CN104087528A - Bacillus pumilus and application thereof in degrading aflatoxin - Google Patents
Bacillus pumilus and application thereof in degrading aflatoxin Download PDFInfo
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- CN104087528A CN104087528A CN201410292766.1A CN201410292766A CN104087528A CN 104087528 A CN104087528 A CN 104087528A CN 201410292766 A CN201410292766 A CN 201410292766A CN 104087528 A CN104087528 A CN 104087528A
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Abstract
The invention provides a Bacillus pumilus and application thereof in degrading aflatoxin. The collection number of the Bacillus pumilus is CGMCC No.9086, and the metabolite can effectively degrade the aflatoxin.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of bacillus pumilus and the application in aflatoxin degradation thereof.
Background technology
Aflatoxin (Aflatoxins) is the similar compound of a class chemical structure, mainly by flavus (Aspergillus flavus) and Aspergillus parasiticus (A.parasiticus)) produce, isolation identification goes out tens kinds of aflatoxin such as B1, B2, G1, G2, M1, M2 at present, and its basic structure is dihydrofuran ring and tonka bean camphor.Aflatoxin is the extremely strong highly toxic substance of a kind of toxicity, and its hazardness is mainly that people and animal livers tissue are had to destruction, can cause liver cancer even dead when serious.
Flavus and Aspergillus parasiticus in air and soil extensively exist, its after the food crop such as infecting peanut, corn, can these crops with and derived product in produce aflatoxin.The most common with aflatoxin B1 in the food of natural contamination, its toxicity and carinogenicity are also the strongest.Due to tremendous economic loss and Health hazard that aflatoxin contamination brings, therefore domestic and international researchist is finding the method for aflatoxin degradation always.
At present, mainly comprise Physical, chemical method and biological process for the method for aflatoxin degradation.Physical is directly removed aflatoxin or aflatoxin is decomposed and change nontoxic intermediate product into, and it comprises extraction process, absorption method, heat treating process, radiation method etc.; Chemical method is mainly to use chemical reagent aflatoxin degradation, and conventional chemical reagent comprises alkali, oxygenant etc.Though Physical and chemical method have certain effect on aflatoxin degradation, but inevitably can destroy the nutritive ingredient of product, the quality of reduction product, also may produce by product and chemical agent residue simultaneously, thereby cause certain harm.
Biological process mainly utilizes antagonistic microbe and meta-bolites (for example enzyme) thereof to carry out aflatoxin degradation, its degradation condition gentleness, neither can destroy the nutritive ingredient of product, also can not produce by product and chemical residual, be a kind of comparatively desirable degradation method.Therefore, from various sources, the bacterial strain of (particularly comparatively safe plant origin) screening efficient degradation aflatoxin, has positive realistic meaning.
Summary of the invention
The invention provides a kind of bacillus pumilus and the application in aflatoxin degradation thereof, the meta-bolites of this bacillus pumilus is aflatoxin degradation effectively.
The invention provides a kind of bacillus pumilus, deposit number is CGMCC No.9086.
Bacillus pumilus provided by the present invention (Bacillus pumilus) is screened and obtains from flower of Greenish Lily, its safety non-toxic of originating, and this bacillus pumilus can produce the meta-bolites of aflatoxin degradation.
The present invention also provides the application of above-mentioned bacillus pumilus in aflatoxin degradation.
Further, described aflatoxin is aflatoxin B1 and/or aflatoxin M 1.
The present invention also provides a kind of method of aflatoxin degradation, comprises the steps:
1) bacillus pumilus described above is fermented, make fermented liquid;
2) described fermented liquid is contacted to for some time with the material that contains aflatoxin.
Bacillus pumilus of the present invention can grow and produce the meta-bolites of aflatoxin degradation under conventional substratum and conventional culture condition.For example, the composition of substratum can comprise conventional carbon source, nitrogenous source, salt etc., and culture condition can be temperature 35-39 DEG C, hunting speed 120-160r/min etc.
