CN104099251A - New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin - Google Patents
New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin Download PDFInfo
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Abstract
The invention discloses a new aspergillus niger strain FS-UV1, the preservation number is CCTCC NO:M2013553. The strain has good degradation effect on aflatoxin, the degradation rate reaches 95.3%, and is increased by 37.8% than that of an original strain; the degradation efficiency of zearalenone reaches 93%, and is increased by 7% than that of the original strain; and the degradation rate of vomitoxin reaches 52.6%, and is increased by 9.5% than that of the original strain. Rat experiments prove that the new strain is a safe and effective degradation method free of toxic and side effects.
Description
Technical field
The present invention relates to a kind of aspergillus niger, particularly a kind of mutagenic obtained, aflatoxin B1 is there is to the bacterial strain of good degradation effect.
Background technology
Mycotoxins is very harmful to grain especially to food, and fungi can biosynthesizing and the metabolism of toxin in storage after food crop field growing and results.Estimate according to Food and Argriculture OrganizationFAO, whole world cereal supply 25% is polluted by mycotoxins and can not be eaten, and the control of mycotoxins is become and ensures the instant cardinal task of food safety.And microbial control has the advantages such as safety, efficient, economy, applicability are strong, environmentally friendly, become the method for preventing and treating that enjoys in recent years high praise.And harm surely belongs to more greatly aflatoxin B1, zearalenone and vomitoxin in mycotoxins.
Since nineteen sixty, the aflatoxicosis event of turkey was broken out in Britain, carry out deeply a large amount of to aflatoxin both at home and abroad and studied widely.Because the reproduction speed of fungi is fast, the quantity of spore is large, it is far away to scatter, therefore the farm crop such as grain are very easily subject to the pollution of Toxigenic fungi and aflatoxin in plantation, results, storage and the course of processing.In 18 kinds of aflatoxin having found, with AFB
1teratogenecity, mutagenicity and carinogenicity the strongest.As far back as 1993, AFB
1be decided to be mankind I class carcinogens by international cancer research institution.
Research shows that various bacteria has biological control effect to aflatoxin.As bacillus, burkholderia, streptomycete, suis and milk-acid bacteria etc. all can suppress to produce the growth of malicious mould, produce toxic action.Smooth Xiao Yuan finds that isolated extension brevibacterium has stronger restraining effect to flavus growth from the traditional cheese of Egypt; From Thailand's zymotic soybean paste, isolated bacillus licheniformis and bacillus pumilis can not only suppress flavus growth, and 74～85% the AFB of can also degrading
1; Cho isolates a bacillus pumilus from Korea S's soy sauce, finds that her withered grass element of this bacterium secretion can suppress the growth of the malicious flavus of product and Aspergillus parasiticus; Elegance is isolated the subtilis that a strain can suppress flavus growth from bean cotyledon; Zhang Ting also isolates and suppresses surfactant peptides and the bacillomycin D that aspergillus spore is sprouted from subtilis.Therefore isolating a kind of bacterial strain tool that aflatoxin is had to good control and a degradation effect is of great significance.
Ultraviolet mutagenesis is most widely used a kind of new approaches of physical mutagenesis, and Guo Jiping adopts ultraviolet mutagenesis to transform aspergillus oryzae K61 bacterial strain, and finishing screen is selected a mutant strain Y29 that strain proteinase activity is high and heritability is stable; Pan Tao screens a strain and stablizes superior strain 10min in ultraviolet mutagenesis process
-5, citric acid output has increased by 5.33%; Zhao Qiong etc. are taking acetobacter xylinum (Acetobacter xylinum) C5 as starting strain, it is carried out to ultraviolet mutagenesis, using irradiation time 3min as ultraviolet mutagenesis dosage, through primary dcreening operation and multiple sieve, obtain 1 strain bacteria cellulose superior strain A.xylinumC544, the output of this mutant strain is 1.5 times of original strain; Visible bacterial strain often can show the characteristic better than former bacterial strain after mutagenesis.Thereby, bacterial strain is carried out to mutagenesis screening and select the good bacterial strain of aflatoxin degradation effect is had a good application prospect.
