CN104099251B - A kind of Aspergillus niger strain and the application in multiple mycotoxin is degraded thereof - Google Patents
A kind of Aspergillus niger strain and the application in multiple mycotoxin is degraded thereof Download PDFInfo
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Abstract
The invention discloses a kind of aspergillus niger (Aspergillus niger) bacterial strain FS UV1, deposit number CCTCC NO:M 2013553.This bacterial strain has good degradation effect to AFB1, and degradation rate reaches 95.3%；More former bacterial strain improves 37.8%；The degradation efficiency of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone is reached 93%, and more former bacterial strain improves 7%, and the degradation rate of vomitoxin is reached 52.6%, and more former bacterial strain improves 9.5%.Rat experiment proves that this bacterial strain is a kind of safely and effectively biodegrading process having no side effect.
Description
Technical field
The present invention relates to a kind of aspergillus niger, a kind of mutagenic obtained, AFB1 is had good degradation effect
Bacterial strain.
Background technology
Mycotoxin is especially very harmful to grain to food, and fungus can store after cereal crops field growing and results
During the biosynthesis of toxin and metabolism.Estimating according to FAO (Food and Agriculture Organization of the United Nation), whole world corn supply 25% is polluted by mycotoxin
And can not eat, the preventing and treating to mycotoxin becomes the guarantee instant cardinal task of food safety.And microbial control has
The advantages such as safe efficient, economy, application are strong, environmentally friendly, become the prevention and controls enjoying high praise in recent years.And it is true
Verticillium toxin endangers and surely belongs to more greatly AFB1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and vomitoxin.
Since nineteen sixty, the aflatoxicosis event of turkey was broken out in Britain, both at home and abroad aflatoxin is carried out greatly
Amount is deeply studied widely.Owing to the reproduction speed of fungus is fast, the quantity of spore is big, it is remote to spread, the therefore crops such as grain
In the pollution planting, gather in the crops, preserve and be highly prone in the course of processing Toxigenic fungi and aflatoxin.At 18 kinds had been found that
In aflatoxin, with AFB1Teratogenecity, mutagenicity and carcinogenecity the strongest.As far back as 1993, AFB1By international cancer
Research institution is set to mankind's I class carcinogen.
Research shows that various bacteria has Biological control effect to aflatoxin.As bacillus, burkholderia, streptomycete,
Streptococcus and lactic acid bacteria etc. all can suppress to produce the growth of poison mycete, produce toxic action.Smooth Xiao Yuan finds to separate from Egyptian tradition cheese
The extension brevibacterium gone out has stronger inhibitory action to Aspergillus flavus growth；Isolated bacillus licheniformis from Thailand's zymotic soybean paste
Aspergillus flavus can not only be suppressed to grow with bacillus pumilis, and the AFB of can also degrade 74～85%1；Cho isolates from Korea S's soy sauce
One bacillus pumilus, finds that her the hay element that this bacterium secretes can suppress to produce poison Aspergillus flavus and the growth of aspergillus parasiticus；Graceful from
Semen Sojae Preparatum is isolated the bacillus subtilis that a strain can suppress Aspergillus flavus to grow；Zhang Ting also isolates suppression from bacillus subtilis
The surfactant peptides of aspergillus spore sprouting and bacillomycin D.Therefore isolate one and aflatoxin is had good preventing and treating and fall
The bacterial strain tool solving effect is of great significance.
Ultraviolet mutagenesis is most widely used a kind of new approaches of physical mutagenesis, and Guo Jiping uses ultraviolet mutagenesis to carry out aspergillus oryzae K61 bacterial strain
Transformation, finishing screen selects the mutant Y29 that a strain prolease activity is high and heritability is stable；Pan Tao sieves during ultraviolet mutagenesis
Choose a strain and stablize superior strain 10min-5, citric acid yield adds 5.33%；Zhao Qiong etc. are with acetobacter xylinum (Acetobacter
Xylinum) C5 is starting strain, and it is carried out ultraviolet mutagenesis, using irradiation time 3min as ultraviolet mutagenesis dosage, through primary dcreening operation and
Multiple sieve, obtains 1 strain Bacterial cellulose superior strain A.xylinumC544, and the yield of this mutant is 1.5 times of original strain；Visible
Bacterial strain often can show the characteristic more more excellent than former bacterial strain after mutation.Thus, bacterial strain is carried out mutagenesis screening selection-breeding
Go out bacterial strain preferable to aflatoxin degradation effect to have a good application prospect.