Further, adopt fermention medium to carry out described fermentation, described fermentation is mainly for generation of the meta-bolites of aflatoxin degradation.In the time of fermentation, the optimum carbon source of this bacillus pumilus is lactose, and optimum nitrogen source is yeast extract paste, and adds a certain amount of MgCl
2be conducive to improve the ability of its aflatoxin degradation.In concrete scheme of the present invention, the formula of described fermention medium is: lactose 0.5-1.0%, yeast extract paste 0.5-1.0%, MgCl
20.05-0.15%, and regulate the pH value of described fermention medium to 7.0-7.5.
Further, the condition of described fermentation can be: leavening temperature 35-39 DEG C, for example 37 DEG C; Hunting speed 120-160r/min, for example 140r/min; Fermentation time 48-96h, for example 72h.
In the present invention, the proportioning between the quality to the described material that contains aflatoxin and the volume of described fermented liquid is strictly restriction not, and fermented liquid usage quantity relatively hour can extend itself and duration of contact of the material that contains aflatoxin relatively.In concrete scheme of the present invention, described in contain aflatoxin the quality of material and the volume of described fermented liquid between proportioning for example can be for 1:(1.2-10), can be further 1:(5-10).
Further, described contact is carried out at the temperature of 35-39 DEG C, for example 37 DEG C; Contact time can determine according to the content of aflatoxin in the usage quantity of fermented liquid and material, can be 48-96h for example duration of contact, described for some time is 48-96h.
In the present invention, described in contain aflatoxin material do not do strict restriction, they can such as, for any material that contains aflatoxin, grain, food, feed etc.Further, described grain can be for being corn, rice, wheat etc., and described food can be soy sauce etc., and described feed can be peanut meal, soybean meal etc.
Bacillus pumilus of the present invention (Bacillus pumilus), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on April 24th, 2014, its address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, and deposit number is CGMCC No.9086.
Bacillus pumilus provided by the invention, source safety non-toxic, and can under relatively simple culture condition, produce the meta-bolites of aflatoxin degradation; The method of aflatoxin degradation provided by the invention, easy and simple to handle, controllability is strong, its degradation condition gentleness to the material that contains aflatoxin, therefore has a good application prospect.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiments of the invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
One, prepare substratum
3g extractum carnis, 5g peptone, 10g NaCl are joined in 1L distilled water, after stirring and dissolving, regulate pH value to 7.2 left and right, then, at 121 DEG C of steam sterilizing 20min, make seed culture medium;
By 5g lactose, 10g yeast extract paste, 1g MgCl
2join in 1L distilled water, after stirring and dissolving, regulate pH value to 7.2 left and right, then, at 121 DEG C of steam sterilizing 20min, make fermention medium.
Two, fermentation
Bacillus pumilus after flat board activation is inoculated in the seed culture medium of above-mentioned preparation, is to cultivate 12h under 37 DEG C, hunting speed (rotating speed) condition that is 140r/min in temperature, makes seed liquor;
Accessing the fermention medium of above-mentioned preparation according to 5% inoculum size, is 37 DEG C in temperature, under the condition that hunting speed is 140r/min, cultivates 72h, makes fermented liquid.
Embodiment 2
By 8g lactose, 8g yeast extract paste, 0.5g MgCl
2join in 1L distilled water, after stirring and dissolving, regulate pH value to 7.2 left and right, then, at 121 DEG C of steam sterilizing 20min, make fermention medium.
Seed liquor prepared by embodiment 1 accesses the fermention medium of above-mentioned preparation according to 3% inoculum size, be 35 DEG C in temperature, under the condition that hunting speed is 160r/min, cultivates 48h, makes fermented liquid.
Embodiment 3
By 10g lactose, 5g yeast extract paste, 1.5g MgCl
2join in 1L distilled water, after stirring and dissolving, regulate pH value to 7.2 left and right, then, at 121 DEG C of steam sterilizing 20min, make fermention medium.