Summary of the invention
The invention provides a kind of Novel black aspergillus (Aspergillus niger) FS-UV1, be preserved in Chinese Typical Representative culture collection center, deposit number CCTCC NO:M2013553 on November 6th, 2013; Classify basic phylogenetic tree as shown in Figure of description 1 with its 18SrDNA total order.
The invention provides a kind of mutagenesis aspergillus niger, this bacterial strain has good degradation effect to aflatoxin B1, and degradation rate reaches 95.3%; More former bacterial strain has improved 37.8%; Degradation efficiency to zearalenone reaches 93%, and more former bacterial strain has improved 7%, and the degradation rate of vomitoxin is reached to 52.6%, and more former bacterial strain has improved 9.5%.
Described aspergillus niger storage conditions is as follows: access 3 ring mutagenesis aspergillus nigers in fermention medium from well-grown solid-state plate culture medium PDA, 28 DEG C of shaking tables (200rpm) are cultivated 36h, get 0.65mL and move in the glycerine pipe that contains the aseptic glycerine of 0.3mL, put into-70 DEG C of Ultralow Temperature Freezers and preserve.
Described solid-state plate culture medium PDA is (w/v): potato 300g, glucose 20g, agar 20g, paraxin 0.1g, distilled water 1L, natural pH, 121 DEG C of autoclaving 15min.
Described liquid fermentation medium PDB is (w/v): potato 300g, glucose 20g, distilled water 1L, natural pH, 121 DEG C of autoclaving 15min.
The culture condition of described mutagenesis aspergillus niger is: by the mutagenesis aspergillus niger access liquid nutrient medium of preservation glycerine pipe, and 28 DEG C, 130rpm, shaking table is cultivated 36h.
The preparation method of described aspergillus niger is: the aspergillus niger parental generation screening from sauce unstrained spirits is inoculated in PDA solid medium, and 28 DEG C of 130rpm, cultivate 36h, get 50ml physiological saline and wash lower spore, and vibration evenly, forms monospore suspension, makes 10
8the bacteria suspension of CFU/ml.Get above-mentioned spore suspension 5ml, through 200w uv irradiating 5min, when microwave is in top grade, heat 80s.By the inoculation after mutagenesis, in PDB substratum, from bacterium colony, the suitable spore of picking makes 10 in a manner described
8cFU/ml bacteria suspension, is inoculated in 30ml substratum, 28 DEG C of 130rpm, and shaking table is cultivated 36h.Draw 10ul fermented liquid and on casein plate, carry out multiple sieve.By bacterial strain and former inoculation after above-mentioned multiple sieve in 30ml substratum, 28 DEG C of 130rpm, shaking table is cultivated 36h.In fermented liquid, add 0.05ppm AFB1,0.5ppm zearalenone and 0.5ppm vomitoxin, more former bacterial strain and the degradation effect of novel mutagenic strain to AFB1, zearalenone and vomitoxin.
The present invention be from sauce unstrained spirits, separate obtain through ultraviolet microwave complex mutation AFB1 is had to the novel strain of good degradation effect, the degradation rate of aflatoxin B1 is reached to 95.3%, more former bacterial strain has improved 37.8% to AFB1; Degradation efficiency to zearalenone reaches 93%, and more former bacterial strain has improved 7%; Degradation rate to vomitoxin reaches 52.6%, and more former bacterial strain has improved 9.5%.Bacterial strain after mutagenesis can be applied food, and agricultural-food in the degraded of multiple mycotoxins, reach and reduce the especially object of aflatoxin B1 content of mycotoxins in the fields such as grain and feed.
Brief description of the drawings
Fig. 1: the phylogenetic tree of Novel black aspergillus FS-UV1
Fig. 2: the degradation effect of Novel black aspergillus FS-UV1 to AFB1, zearalenone, vomitoxin
Fig. 3: the enzymic activity of Novel black aspergillus FS-UV1
Fig. 4: toxin rat experiment slice map in Novel black aspergillus FS-UV1 degrading maize slurry
A: feed group; B: standard substance group; C: peanut meal group; D: experimental group.
