CN103937681A - Food-grade aspergillus niger strain and application of strain in zearalenone degradation - Google Patents
Food-grade aspergillus niger strain and application of strain in zearalenone degradation Download PDFInfo
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- CN103937681A CN103937681A CN201410147140.1A CN201410147140A CN103937681A CN 103937681 A CN103937681 A CN 103937681A CN 201410147140 A CN201410147140 A CN 201410147140A CN 103937681 A CN103937681 A CN 103937681A
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Abstract
The invention discloses a food-grade aspergillus niger strain FS-Z1 which is separated out from sauce grains and capable of preventing and treating zearalenone toxin. The collection number of the food-grade aspergillus niger strain FS-Z1 is CCTCC NO: M 2013703. The strain has excellent degradation effect on the zearalenone and the degradation rate reaches 89.56%. Besides, animal experiments in rats verify that the zearalenone in the corn steep liquor degraded by using an aspergillus niger fermentation liquor has no toxicity.
Description
Technical field
The present invention relates to a strain aspergillus niger, particularly from sauce unstrained spirits, screening and separating goes out can control of maize zeranol toxin food grade strain.
Background technology
Zearalenone (zearalenone, ZEA) is a kind of oestrogen-like hormone sample mycotoxins being produced by sickle-like bacteria, is to pollute in the world the most a kind of Fusarium toxin, and whole world cereal and feed are generally subject to the severe contamination of zearalenone.In China, bad grain harvest condition and Oil processing process have increased the weight of the pollution of ZEA, and being widely used of low-quality corn and food processing byproducts, particularly unconventional raw material, makes the pollution level of the mycotoxins such as ZEA far beyond abroad.The main toxicity of ZEA and metabolite thereof is owing to their estrus hormone activity and anabolic activity.Studies confirm that in a large number, ZEA and metabolite thereof can cause the mycotoxicosis of a lot of animals, affect internal organs and grow, and also can cause animal reproduction obstacle, digestive system function disorder, to livestock industry, bring very large financial loss.
At present, for the method for removing zearalenone in feed and food, according to its action principle, can divide chemical method, Physical and biological process three classes.Wherein physics detoxicity method also can cause the loss of animal-feed nutritive substance when removing zearalenone toxin; And although chemical detoxication method can be sloughed some more serious grain troughs that go mouldy, but time-consuming taking a lot of work, and nutritive ingredient is caused to great destruction, also may produce some chemical substances harmful to animal health, it should be noted that European Union does not allow applied chemistry method in food production to eliminate mycotoxins.Because traditional physics, chemical process removal endotoxin contamination exist inevitable limitation, utilize biological degradation method to remove ZEN toxin and become important research direction, the toxophore that biological degradation refers to mycotoxin molecule by the secondary metabolite of microorganisms or secreted born of the same parents, extracellular enzyme decomposes and destroys, and produces nontoxic degraded product process simultaneously.The method of this detoxification can not destroyed other compositions in the middle of raw material, can not reduce nutritive value.Therefore, biological degradation method is to remove the effective and the most feasible method of ZEN.
Research shows that multiple-microorganism has biological control effect to zearalenone.As Bacillus subtillis, acinetobacter, pseudomonas putida, micrococcus luteus, trichosporon, the microorganisms such as rhodotorula glutinis have stronger detoxification to zearalenone; Kakeya H etc. is studied being separated to Gliocladium roseum (C.rosea IF07063) bacterial strain, research finds that this bacterial strain can be converted into ZEN 1-(3,5 dihydroxyphenyls)-10 '-hydroxyl-1 ' trans undecylene-6 '-one, this degraded product is without any estrogen activity.Yu Yuanshan is successfully separated to a strain from agricultural soil can take ZEN and be the acinetobacter SM04 of sole carbon source and energy growth, and the supernatant fermented liquid of this bacterial strain has the ability of efficient degradation ZEN.Therefore isolating a kind of safe bacterial strain tool that zearalenone toxin is had to good control and a degradation effect is of great significance.
Summary of the invention
The invention provides strain food grade aspergillus niger FS-Z1 (Aspergillus niger) FS-Z1 bacterial strain, on December 24th, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number CCTCC M2013703; With its 18SrDNA total order, classify basic phylogenetic tree as shown in Figure of description 1.This bacterial strain has good degradation effect to zearalenone, and degradation rate reaches 89.56%.
