CN103966147A - Bacillus amyloliquefacien for degrading aflatoxin B1 in peanut meal - Google Patents
Bacillus amyloliquefacien for degrading aflatoxin B1 in peanut meal Download PDFInfo
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Abstract
The invention discloses a bacillus amyloliquefacien for degrading aflatoxin B1 in peanut meal, and belongs to the technical field of applied microbiology. According to the invention, a bacillus amyloliquefacien capable of significantly inhibiting the growth of Aspergillus flavus and efficiently degrading aflatoxin B1 can be obtained by screening and the content of aflatoxin B1 in detoxified peanut meal is lower than the national limited standard and achieves the safe feeding level after the bacillus amyloliquefacien is applied to moldy peanut meal. Furthermore, the detoxification mechanism of the strain is the degradation effect of extracellular metabolites, and generation of the active substance is of a non-induced type, which is an inherent attribute of the strain. The features of the strain provide the possibility for biocontrol of Aspergillus flavus and aflatoxin B1 in industries such as feed and foods and the strain has very high application values.
Description
Technical field
The present invention relates to AFB in a strain degraded peanut meal
1bacillus amyloliquefaciens, belong to using microbe technical field.
Background technology
Peanut meal is a kind of good vegetable protein feed, and annual output is at 3,000,000 tons, wide material sources; Rich in protein; Metabolizable energy is also the highest in dregs of rice class feed; Contain 8 kinds of indispensable amino acids simultaneously; But peanut meal very easily pollutes aflatoxin.
Aflatoxin is the strong toxicity secondary metabolite that a class is mainly produced by flavus (Aspergillus flavus) and Aspergillus parasiticus (Aspergillus parasiticus).1993, international cancer tissue (International Agency for Research onCancer, IARC) was classified as one-level carcinogens.Aflatoxin is mainly by harm animal and HUMAN HEALTH such as carcinogenic, teratogenesis, mutagenic " three cause " effect and immunosuppression.The target organ of its harm is mainly liver.The aflatoxin having identified at present has kind more than 20, and AFB
1being the most common in food, feed, is also toxicity maximum.
At present mainly contain Physical, chemical method and biological process for removing the method for aflatoxin.And biological process is with its treatment condition gentleness, does not destroy product quality, and can increase product nutritive value and extensively be approved.
Mainly contain at present the adsorption of the somatic cells such as milk-acid bacteria and yeast contratoxin for the biological detoxification method of aflatoxin, but its mechanism of action be thalline and aflatoxin by hydrophobic interaction with non covalent bond form in conjunction with forming thalline-aflatoxin complex body, this adsorption is physical properties, reversible, possibility desorption is attached under certain condition, aflatoxin still has the toxicity of itself, is not effectively removed.The enzyme that microorganism fermentation produces or the Degradation of plant milk extract contratoxin, as the metabolic enzyme that the orange myxococcus of being reported, mycobacterium, Rhodococcus, stenotrophomonas maltophilia, armillariella tabescens, white-rot fungi etc. are produced, the horseradish oxydase in radish etc. has the effect of obvious degradation aflatoxin.Be to be confined to the laboratory study stage but relevant research is mostly, the research of industrial applications is also few.
A strain provided by the invention can reduce AFB in peanut meal
1bacillus amyloliquefaciens and application, bacterial strain not only significantly suppresses flavus growth in process of growth, reduces AFB from source
1produce; And existing AFB in can efficient degradation peanut meal
1.The detoxification mechanism of bacterial strain is the Degradation of born of the same parents' extra-metabolite, produces by non-inducing action, has very high industrial application value.
Summary of the invention
First technical problem that the present invention will solve is to provide a bacillus amyloliquefaciens (Bacillus amyloliquefaciens), this bacterium has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 8th, 2014, deposit number is CGMCC No.9021, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.Described bacillus amyloliquefaciens can suppress the growth of flavus, and aflatoxin degradation.
The preferred AFB of described aflatoxin
1.