Summary of the invention
The invention provides a kind of Novel black aspergillosis (Aspergillus niger), be preserved in China on November 6th, 2013
Type Tissue Collection, deposit number CCTCC NO:M 2013553；Based on its 18SrDNA complete sequence it is
System grows tree as shown in Figure of description 1.
The invention provides a kind of mutation aspergillus niger, this bacterial strain has good degradation effect to AFB1, and degradation rate reaches
To 95.3%；More former bacterial strain improves 37.8%；The degradation efficiency of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone is reached 93%, and more former bacterial strain improves 7%,
The degradation rate of vomitoxin is reached 52.6%, and more former bacterial strain improves 9.5%.
Described aspergillus niger storage conditions is as follows: access from well-grown solid state flat panel culture medium PDA 3 ring mutation aspergillus nigers to
In fermentation medium, 28 DEG C of shaking tables (200rpm) cultivate 36h, take 0.65mL and move into the glycerol containing 0.3mL sterile glycerol
Guan Zhong, puts into-70 DEG C of ultra cold storage freezers and preserves.
Described solid state flat panel culture medium PDA is (w/v): Rhizoma Solani tuber osi 300g, glucose 20g, agar 20g, chloromycetin 0.1g,
Distilled water 1L, natural pH, 121 DEG C of autoclaving 15min.
Described liquid fermentation medium PDB is (w/v): Rhizoma Solani tuber osi 300g, glucose 20g, distilled water 1L, natural pH,
121 DEG C of autoclaving 15min.
The condition of culture of described mutation aspergillus niger is: the mutation aspergillus niger of preservation glycerol pipe accesses fluid medium, 28 DEG C,
130rpm, 36h cultivated by shaking table.
The preparation method of described aspergillus niger is: the aspergillus niger parental generation screened from beans unstrained spirits be inoculated in PDA solid medium,
28 DEG C of 130rpm, cultivate 36h, take 50ml normal saline and wash lower spore, shaken well, form monospore suspension, make
108The bacteria suspension of CFU/ml.Take above-mentioned spore suspension 5ml, through 200w ultra-vioket radiation 5min, heating 80s when microwave is in top grade.
By the inoculation after mutation in PDB culture medium, from bacterium colony, the suitable spore of picking makes 10 in a manner described8CFU/ml bacterium is hanged
Liquid, is inoculated in 30ml culture medium, 28 DEG C of 130rpm, and 36h cultivated by shaking table.Draw 10ul fermentation liquid enterprising in casein plate
The multiple sieve of row.By in bacterial strain after above-mentioned multiple sieve and former inoculation to 30ml culture medium, 28 DEG C of 130rpm, 36h cultivated by shaking table.To
Fermentation liquid adds 0.05ppm AFB1,0.5ppm 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and 0.5ppm vomitoxin, relatively former bacterial strain and novel lure
Become the bacterial strain degradation effect to AFB1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and vomitoxin.
The present invention be from beans unstrained spirits isolated through ultraviolet microwave complex mutation, AFB1 is had the novel of good degradation effect
Bacterial strain, reaches 95.3% to the degradation rate of AFB1, and more former bacterial strain improves 37.8% to AFB1；To Gibberella zeae alkene
The degradation efficiency of ketone reaches 93%, and more former bacterial strain improves 7%；The degradation rate of vomitoxin is reached 52.6%, and more former bacterial strain carries
High by 9.5%.Bacterial strain after mutation can apply food, agricultural product, the degraded of multiple mycotoxin in the field such as grain and feedstuff
In, reduce the purpose of mycotoxin especially AFB1 content.
Accompanying drawing explanation
Fig. 1: the phylogenetic tree of Novel black aspergillosis FS-UV1
Fig. 2: Novel black aspergillosis FS-UV1 is to AFB1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, the degradation effect of vomitoxin
Fig. 3: the enzymatic activity of Novel black aspergillosis FS-UV1
Toxin rat experiment slice map in Fig. 4: Novel black aspergillosis FS-UV1 degrading maize slurry
A: feedstuff group；B: standard substance group；C: Semen arachidis hypogaeae dregs group；D: experimental group.
Detailed description of the invention
Embodiment 1: the screening technique of aspergillus niger in beans unstrained spirits
The non-product aeontium shape isolated qualitatively in beans unstrained spirits by the PDA culture medium containing beta-schardinger dextrin-from the beans after fermentation 4d is true
Bacterium, then carries out quantitative analysis by ELISA method to the Toxin producing C of institute's isolated strains, finally filters out the non-mycete producing poison.