Described aspergillus niger is that separation obtains from sauce unstrained spirits, its storage conditions is as follows: from well-grown solid-state plate culture medium, access 3 ring aspergillus niger spores in shake-flask culture base, 28 ℃ of shaking tables (150rpm) are cultivated 36h, get 0.65mL and move in the glycerine pipe that contains the aseptic glycerine of 0.3mL, put into-70 ℃ of Ultralow Temperature Freezers and preserve.
Described solid plate substratum PDA is (w/v): potato 300g, glucose 20g, agar 20g, distilled water 1L, natural pH, 121 ℃ of autoclaving 15min.
Described liquid nutrient medium PDB is (w/v): potato 300g, glucose 20g, distilled water 1L, natural pH, 121 ℃ of autoclaving 15min.
The culture condition of described aspergillus niger is: by the aspergillus niger access solid medium activation 3d of preservation glycerine pipe, and then be linked into liquid nutrient medium, and 28 ℃, 150rpm, shaking table is cultivated.
The screening conditions of described aspergillus niger are: the aspergillus niger screening from sauce unstrained spirits is inoculated into PDA solid medium, cultivates 36h for 28 ℃, get 50ml physiological saline and wash lower spore, vibration evenly, forms monospore suspension, makes 10
8the bacteria suspension of CFU/ml.Inoculum size by 2% is inoculated in the aseptic PDB substratum of 60ml, 28 ℃, 150rpm, shaking table is cultivated 5d, in fermented liquid, add zearalenone, make its final concentration reach 2ppm, the aseptic PDB nutrient solution of usining adds zearalenone as blank, respectively at detect the concentration of zearalenone after 48h by HPLC detection method.
The present invention also provides the application method of a kind of described aspergillus niger in degrading zearalenone, and the suitableeest degradation condition is bacterium liquid inoculum size 2%, 28 ℃ of degradation temperatures, degraded pH=5, rotating speed 150r/min, degradation time 48h.
Described aspergillus niger also belongs to protection scope of the present invention in the application of food, field of fodder.
The present invention be from sauce unstrained spirits separated obtain zearalenone is had to the safe bacterial strain of good degradation effect, degradation efficiency to zearalenone reaches 89.56%, this bacterial strain also can be applied food, agricultural-food, in the field such as grain and feed, in the degraded of multiple mycotoxins, reach and reduce the especially object of zearalenone content of mycotoxins.
Accompanying drawing explanation
Fig. 1: the phylogenetic tree of food grade aspergillus niger FS-Z1 (sample)
Fig. 2: the degradation effect of food grade aspergillus niger FS-Z1 to zearalenone in corn steep liquor
Fig. 3: toxicity (the 1-liver after rat animal experimental verification zearalenone is degraded; 2-kidney)
A: physiological saline group; B: corn steep liquor group; C: degraded group.D: nutrient solution group;
Embodiment
Embodiment 1: the screening method of aspergillus niger in sauce unstrained spirits
From the sauce fermentation 4d, with the PDA substratum containing beta-cyclodextrin, isolate qualitatively the malicious filamentous fungus of non-product in sauce unstrained spirits, 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.To suppressing flavus toxigenic bacterium, carry out multiple sieve, filter out the bacterial strain that can prevent and treat mycotoxins.
By colony characteristics, observe and individual morphology is observed, and by the 18S rDNA of bacterial strain and GenBank database (
http:// blast.ncbi.nlm.nih.gov/Blast.cgi) middle known array contrast, 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 with on December 24th, 2013, deposit number CCTCC M2013703, and preservation address is Wuhan, China university.
Embodiment 2: the degraded of aspergillus niger to zearalenone
The bacterial strain FS-Z1 that separation is obtained is inoculated in the aseptic PDB substratum of 60ml by 2% inoculum size, 28 ℃ of 150rpm, and shaking table is cultivated 5d.In fermented liquid, add zearalenone to make its final concentration reach 2ppm, the aseptic PDB nutrient solution of usining adds zearalenone as blank, cultivate respectively after 48h, from two groups of samples, draw 2ml liquid and add 4ml trichloromethane, extract 3 times, after nitrogen dries up, add again 1ml moving phase [acetonitrile: water=50:50 (v/v)] and redissolve, excessively after organic filter membrane of 0.22 μ m, by high performance liquid chromatography (HPLC), detect.