Described bacillus amyloliquefaciens has following characteristic:
(1) more easily cultivate
On broth agar culture medium, colony shape is irregular, has gauffer, and edge is irregular, canescence, central uplift, bacterium colony thickness.While growth, form white wrinkle mould in broth culture.The gram stain microscopy positive, thalline central part has gemma.In broth culture, cultivate 12h and can reach rapidly 10
9cFU/mL, and culture medium prescription is simple.
(2) can suppress flavus growth
Bacillus amyloliquefaciens can significantly suppress flavus growth in process of growth, reduces AFB from source
1generation.
(3) aflatoxin degradation B
1composition be extracellular products, and non-induction produce
This bacterial strain is by extracellular enzyme aflatoxin degradation, but not thalline adsorption; And what extracellular enzyme stopped up flavus is degraded to non-inducibility.
Second technical problem that the present invention will solve is to provide the described bacillus amyloliquefaciens of a kind of application and reduces AFB in peanut meal
1method, be the bacillus amyloliquefaciens of inoculating activated cultivation in the peanut meal after sterilizing.
The preferred following steps of described application method: pollution is had to AFB
1peanut meal sample pulverize, dry, weigh 20～50g, add in 250mL fermentation flask high pressure steam sterilization; The bacillus amyloliquefaciens seed liquor that activates, spreads cultivation, with 5～15% inoculum sizes, in the cooled peanut meal of material-water ratio 1:0.5～1 access, is stirred; At 35～40 DEG C of condition bottom fermentation 60～72h of leavening temperature, turn once every 6h, it is mixed again; Peanut meal after fermentation is dried, pulverized.
A strain provided by the invention can reduce AFB in peanut meal
1the bacillus amyloliquefaciens of content and the growth of inhibition flavus, has not only suppressed flavus and has produced aflatoxin from source, and aflatoxin has been realized to the dual function of efficient degradation.This bacterium bacterium is not by thalline adsorption, but has really realized AFB in enzymolysis peanut meal
1, more safe and reliable.The non-induction type Degradation of bacterial strain extracellular enzyme, is very beneficial for industrialized utilization simultaneously.This bacterium application process is simple, easy to operate, and biological degradation method mild condition is not destroyed product quality, can make to pollute peanut meal and be utilized effectively, and can increase product nutritive value, has very high industrial application value.
Biomaterial preservation
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 8th, 2014, deposit number is CGMCC No.9021, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
Brief description of the drawings
Fig. 1 is bacillus amyloliquefaciens inhibition to flavus growth in process of growth.
Embodiment
The characteristic of embodiment 1 bacillus amyloliquefaciens CGMCC NO.9021
(1) more easily cultivate
Bacillus amyloliquefaciens colony shape on broth agar culture medium is irregular, has gauffer, and edge is irregular, canescence, central uplift, bacterium colony thickness.While growth, form white wrinkle mould in broth culture.The gram stain microscopy positive, thalline central part has gemma.In broth culture, cultivate 12h and can reach rapidly 10
9cFU/mL, and culture medium prescription is simple, and industrial applications will be more economically, practicality.
(2) can suppress flavus growth
Bacillus amyloliquefaciens significantly suppresses flavus (as flavus CGMCC NO.3.4408, CGMCCNo.3.4410) growth in process of growth, has reduced AFB from source
1produce.As shown in Figure 1, filter paper growth around has bacillus amyloliquefaciens, centre to present yellow-green colour repressed to be flavus (CGMCC NO.3.4408), to have obvious inhibition zone.Bacillus amyloliquefaciens has restraining effect clearly to flavus, is because the metabolism in process of growth of this bacterial strain produces active substance, suppresses the growth of flavus.