Embodiment
Embodiment 1: the screening method of aspergillus niger in sauce unstrained spirits
From the sauce fermentation 4d, isolate qualitatively the malicious filamentous fungus of non-product in sauce unstrained spirits with the PDA substratum containing beta-cyclodextrin, then by ELISA method, the product poison ability of institute's isolated strains is carried out to quantitative analysis, finally filter out the malicious mould of non-product.
Carry out multiple sieve to suppressing flavus toxigenic bacterium, filter out the AFB that can degrade
1bacterial strain, degradation rate is 58%.Filter out AFB
1control bacterial strain.
Observe and individual morphology observation by colony characteristics, and by known array contrast in the 18S rDNA of bacterial strain and GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), find that itself and aspergillus niger (Aspergillus niger) homology reach 100%.In conjunction with morphological specificity and the molecular biology identification of bacterial strain, can determine that bacterial strain is aspergillus niger.This bacterial strain is preserved in Chinese Typical Representative culture collection center, and deposit number is: CCTCC NO:M2013553.
Embodiment 2: ultraviolet microwave complex mutation aspergillus niger preparation method
Get above-mentioned spore suspension 5ml, through 200w uv irradiating 5min, when microwave is in top grade, heat 80s.By the inoculation after mutagenesis, in PDB substratum, from bacterium colony, the suitable spore of picking makes 10 in a manner described
8cFU/ml bacteria suspension, is inoculated in 30ml substratum, 28 DEG C of 130rpm, and shaking table is cultivated 36h.Draw 10ul fermented liquid and on casein plate, carry out multiple sieve.Observe by colony characteristics, individual morphology is observed, and by known array contrast in the 18SrDNA of bacterial strain and GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi), find that itself and aspergillus niger (Aspergillus niger) homology reach 100%.In conjunction with morphological specificity and the molecular biology identification of bacterial strain, can determine that bacterial strain is aspergillus niger.The evolutionary tree of Novel black aspergillus FS-UV1 is as Fig. 1.This bacterial strain is preserved in Chinese Typical Representative culture collection center on November 6th, 2013, deposit number CCTCC NO:M2013553, and preservation address is Wuhan, China Wuhan University.
Embodiment 3: the degraded of Novel black aspergillus to multiple mycotoxins
By above-mentioned bacterial strains FS-UV1 and former inoculation in 30mlPDB substratum, 28 DEG C of 130rpm, shaking table is cultivated 36h.In fermented liquid, add 0.05ppmAFB1,0.5ppm zearalenone and 0.5ppm vomitoxin, more former bacterial strain and the degradation effect of novel mutagenic strain to AFB1, zearalenone and vomitoxin.
1) degraded of Novel black aspergillus FS-UV1 to AFB1
By complex mutation bacterial strain FS-UV1 and former inoculation in 30mlPDB substratum, 28 DEG C of 130rpm, shaking table is cultivated 36h.In fermented liquid, add 0.05ppm aflatoxin B1, therefrom draw 1ml and add 3ml trichloromethane, extracting twice, nitrogen dries up, and adds trifluoroacetic acid 100ul, normal hexane 200ul, derivatize 30min, application high performance liquid chromatography detects.Chromatographic condition is C18 post 6mm × 150mm × 5um; Moving phase acetonitrile: water=20:80 detected temperatures: 30 DEG C; Flow velocity: 1ml/min; Detect wavelength: excitation wavelength: 360nm; Emission wavelength: 440nm.