Chromatogram testing conditions is: chromatographic column: C18 post 6mm * 150mm * 5um; Moving phase: acetonitrile: water=50:50; Detected temperatures: 25 ℃; Flow velocity: 1ml/min; Detect wavelength: excitation wavelength: 274nm; Emission wavelength: 440nm; Sample size: 20 μ L; Elution time 20min; The retention time of zearalenone is at 6.5 ± 0.5min.
Result demonstration, the degradation rate of zearalenone reaches 89.56%.
Embodiment 3: the control of aspergillus niger FS-Z1 to the corn steep liquor of zearalenone endotoxin contamination
The bacterial strain FS-Z1 that separation is obtained is inoculated in the aseptic PDB substratum of 60ml by 2% inoculum size, 28 ℃ of 150rpm, and shaking table is cultivated 5d.With four layers of sterile gauze, the mycelia of aspergillus niger is filtered, obtain the fermented liquid of aspergillus niger.Fermented liquid is mixed to [1:1 (v/v)] vibration with corn steep liquor evenly, the aseptic PDB substratum of usining mixes as blank with corn steep liquor equal-volume, under the condition of 28 ℃ of 150rpm, cultivate 48h, through glass fiber filter paper, filter 1～2 time, clarify to filtrate, extract as stated above the zearalenone in sample, by high performance liquid chromatography, detect the content of residual zearalenone toxin.
Through high effective liquid chromatography for measuring, find that the concentration of zearalenone in 0h corn steep liquor is 29.06ppm, and 48h later in corn steep liquor the content of toxin be only 11.62ppm, degradation rate reaches 60%(Fig. 2).
Embodiment 4: after rat animal experimental verification zearalenone is degraded, toxicity changes
Fasting 12h before rat oral gavage, freely drinks water, and claims weight record, carries out mark, is divided at random 4 groups, that is: gavage physiological saline group (is called for short: physiological saline group A); The corn steep liquor group that gavage is polluted by zearalenone (is called for short: corn steep liquor group B); Gavage pollutes corn steep liquor and fermentation of Aspergillus niger liquid is cultivated 48h experimental group (abbreviation: degraded group C) altogether; The aseptic PDB nutrient solution of gavage group (is called for short: nutrient solution group D); , every group each 8.Each is organized mouse gavage of same time of every day and processes, and free choice feeding drinking-water, continues 30 days, and finally de-neck is put to death.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 figure.Found that, corn steep liquor group B, because zearalenone content of toxins is high, causes the liver kidney apparent damage of rat: hepatic cell fattydegeneration, focal necrosis; There is obvious atrophy in renal glomerulus, nephrocyte steatosis; And rats'liver kidney and the control group A of degraded group C, control group D does not all find obvious tissue damage, the zearalenone toxin of this explanation fermentation of Aspergillus niger liquid in can degrading maize slurry, and its toxicity is disappeared.Therefore, this aspergillus niger is the bacterial strain of safety non-toxic by product to the degraded of zearalenone, 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 (6)
1. strain food grade aspergillus niger FS-Z1 (Aspergillus niger) FS-Z1 bacterial strain, is preserved in Chinese Typical Representative culture collection center on December 24th, 2013, and deposit number is: CCTCC M2013703, preservation address is Wuhan, China Wuhan University.
2. aspergillus niger according to claim 1, is characterized in that this bacterial strain is through primary dcreening operation, to sieve again the isolated bacterial strain that can prevent and treat aflatoxin B1, zearalenone from sauce unstrained spirits.
3. the aspergillus niger described in claim 2, is characterized in that this bacterial strain can reach 89.56% to the degradation rate of zearalenone.
4. the application in degrading zearalenone according to the arbitrary described aspergillus niger of claim 1-3.
5. the method described in claim 4, is characterized in that this bacterial strain is bacterium liquid inoculum size 2% to the suitableeest degradation condition of zearalenone, 28 ℃ of degradation temperatures, degraded pH=5, rotating speed 150r/min, degradation time 48h.
6. the application in food, field of fodder according to the arbitrary described aspergillus niger of claim 1-3.
