-80 solution to be added into a sterilized 50mL of PDB culture medium, adding the aflatoxin subjected to filtration sterilization to enable the concentration of the aflatoxin in a culture solution to be 500ppb, fully and uniformly mixing, culturing in a shaking table at 28 ℃ at 180rpm, sampling, extracting the toxin and detecting on 3 days, 6 days and 9 days of culture respectively. Negative control experiment: taking JZ2 spore liquid (1 × 10)6cfu/mL)1mL of the culture medium was added to a sterilized 50mL LPDB medium, mixed well, cultured at 180rpm in a shaker at 28 ℃ and sampled, toxin-extracted and tested on days 3, 6 and 9 of the culture, respectively.
4. JZ2 treatment of aflatoxin B1Measurement of degradation Capacity
Filtering the fermentation liquor by using qualitative filter paper, taking 1mL of filtrate, adding 1mL of trichloromethane, carrying out vortex oscillation for 1min, centrifuging at 8000rpm for 4min, then removing the lower-layer solution into a clean test tube, continuously adding 1mL of trichloromethane into the lower-layer solution, repeating the operation twice, then carrying out nitrogen blowing at 50 ℃, redissolving by using a mobile phase, filtering by using a 0.22 mu m filter membrane, detecting the content of aflatoxin by using HPLC (high performance liquid chromatography), and calculating the degradation rate.
HPLC assay conditions mobile phase methanol (chromatographic grade): water is 1:1, and ultrasonic degassing is carried out for 5min after ultrafiltration; flow rate: 1 mL/min; agilent C18Chromatography column (4.6 mm. times.250 mm, 5 μm); a detector: a fluorescence detector with an excitation wavelength of 360nm and an emission wavelength of 440 nm; sample introduction amount: 20 μ L.
The results are shown in Table 2. The results show that the aspergillus flavus JZ2 pair not producing toxin to aflatoxin B1Has certain degradation effect, the degradation effect reaches the maximum in 9 days, and the degradation rate is 80.9 percent.
TABLE 2 degradation rate of aflatoxin B1 by non-toxigenic Aspergillus flavus JZ2
Example 3 No productAspergillus flavus JZ2 degradation of aflatoxin B1Analysis of the product of (1)
Analyzing aflatoxin B by adopting LC-qTOF/MS1The degradation product of (a). Aflatoxin B1Firstly, separating degradation products by using a separation column, and ionizing separated components by using an MS ion source; after the first-stage mass spectrum, TOF is reached through the mass spectrum analysis without CID, the CID mode is used for MS/MS analysis, and parent ions are cracked in CID to obtain a cracking path.
LC detection conditions were mobile phase acetonitrile (chromatographic grade): 0.1% formic acid water solution 70:30, ultrafiltering, ultrasonic degassing for 5 min; flow rate: 0.4mL/min for 12 min; agilent reinforced type C18Chromatography column (2.1 mm. times.150 mm, 5 μm); sample introduction amount: 2 μ L.
Mass spectrometry conditions: the compound analysis mode is a cation mode; the capillary and collision induced dissociation voltages are 3500V and 175V respectively; skimmer voltage 65V; the gas flow rate is 10L/min; spray pressure was 40 psi; the collision gas is nitrogen; scanning at the speed of 1.4 spectrum per second in the range of nuclear mass ratio m/z 100-1000 to obtain the mass spectrum of the fragmentation ions; mass HunterWorkstation software V B.04.00 is used for Mass spectrometry; 121.0508 and 922.0097 as reference ions for maintaining mass spectral accuracy.
As can be seen from FIGS. 4 and 5, the results show that Aspergillus flavus JZ2 does not produce toxin after being cultured and is used for treating aflatoxin B1Has certain degradation effect, and derives a degradation product with a molecular formula of C15H20O5The relative molecular mass was 281.14.