(3) aflatoxin degradation B
1composition be extracellular products, and non-induction produce
As shown in table 1,2, the extracellular fluid after bacillus amyloliquefaciens liquid fermenting has obvious degradation AFB
1effect, and its somatic cells, intracellular fluid are to AFB
1degradation effect is very micro-; Extracellular fluid is heated or adds proteolytic enzyme-K to process, and the action effect of extracellular fluid almost disappears; And having, without AFB
1in the situation that substrate exists, the extracellular fluid after fermentation is to AFB
1not obviously difference of Degradation.Above phenomenon illustrates the Degradation that this bacterial strain is extracellular enzyme to the mechanism of degradation of aflatoxin, but not the adsorption of thalline, and this enzyme be produced as non-inducibility, these are all conducive to the industrial applications of this bacterial strain.
Table 1
Table 2
Embodiment 2 bacillus amyloliquefaciens CGMCC NO.9021 are to low levels AFB in peanut meal
1degradation
Bacillus amyloliquefaciens on picking inclined-plane, in broth culture, carries out one-level activation in 24 hours, then spreads cultivation 12 hours through the broth culture secondary containing 3% (m/v) peanut meal under 37 DEG C, 200r/min oscillating condition.Pollution is had to AFB
1peanut meal sample pulverize, dry, accurately weigh 50g, add in 250mL fermentation flask high pressure steam sterilization.The bacillus amyloliquefaciens seed liquor that secondary is spread cultivation accesses in cooled peanut meal with 10% (v/w) inoculum size, and adjustment material-water ratio is 1:0.8, under aseptic condition, stirs, and bottleneck wraps up with 8 layers of gauze.Postvaccinal fermentation flask is placed in to 37 DEG C of constant incubators, and fermentation 72h, stirs the peanut meal in fermentation flask every 6h between yeast phase.By the 60 DEG C of oven dry of peanut meal after fermentation, pulverizing.Accurately take 20g, extract wherein AFB with solid phase extraction
1, utilize its content of high-performance liquid chromatogram determination.
AFB in the peanut meal sample that the present embodiment adopts
1content is 12.32 μ g/kg, and after the detoxification of bacillus amyloliquefaciens solid state fermentation, its content is reduced to 1.45 μ g/kg, and virus elimination rate reaches 88.24%.Do not inoculate AFB in the control group of bacillus amyloliquefaciens
1content is 16.52 μ g/kg.
Embodiment 3 bacillus amyloliquefaciens CGMCC NO.9021 are to high-content AFB in peanut meal
1degradation
Bacillus amyloliquefaciens on picking inclined-plane, in broth culture, carries out one-level activation in 24 hours, then spreads cultivation 12 hours through the broth culture secondary containing 3% (m/v) peanut meal under 37 DEG C, 200r/min oscillating condition.Pollution is had to AFB
1peanut meal sample pulverize, dry, accurately weigh 50g, add in 250mL fermentation flask high pressure steam sterilization.The bacillus amyloliquefaciens seed liquor that secondary is spread cultivation is with 10% inoculum size, and 1:0.8 material-water ratio accesses in cooled peanut meal, under aseptic condition, stirs, and bottleneck wraps up with 8 layers of gauze.Postvaccinal fermentation flask is placed in to 37 DEG C of constant incubators, and fermentation 72h, stirs the peanut meal in fermentation flask every 6h between yeast phase.By the 60 DEG C of oven dry of peanut meal after fermentation, pulverizing.Accurately take 20g, extract wherein AFB with solid phase extraction
1, utilize its content of high-performance liquid chromatogram determination.
AFB in the peanut meal sample that the present embodiment adopts
1content is 145.17 μ g/kg, and after the degraded of bacillus amyloliquefaciens solid state fermentation, its content is reduced to 27.01 μ g/kg, and virus elimination rate reaches 81.39%.Do not inoculate AFB in the control group of bacillus amyloliquefaciens
1content is 141.14 μ g/kg.