2) degraded of Novel black aspergillus FS-UV1 to zearalenone
By complex mutation bacterial strain FS-UV1 and former inoculation in 30mlPDB substratum, 28 DEG C of 130rpm, shaking table is cultivated 36h.In fermented liquid, add 0.5ppm zearalenone standard substance, more therefrom draw 1ml and add 3ml trichloromethane, extracting twice, nitrogen dries up, and high performance liquid chromatography is carried out content detection.Chromatographic condition is chromatographic column: C18 post 6mm × 150mm × 5um; Moving phase: acetonitrile: water=50:50; Detected temperatures: 25 DEG C; Flow velocity: 1ml/min; Detect wavelength: excitation wavelength: 240nm; Emission wavelength: 440nm
3) degraded of Novel black aspergillus FS-UV1 to vomitoxin
Bacterial strain after above-mentioned mutagenesis and former inoculation, in 30mlPDB substratum, are cultivated to 36h for 28 DEG C.In fermented liquid, add 0.5ppm vomitoxin standard substance, draw 10ml, more therefrom draw 1ml and add 3ml trichloromethane to carry out extracting twice, nitrogen dries up, and high performance liquid chromatography is carried out content detection.Chromatographic condition: chromatographic condition is chromatographic column: C18 post 6mm × 150mm × 5um; Moving phase: methyl alcohol: water=20:80; Detected temperatures: 30 DEG C; Flow velocity: 0.8ml/min; Detect wavelength: 218nm.
Result as shown in Figure 2, Novel black aspergillus FS-UV1(CCTCC NO:M2013553) degradation rate of aflatoxin B1 is reached to 95.3%, more former bacterial strain has improved 37.8% to AFB1; Degradation efficiency to zearalenone reaches 93%, and more former bacterial strain has improved 7%; Degradation rate to vomitoxin reaches 52.6%, and more former bacterial strain has improved 9.5%.
Embodiment 4: the control of aflatoxin B1 in peanut meal, cottonseed meal that Novel black aspergillus FS-UV1 contratoxin pollutes
Get appropriate complex mutation aspergillus niger FS-UV1(CCTCC NO:M2013553) spore inoculating (aflatoxin B1 content 68 μ g/mL) in the cottonseed meal or peanut meal of aflatoxin contamination, 28 DEG C of fermentation culture 7d, getting 40g cottonseed meal or peanut meal pulverizes sample and is dissolved in 100ml acetonitrile-water (9:1) solution, high speed homogenization device stirs and extracts 2min, qualitative filter paper filters, pipette 10ml filtrate and add 40ml water dilution to mix, glass filter paper filtering 1～2 time to filtrate is clarified.Get appropriate filtrate and chloroform by twice of 1:2 hybrid extraction.Nitrogen dries up, and is dissolved in 1.0ml moving phase, to be measured.Through high effective liquid chromatography for measuring, find that in cottonseed meal or peanut meal, aflatoxin B1 residual quantity is lower than 2 μ g/mL, degradation rate reaches 98%.
Embodiment 5: degraded product rat animal verification experimental verification
Rat feed before fasting 12h, freely drink water, claim weight record, carry out mark, be divided at random 4 groups, that is: the normal diet group of feeding A(is called for short: blank group); Gavage aflatoxin B1 standard substance group B(is called for short: control group); The peanut meal group C(that feeds aflatoxin-contaminated is called for short: peanut meal group); Feed and pollute peanut meal and fermentation of Aspergillus niger liquid and cultivate altogether 48h experimental group D(and be called for short: degraded group), every group each 8.Processings of feeding of each group mouse same time of every day, free choice feeding drinking-water, continues 30 days, finally de-neck execution.Take out liver and the kidney of rat, through paraffin embedding, section, HE dyeing, 100 × and 400 × its tissue slice of optical microphotograph Microscopic observation Fig. 4 (left side is liver, and the right side is kidney).Found that, peanut meal group, because content of toxins is high, causes the liver kidney apparent damage of rat; And the rats'liver kidney of degraded group is not found obvious tissue damage, the aflatoxin that this explanation fermentation of Aspergillus niger liquid can be degraded in peanut meal, and its toxicity is disappeared.Therefore, the degraded of this aspergillus niger contratoxin is the bacterial strain of safety non-toxic by product, for animal-feed provides a kind of poison-removing method of highly effective and safe.
Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.
Claims (8)
1. Novel black aspergillus (Aspergillus niger) FS-UV1, is preserved in Chinese Typical Representative culture collection center on November 6th, 2013, and deposit number is: CCTCC NO:M2013553, preservation address is Wuhan, China Wuhan University.
2. aspergillus niger according to claim 1, is characterized in that preventing and treating the bacterial strain of aflatoxin B1, zearalenone, vomitoxin.