To Aspergillus flavus toxigenic bacterium can be suppressed to carry out multiple sieve, filter out the AFB that can degrade1Bacterial strain, degradation rate is 58%.Filter out
AFB1Preventing and treating bacterial strain.
Observed by colony characteristics and individual morphology is observed, and by the 18S rDNA of bacterial strain and GenBank data base
Known array contrast in (http://blast.ncbi.nlm.nih.gov/Blast.cgi), finds itself and aspergillus niger (Aspergillus niger)
Homology reaches 100%.Morphological characteristic and molecular biology identification in conjunction with bacterial strain, it may be determined that bacterial strain is aspergillus niger.The preservation of this bacterial strain
In China typical culture collection center, deposit number is: CCTCC M 2013703.
Embodiment 2: ultraviolet microwave complex mutation aspergillus niger preparation method
Take above-mentioned spore suspension 5ml, through 200w ultra-vioket radiation 5min, heating 80s when microwave is in top grade.By the bacterial strain after mutation
Being inoculated in PDB culture medium, from bacterium colony, the suitable spore of picking makes 10 in a manner described8CFU/ml bacteria suspension, is inoculated into 30ml
In culture medium, 28 DEG C of 130rpm, 36h cultivated by shaking table.Draw 10ul fermentation liquid on casein plate, carry out multiple sieve.Pass through bacterium
Fall observation of characteristics, and individual morphology is observed, and by the 18S rDNA of bacterial strain and GenBank data base
Known array contrast in (http://blast.ncbi.nlm.nih.gov/Blast.cgi), finds itself and aspergillus niger (Aspergillus niger)
Homology reaches 100%.Morphological characteristic and molecular biology identification in conjunction with bacterial strain, it may be determined that bacterial strain is aspergillus niger.Novel black aspergillosis
The cladogram of FS-UV1 such as Fig. 1.This bacterial strain is preserved in China typical culture collection center on November 6th, 2013, protects
Hiding numbering CCTCC NO:M 2013553, preservation address is Wuhan, China Wuhan University.
Embodiment 3: the Novel black aspergillosis degraded to multiple mycotoxin
By in above-mentioned bacterial strains FS-UV1 and former inoculation to 30mlPDB culture medium, 28 DEG C of 130rpm, 36h cultivated by shaking table.To
Fermentation liquid adds 0.05ppmAFB1,0.5ppm 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and 0.5ppm vomitoxin, relatively former bacterial strain and novel lure
Become the bacterial strain degradation effect to AFB1,6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone and vomitoxin.
1) the Novel black aspergillosis FS-UV1 degraded to AFB1
By in complex mutation bacterial strain FS-UV1 and former inoculation to 30mlPDB culture medium, 28 DEG C of 130rpm, 36h cultivated by shaking table.
In fermentation liquid, add 0.05ppm AFB1, therefrom draw 1ml and add 3ml chloroform, being extracted twice, nitrogen
Air-blowing is done, and adds trifluoroacetic acid 100ul, normal hexane 200ul, derivatization 30min, and application high performance liquid chromatography detects.
Chromatographic condition is C18 post 6mm × 150mm × 5um；Flowing phase acetonitrile: water=20:80 detects temperature: 30 DEG C；Flow velocity:
1ml/min；Detection wavelength: excitation wavelength: 360nm；Launch wavelength: 440nm.
2) the Novel black aspergillosis FS-UV1 degraded to 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone
By in complex mutation bacterial strain FS-UV1 and former inoculation to 30mlPDB culture medium, 28 DEG C of 130rpm, shaking table is trained
Support 36h.In fermentation liquid, add 0.5ppm 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone standard substance, more therefrom draw 1ml and add 3ml chloroform,
Being extracted twice, nitrogen dries up, and high performance liquid chromatography carries out content detection.Chromatographic condition is chromatographic column: C18 post 6mm × 150mm
×5um；Flowing phase: acetonitrile: water=50:50；Detection temperature: 25 DEG C；Flow velocity: 1ml/min；Detection wavelength: excitation wavelength:
240nm；Launch wavelength: 440nm
3) the Novel black aspergillosis FS-UV1 degraded to vomitoxin
By in bacterial strain after above-mentioned mutation and former inoculation to 30mlPDB culture medium, cultivate 36h for 28 DEG C.Add in fermentation liquid
Enter 0.5ppm vomitoxin standard substance, draw 10ml, more therefrom absorption 1ml addition 3ml chloroform is extracted twice,
Nitrogen dries up, and high performance liquid chromatography carries out content detection.Chromatographic condition: chromatographic condition is chromatographic column: C18 post 6mm × 150mm
×5um；Flowing phase: methanol: water=20:80；Detection temperature: 30 DEG C；Flow velocity: 0.8ml/min；Detection wavelength: 218nm.