In sum, this bacterium is not only to low AFB
1the peanut meal sample of content (12.32 μ g/kg) has 88.24% virus elimination rate; And to higher AFB
1the peanut meal sample of content (145.17 μ g/kg) also has 81.39% virus elimination rate, its AFB after detoxification
1content is less than 30 μ g/kg, meets country to AFB in feed
1the limitation of content requires (≤50 μ g/kg), has reached feeding safety standards, is applicable to being applied to the industrialization detoxification treatment to aflatoxin B1 in moldy feed.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, therefore protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (7)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens), this bacterium has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 8th, 2014, deposit number is CGMCC No.9021, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
2. bacillus amyloliquefaciens according to claim 1, is characterized in that, can suppress the growth of flavus, and aflatoxin degradation B
1.
3. described in an application rights requirement 1, bacillus amyloliquefaciens reduces AFB
1the method of content, is characterized in that, is the described bacillus amyloliquefaciens of inoculation, suppresses growth or the aflatoxin degradation B of aflatoxin generation bacterium
1.
4. application rights requires bacillus amyloliquefaciens described in 1 to suppress the method that flavus grows, and it is characterized in that, is to flavus growth Inoculation bacillus amyloliquefaciens.
5. described in an application rights requirement 1, bacillus amyloliquefaciens reduces AFB in peanut meal
1the method of content, is characterized in that, is the bacillus amyloliquefaciens of inoculating activated cultivation in the peanut meal after sterilizing.
6. described in an application rights requirement 1, bacillus amyloliquefaciens is prepared AFB
1the method of peanut meal that content reduces, is characterized in that, is the bacillus amyloliquefaciens of inoculating activated cultivation in the peanut meal after sterilizing.
7. according to the method described in claim 5 or 6, it is characterized in that, concrete steps comprise: pollution is had to AFB
1peanut meal sample pulverize, dry, weigh 20～50g, add in 250mL fermentation flask high pressure steam sterilization; The bacillus amyloliquefaciens seed liquor that activates, spreads cultivation is accessed to cooled peanut meal with 5～15% inoculum sizes, and adjustment material-water ratio is 1:0.5～1, stirs; At 35～40 DEG C of condition bottom fermentation 60～72h of leavening temperature, turn once every 6h, it is mixed again; Peanut meal after fermentation is dried, pulverized.
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Cited By (15)
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| CN104397355A (en) * | 2014-12-04 | 2015-03-11 | 嘉兴学院 | Method for removing aflatoxin from feed through fermentation and preparation method of fermented feed |
| CN105112394A (en) * | 2015-09-11 | 2015-12-02 | 江南大学 | Separation and purification method of lyase capable of degrading aflatoxin B1 |
| CN105166602A (en) * | 2015-08-07 | 2015-12-23 | 山东省花生研究所 | Method for degrading aflatoxins in peanut meal by utilizing solid fermentation of fructificatio amaurodermatis rudae |
| CN106065396A (en) * | 2015-11-24 | 2016-11-02 | 沈阳农业大学 | A kind of vegetable bacillus cereus and cultural method thereof and application |
| CN106720931A (en) * | 2016-12-06 | 2017-05-31 | 江南大学 | A kind of peanut protein of low SNSP and preparation method thereof |
| CN108251458A (en) * | 2018-01-25 | 2018-07-06 | 青岛农业大学 | A kind of degradation agent of aflatoxin, preparation method and application |
| CN108456648A (en) * | 2018-03-04 | 2018-08-28 | 河南亿万中元生物技术有限公司 | A kind of method of aflatoxin B1 in degradation peanut meal |