3. aspergillus niger claimed in claim 2, is characterized in that described aspergillus niger obtains through ultraviolet microwave complex mutation.
4. aspergillus niger according to claim 3, is characterized in that described mutagenic condition is ultraviolet lamp 200w, 5min, the high-grade heating of microwave 80s.
5. be applied to aflatoxin in the peanut meal, cottonseed meal of degraded endotoxin contamination according to the arbitrary described aspergillus niger of claim 1-4.
6. be applied to zearalenone in the peanut meal, cottonseed meal of degraded endotoxin contamination according to the arbitrary described aspergillus niger of claim 1-4.
7. be applied to vomitoxin in the peanut meal, cottonseed meal of degraded endotoxin contamination according to the arbitrary described aspergillus niger of claim 1-4.
8. the application in food, field of fodder according to the arbitrary described aspergillus niger of claim 1-4.
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| CN104673682A (en) * | 2015-02-15 | 2015-06-03 | 华南农业大学 | Aspergillus niger and application thereof in biological prevention and control of aflatoxin |
| CN104789479A (en) * | 2014-07-25 | 2015-07-22 | 江南大学 | Preparation of novel Aspergillus niger immobilized cell and degradation effect of Aspergillus niger immobilized cell on aflatoxin B1 |
| CN106520593A (en) * | 2016-10-09 | 2017-03-22 | 内蒙古和美科盛生物技术有限公司 | Microbial preparation capable of reducing zearalenone and vomitoxin in silage |
| CN108034598A (en) * | 2017-11-24 | 2018-05-15 | 河南德邻生物制品有限公司 | It is a kind of can simultaneous degrading aspergillus flavus toxin and zearalenone formula |
| CN108541817A (en) * | 2018-05-25 | 2018-09-18 | 河南牧鹤(集团)饲料有限公司 | One broad sow compound premix |
| CN109363058A (en) * | 2017-09-29 | 2019-02-22 | 海南泓缘生物科技股份有限公司 | The biodegradation method of vomitoxin in a kind of pair of corn pulp |
| CN110583964A (en) * | 2019-09-23 | 2019-12-20 | 江南大学 | Biological removal method for efficiently removing four aflatoxins in peanut meal |
| CN111011707A (en) * | 2019-11-28 | 2020-04-17 | 国家粮食和物资储备局科学研究院 | Preparation method of flavor detoxified corn steep liquor |
| CN111117900A (en) * | 2020-02-03 | 2020-05-08 | 河南工业大学 | Aflatoxin B capable of being efficiently degraded1And application thereof |
| CN112535253A (en) * | 2020-12-08 | 2021-03-23 | 江南大学 | Biological detoxification method for efficiently removing patulin in apple pomace |
| CN112553087A (en) * | 2020-12-17 | 2021-03-26 | 江南大学 | Aspergillus niger capable of rapidly degrading zearalenone and application thereof |
| CN112831439A (en) * | 2021-01-29 | 2021-05-25 | 山东省花生研究所 | Application of burkholderia in aflatoxin degradation |
| CN113265354A (en) * | 2021-05-19 | 2021-08-17 | 科润生科技发展有限公司 | Streptomyces rochei capable of simultaneously degrading aflatoxin and vomitoxin and preparation thereof |
| CN113373060A (en) * | 2021-06-18 | 2021-09-10 | 江南大学 | Aspergillus niger capable of rapidly and simultaneously degrading three mycotoxins and application thereof |
| CN113736668A (en) * | 2021-09-15 | 2021-12-03 | 江南大学 | Aspergillus niger strain and application thereof in ochratoxin A degradation |
| CN114717212A (en) * | 2022-04-01 | 2022-07-08 | 华中农业大学 | Glutathione mercaptotransferases as agents for controlling AFB1Application of detoxification enzyme causing duck liver injury |
| CN114752592A (en) * | 2022-05-12 | 2022-07-15 | 浙江师范大学 | Microbial inoculum for fungus bran feed and preparation method thereof |
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| CN104673682A (en) * | 2015-02-15 | 2015-06-03 | 华南农业大学 | Aspergillus niger and application thereof in biological prevention and control of aflatoxin |
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