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Cited By (9)
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| CN105838614A (en) * | 2015-12-31 | 2016-08-10 | 湖南圣雅凯生物科技有限公司 | Method for degrading zearalenone through aspergillus niger |
| CN106858042A (en) * | 2017-03-31 | 2017-06-20 | 临沂众客饲料有限公司 | A kind of pretreated corn pulp and its direct-injection application method in animal and fowl fodder |
| CN106962615A (en) * | 2017-03-24 | 2017-07-21 | 中国科学院亚热带农业生态研究所 | A kind of method of the few spore mutation degrading zearalenone of utilization Rhizopus microsporus |
| CN110583964A (en) * | 2019-09-23 | 2019-12-20 | 江南大学 | Biological removal method for efficiently removing four aflatoxins in peanut meal |
| CN111685259A (en) * | 2020-04-26 | 2020-09-22 | 国家食品安全风险评估中心 | Method for reducing zearalenone |
| CN111820363A (en) * | 2019-04-17 | 2020-10-27 | 海南泓缘生物科技股份有限公司 | Biodegradation method for gibberellin ketene toxin in DDGS |
| 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 |
| CN113736668A (en) * | 2021-09-15 | 2021-12-03 | 江南大学 | Aspergillus niger strain and application thereof in ochratoxin A degradation |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838614A (en) * | 2015-12-31 | 2016-08-10 | 湖南圣雅凯生物科技有限公司 | Method for degrading zearalenone through aspergillus niger |
| CN105838614B (en) * | 2015-12-31 | 2019-11-05 | 湖南微草生物饲料科技有限公司 | A method of utilizing black-koji mould degrading zearalenone |
| CN106962615A (en) * | 2017-03-24 | 2017-07-21 | 中国科学院亚热带农业生态研究所 | A kind of method of the few spore mutation degrading zearalenone of utilization Rhizopus microsporus |
| CN106858042A (en) * | 2017-03-31 | 2017-06-20 | 临沂众客饲料有限公司 | A kind of pretreated corn pulp and its direct-injection application method in animal and fowl fodder |
| CN106858042B (en) * | 2017-03-31 | 2020-03-13 | 临沂众客饲料有限公司 | Pretreated corn steep liquor and direct injection use method thereof in livestock and poultry feed |
| CN111820363A (en) * | 2019-04-17 | 2020-10-27 | 海南泓缘生物科技股份有限公司 | Biodegradation method for gibberellin ketene toxin in DDGS |
| CN110583964A (en) * | 2019-09-23 | 2019-12-20 | 江南大学 | Biological removal method for efficiently removing four aflatoxins in peanut meal |
| CN111685259A (en) * | 2020-04-26 | 2020-09-22 | 国家食品安全风险评估中心 | Method for reducing zearalenone |
| 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 |
| CN113736668A (en) * | 2021-09-15 | 2021-12-03 | 江南大学 | Aspergillus niger strain and application thereof in ochratoxin A degradation |
| CN113736668B (en) * | 2021-09-15 | 2023-06-13 | 江南大学 | Aspergillus niger strain and application thereof in ochratoxin A degradation |
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Application publication date: 20140723 Assignee: GUANGZHOU GRG METROLOGY & TEST Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2022320000002 Denomination of invention: A food grade Aspergillus niger and its application in the degradation of zearalenone Granted publication date: 20160629 License type: Common License Record date: 20220106 Application publication date: 20140723 Assignee: Kaifeng Longda Vegetable Oil Co.,Ltd. Assignor: Jiangnan University Contract record no.: X2021320000195 Denomination of invention: A food grade Aspergillus niger and its application in the degradation of zearalenone Granted publication date: 20160629 License type: Common License Record date: 20220104 Application publication date: 20140723 Assignee: SHANGHAI XIONGTU BIOTECHNOLOGY CO.,LTD. Assignor: Jiangnan University Contract record no.: X2021320000193 Denomination of invention: A food grade Aspergillus niger and its application in the degradation of zearalenone Granted publication date: 20160629 License type: Common License Record date: 20220104 Application publication date: 20140723 Assignee: NANJING MICRODETECTION BIOLOGICAL TECHNOLOGY CO.,LTD. Assignor: Jiangnan University Contract record no.: X2021320000194 Denomination of invention: A food grade Aspergillus niger and its application in the degradation of zearalenone Granted publication date: 20160629 License type: Common License Record date: 20220104 |