| CN109744367A (en) * | 2019-03-22 | 2019-05-14 | 东莞市澹一生物科技有限公司 | The method and feedstuff of resource utilization wheat vinasse production feedstuff |
| CN109744368A (en) * | 2019-03-22 | 2019-05-14 | 东莞市澹一生物科技有限公司 | Utilize the method and feedstuff of wheat vinasse production feedstuff |
| CN110042072A (en) * | 2019-04-08 | 2019-07-23 | 山东鲁花集团有限公司 | A kind of aflatoxin degradation B1Bacterial strain and its application |
| CN110583964A (en) * | 2019-09-23 | 2019-12-20 | 江南大学 | Biological removal method for efficiently removing four aflatoxins in peanut meal |
| CN113632878A (en) * | 2021-08-09 | 2021-11-12 | 云南师范大学 | Enzyme-bacterium composite biological agent for degrading mycotoxin in fermented unconventional feed and application |
| CN113995082A (en) * | 2021-10-17 | 2022-02-01 | 河南工业大学 | Aflatoxin B1Preparation of the degradation agent |
| CN114097459A (en) * | 2021-10-19 | 2022-03-01 | 中国农业科学院油料作物研究所 | Method for preventing and controlling aspergillus flavus and toxin thereof and increasing number of root nodules with nitrogenase activity of leguminous crops and application of method |
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| CN104397355A (en) * | 2014-12-04 | 2015-03-11 | 嘉兴学院 | Method for removing aflatoxin from feed through fermentation and preparation method of fermented feed |
| CN105166602A (en) * | 2015-08-07 | 2015-12-23 | 山东省花生研究所 | Method for degrading aflatoxins in peanut meal by utilizing solid fermentation of fructificatio amaurodermatis rudae |
| CN105112394A (en) * | 2015-09-11 | 2015-12-02 | 江南大学 | Separation and purification method of lyase capable of degrading aflatoxin B1 |
| CN106065396A (en) * | 2015-11-24 | 2016-11-02 | 沈阳农业大学 | A kind of vegetable bacillus cereus and cultural method thereof and application |
| CN106720931A (en) * | 2016-12-06 | 2017-05-31 | 江南大学 | A kind of peanut protein of low SNSP and preparation method thereof |
| CN108251458A (en) * | 2018-01-25 | 2018-07-06 | 青岛农业大学 | A kind of degradation agent of aflatoxin, preparation method and application |
| CN108251458B (en) * | 2018-01-25 | 2019-02-26 | 青岛农业大学 | A kind of degradation agent of aflatoxin, preparation method and application |
| CN108456648A (en) * | 2018-03-04 | 2018-08-28 | 河南亿万中元生物技术有限公司 | A kind of method of aflatoxin B1 in degradation peanut meal |
| CN109744367A (en) * | 2019-03-22 | 2019-05-14 | 东莞市澹一生物科技有限公司 | The method and feedstuff of resource utilization wheat vinasse production feedstuff |
| CN109744368A (en) * | 2019-03-22 | 2019-05-14 | 东莞市澹一生物科技有限公司 | Utilize the method and feedstuff of wheat vinasse production feedstuff |
| CN110042072A (en) * | 2019-04-08 | 2019-07-23 | 山东鲁花集团有限公司 | A kind of aflatoxin degradation B1Bacterial strain and its application |
| CN110042072B (en) * | 2019-04-08 | 2022-03-25 | 山东鲁花集团有限公司 | Aflatoxin B degradation1And application thereof |
| CN110583964A (en) * | 2019-09-23 | 2019-12-20 | 江南大学 | Biological removal method for efficiently removing four aflatoxins in peanut meal |
| CN113632878A (en) * | 2021-08-09 | 2021-11-12 | 云南师范大学 | Enzyme-bacterium composite biological agent for degrading mycotoxin in fermented unconventional feed and application |
| CN113995082A (en) * | 2021-10-17 | 2022-02-01 | 河南工业大学 | Aflatoxin B1Preparation of the degradation agent |
| CN114097459A (en) * | 2021-10-19 | 2022-03-01 | 中国农业科学院油料作物研究所 | Method for preventing and controlling aspergillus flavus and toxin thereof and increasing number of root nodules with nitrogenase activity of leguminous crops and application of method |
| CN114644995A (en) * | 2022-02-17 | 2022-06-21 | 华南农业大学 | Bacillus amyloliquefaciens for preventing and controlling aflatoxin B1 pollution and application thereof |
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