Example 4 non-toxigenic Aspergillus flavus JZ2 on Aflatoxin G1、G2Analysis of the ability to degrade
1. Aflatoxin G1And G2Preparation of standard substance
1mg of aflatoxin G1(AFG1) And G2(AFG2) The standard substance is dissolved in 2mL of chromatographic pure methanol to obtain AFG with the concentration of 500ppm1And AFG2And (3) solution.
2. Cultivation of bacterial species
Yellow rice cake without producing toxinInoculating mildew JZ2 on MEA slant test tube culture medium, culturing at 28 deg.C for 5 days, dipping spores on the culture medium with cotton swab in sterile 0.1% Tween-80, shaking with oscillator, and adjusting the spore concentration to 1 × 10 with blood counting plate6cfu/mL。
3. Aflatoxin G of aspergillus flavus JZ2 without producing toxin1And G2Degradation of
Taking JZ2 spore liquid (1 × 10)6cfu/mL)1mL in sterilized 50mL PDB Medium, Filter sterilized Aflatoxin G was added1And G2The concentration of the cells in the culture solution was adjusted to 500ppb, the cells were cultured in a shaker at 28 ℃ and 180rpm, and the cells were sampled, tested and tested on days 3, 6 and 9 of the culture and recorded as test groups. Positive control experiment: adding sterile 0.1% Tween-80 solution 1mL into sterilized 50mL PDB culture medium, and adding filter sterilized aflatoxin G1And G2The concentration of the extract in the culture medium was adjusted to 500ppb, the mixture was thoroughly mixed and cultured at 180rpm in a shaker at 28 ℃ and then sampled, toxin-extracted and tested on days 3, 6 and 9 of the culture. Negative control experiment: taking JZ2 spore liquid (1 × 10)6cfu/mL)1mL of the culture solution was added to 50mL of a sterilized PDB medium, mixed well, cultured at 180rpm in a shaker at 28 ℃ and sampled, toxin-extracted and examined on days 3, 6 and 9 of the culture, respectively.
4. JZ2 treatment of aflatoxin G1And G2Measurement of degradation Capacity
Filtering the fermentation liquid with qualitative filter paper, collecting filtrate 1mL, adding chloroform 1mL, vortex oscillating for 1min, centrifuging at 8000rpm for 4min, removing lower layer solution in clean test tube, adding chloroform 1mL, repeating the above steps twice, blowing nitrogen at 50 deg.C, re-dissolving with mobile phase, filtering with 0.22 μm filter membrane, and detecting aflatoxin G with HPLC1And G2Content and calculating degradation rate.
HPLC assay conditions mobile phase methanol (chromatographic grade): water is 1:1, and ultrasonic degassing is carried out for 5min after ultrafiltration; flow rate: 1 mL/min; agilent C18Chromatography column (4.6 mm. times.250 mm, 5 μm); a detector: fluorescence detector, excitation wavelength 360nm, emissionThe wavelength is 440 nm; sample introduction amount: 20 μ L.
The results are shown in Table 3. The result shows that the aspergillus flavus JZ2 without producing toxin is resistant to aflatoxin G1And G2Has obvious degradation effect, the degradation effect reaches the maximum in 9 th day, and the degradation rates are 84.5 percent and 85.1 percent respectively.
TABLE 3 non-toxigenic Aspergillus flavus JZ2 on Aflatoxin G1And G2Degradation of
Example 5 analysis of the ability of non-toxigenic Aspergillus flavus JZ2 fermentation broth to degrade class B or class G aflatoxins
1. Aflatoxin B1、B2、G1And G2Preparation of standard substance
1mg of aflatoxin B1、B2、G1And G2The sample was dissolved in 2mL of chromatographically pure methanol to give a stock solution with a concentration of 500ppm, and then 100. mu.L of the toxin stock solution was dissolved in 5mL of chromatographically pure methanol to give a degradation addition solution with a concentration of 10 ppm.