Result is as in figure 2 it is shown, the Novel black aspergillosis FS-UV1 (the CCTCC NO:M 2013553) fall to AFB1
Solution rate reaches 95.3%, and more former bacterial strain improves 37.8% to AFB1；The degradation efficiency of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone is reached 93%,
More former bacterial strain improves 7%；The degradation rate of vomitoxin is reached 52.6%, and more former bacterial strain improves 9.5%.
Embodiment 4: the preventing and treating of AFB1 in the Semen arachidis hypogaeae dregs of Novel black aspergillosis FS-UV1 contratoxin pollution, cottonseed meal
Take appropriate complex mutation aspergillus niger FS-UV1 (CCTCC NO:M 2013553) spore inoculating in aflatoxin contamination
Cottonseed meal or Semen arachidis hypogaeae dregs in (AFB1 content 68 μ g/mL), 28 DEG C of fermentation culture 7d, take 40g cottonseed meal or
Semen arachidis hypogaeae dregs is pulverized sample and is dissolved in 100ml acetonitrile-water (9:1) solution, and 2min, qualitative filter paper mistake are extracted in the stirring of high speed homogenization device
Filter, pipettes 10ml filtrate and adds the dilution mixing of 40ml water, and glass filter paper filters 1～2 time and clarifies to filtrate.Take appropriate filtrate with
Chloroform presses 1:2 hybrid extraction twice.Nitrogen dries up, and is dissolved in 1.0ml flowing phase, to be measured.Through high effective liquid chromatography for measuring, send out
In existing cottonseed meal or Semen arachidis hypogaeae dregs, AFB1 residual quantity is less than 2 μ g/mL, and degradation rate reaches 98%.
Embodiment 5: catabolite rat animal verification experimental verification
Rat feeds front fasting 12h, freely drinks water, and claims weight record, carries out labelling, be randomly divided into 4 groups, it may be assumed that raise
Feed normal diet group A (being called for short: blank group)；Gavage AFB1 standard substance group B (is called for short: matched group)；Feed by
Semen arachidis hypogaeae dregs group C (being called for short: Semen arachidis hypogaeae dregs group) of aflatoxin contamination；Feed pollution Semen arachidis hypogaeae dregs and fermentation of Aspergillus niger liquid co-cultures
48h experimental group D (is called for short: degraded group), often organizes each 8.Each group mice every day same time feeds process, and free choice feeding is drunk
Water, continues 30 days, finally takes off neck and puts to death.Taking out liver and the kidney of rat, through paraffin embedding, section, HE dyes,
100 × and 400 × optical microphotograph Microscopic observation its tissue slice Fig. 4 (left side is liver, and the right side is kidney).It was found that flower
Raw dregs of rice group is high due to content of toxins, causes the Liver and kidney apparent damage of rat；And the rat Liver and kidney of degraded group does not finds significantly to organize
Damaging, this explanation fermentation of Aspergillus niger liquid can be degraded the aflatoxin in Semen arachidis hypogaeae dregs, and makes its lack of toxicity.Therefore, this is black
The degraded of aspergillosis contratoxin is the bacterial strain of safety non-toxic by-product, provides the poison-removing method of a kind of highly effective and safe for animal feed.
It is understood that for those of ordinary skills, can be according to technical scheme and inventive concept thereof
In addition equivalent or change, and all these change or replace the protection domain that all should belong to appended claims of the invention.
Claims (5)
1. aspergillus niger (Aspergillus niger) FS-UV1, is preserved in Chinese Typical Representative culture on November 6th, 2013
Preservation center, deposit number is: CCTCC NO:M 2013553, and preservation address is Wuhan, China, Wuhan University.
Aspergillus niger the most according to claim 1 is applied to AFB1 in the degraded Semen arachidis hypogaeae dregs of endotoxin contamination, cottonseed meal.
Aspergillus niger the most according to claim 1 is applied to 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone in the degraded Semen arachidis hypogaeae dregs of endotoxin contamination, cottonseed meal.
Aspergillus niger the most according to claim 1 is applied to vomitoxin in the degraded Semen arachidis hypogaeae dregs of endotoxin contamination, cottonseed meal.
Aspergillus niger the most according to claim 1 is in food, the application of field of fodder.
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