2. Cultivation of bacterial species
Inoculating Aspergillus flavus JZ2 not producing toxin on MEA slant test tube culture medium, culturing at 28 deg.C for 5 days, dipping spores on the culture medium in sterile 0.1% Tween-80 with cotton swab, shaking with oscillator, and regulating spore concentration to 1 × 10 with blood counting plate6cfu/mL。
3. Culture of non-toxigenic aspergillus flavus JZ2
Taking JZ2 spore liquid (1 × 10)6cfu/mL)1mL in a sterilized 50mL PDB culture medium, culturing for 5 days at 180rpm in a shaking table at 28 ℃, taking a fermentation system, and centrifuging for 5min at 10000rpm to obtain fermentation liquor.
4. Transferring 990 μ L of the above fermentation broth (PDB liquid culture medium as control of the fermentation broth) and 10 μ L of aflatoxin B with a concentration of 10ppm1、B2、G1And G2Standard solution mix, end of four aflatoxinsThe concentration is 100ppb, and the mixture is added into a 1.5mL sterilized centrifuge tube, reacted for 72h at 30 ℃ and 200rpm, sampled, extracted with toxin and detected.
5. Treatment of aflatoxin B by JZ2 fermentation liquor1、B2、G1And G2Measurement of degradation Capacity
Filtering the degraded mixed solution with qualitative filter paper, collecting filtrate 1mL, adding chloroform 1mL, vortex oscillating for 1min, centrifuging at 8000rpm for 4min, removing lower layer solution, adding chloroform 1mL, repeating the above steps twice, blowing nitrogen at 50 deg.C, redissolving with mobile phase, filtering with 0.22 μm filter membrane, and detecting aflatoxin B with HPLC1、B2、G1And G2Content and calculating degradation rate.
HPLC assay conditions mobile phase methanol (chromatographic grade): water is 1:1, and ultrasonic degassing is carried out for 5min after ultrafiltration; flow rate: 1 mL/min; agilent C18Chromatography column (4.6 mm. times.250 mm, 5 μm); a detector: a fluorescence detector with an excitation wavelength of 360nm and an emission wavelength of 440 nm; sample introduction amount: 20 μ L.
The results are shown in Table 4. The result shows that the aspergillus flavus JZ2 fermentation liquor without producing toxin is opposite to the aflatoxin B1、B2、G1And G2Has obvious degradation effect, and the degradation rates are respectively 98.7%, 96.5%, 99.4% and 98.6%.
TABLE 4 non-toxigenic Aspergillus flavus JZ2 fermentation broth versus Aflatoxin B1、B2、G1And G2Degradation of
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. Aspergillus flavus producing no toxinAspergillus flavus) JZ2, wherein the accession number is CGMCCNo.12865.
2. Use of non-toxigenic aspergillus flavus JZ2 as claimed in claim 1 for degrading aflatoxins.
3. The use according to claim 2, wherein aflatoxin is degraded by non-toxigenic aspergillus flavus JZ2, or a bacterial suspension thereof, or a fermentation broth thereof.
4. Use according to claim 2 or 3, wherein the aflatoxin is a group B aflatoxin and/or a group G aflatoxin.
5. Use of non-toxigenic aspergillus flavus JZ2 as claimed in claim 1 in the preparation of a product for degrading aflatoxin.
6. The use according to claim 5, wherein the product comprises Aspergillus flavus JZ2, or a bacterial suspension thereof, or a fermentation broth thereof, which is not toxigenic.
7. A method for degrading aflatoxin, which is characterized in that the aflatoxin is degraded by using the non-toxigenic aspergillus flavus JZ2 or a bacterial suspension thereof or a fermentation liquor thereof, which is disclosed in claim 1.
8. The method of claim 7, wherein the non-toxigenic Aspergillus flavus JZ2, or a bacterial suspension thereof, or a fermentation broth thereof, is mixed with the sample containing aflatoxin.
9. An agent for degrading flavomycotoxins, which comprises the non-toxigenic Aspergillus flavus JZ2 or a bacterial suspension thereof or a fermentation broth thereof according to claim 1.
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| CN113355246B (en) * | 2021-06-16 | 2022-05-24 | 中国农业科学院油料作物研究所 | Aspergillus flavus SX0104 without producing toxicity and application thereof |
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