Seed liquor prepared by embodiment 1 accesses the fermention medium of above-mentioned preparation according to 1% inoculum size, be 39 DEG C in temperature, under the condition that hunting speed is 120r/min, cultivates 96h, makes fermented liquid.
The degraded of test example 1 to aflatoxin B1
Get fermented liquid 1.8mL prepared by embodiment 1-3, added respectively in the centrifuge tube of sterilizing, be the aflatoxin B1 (purchased from Sigma company) of 1mg/L to adding respectively 0.2mL concentration in each centrifuge tube again, mixing and being placed on temperature is in the constant incubator of 37 DEG C, in 48h, 72h and 96h sampling, adopt aflatoxin B1 test kit (purchased from biological company limited of Huaan, Beijing wheat section) to measure the content of aflatoxin B1 respectively; Respectively using aseptic fermention medium as blank, result is as shown in table 1 simultaneously.
The degraded of test example 2 to aflatoxin M 1
Get fermented liquid 1.8mL prepared by embodiment 1-3, added respectively in the centrifuge tube of sterilizing, be the aflatoxin M 1 (purchased from Sigma company) of 1mg/L to adding respectively 0.2mL concentration in each centrifuge tube again, mixing and being placed on temperature is in the constant incubator of 37 DEG C, in 48h, 72h and 96h sampling, adopt aflatoxin M 1 test kit (purchased from biological company limited of Huaan, Beijing wheat section) to measure the content of aflatoxin M 1 respectively; Respectively using aseptic fermention medium as blank, result is as shown in table 1 simultaneously.
The degraded of table 1 fermented liquid to aflatoxin B1 and M1
As seen from the results in Table 1:
The fermented liquid of bacillus pumilus of the present invention is degrading aflatoxin B 1 and M1 effectively, and degradation rate increases along with the prolongation of duration of contact, particularly in the time of contact 96h, to degradation rate >=90% of aflatoxin B1, to degradation rate >=85% of aflatoxin M 1.
The degraded of test example 3 to aflatoxin B1 in peanut meal
Get peanut meal sample, after being pulverized, be divided into three parts, every part of 5g, the fermented liquid of preparing with 50mL embodiment 1-3 respectively mixes, the constant incubator that mixed sample is placed in to 37 DEG C is placed after 48h, in the centrifugal 5min of 4000r/min, get supernatant liquor 5ml, add 25ml methylene dichloride, after concussion 10min, in the centrifugal 5min of 4000r/min, take off a layer methylene dichloride 5ml, after nitrogen dries up at 50 DEG C, volatilize thing with the dissolve with methanol solution that 1mL concentration is 10%, adopt the content of aflatoxin B1 kit measurement aflatoxin B1; Simultaneously using aseptic fermention medium as blank.
After testing, the fermented liquid that prepared by embodiment 1-3 is respectively 78.8%, 74.5% and 76.2% to the degradation rate of aflatoxin B1 in peanut meal.
The degraded of test example 4 to aflatoxin B1 in corn
Get mouldy corn sample, after being pulverized, cross 20 mesh sieves, be divided into three parts by sieving thing, every part of 5g, the fermented liquid of preparing with 25mL embodiment 1-3 respectively mixes, and the constant incubator that mixed sample is placed in to 35 DEG C is placed after 72h, in the centrifugal 5min of 4000r/min, get supernatant liquor 5ml, adopt method described in test example 3 to measure the content of aflatoxin B1; Simultaneously using aseptic fermention medium as blank.
After testing, the fermented liquid that prepared by embodiment 1-3 is respectively 69.2%, 63.9% and 66.5% to the degradation rate of aflatoxin B1 in corn.
The degraded of test example 5 to aflatoxin B1 in soy sauce
Get soy sample, be divided into three parts, every part of 5g, the fermented liquid of preparing with 10mL embodiment 1-3 respectively mixes, the constant incubator that mixed sample is placed in to 39 DEG C is placed after 96h, in the centrifugal 5min of 4000r/min, get supernatant liquor 5ml, adopt method described in test example 3 to measure the content of aflatoxin B1; Simultaneously using aseptic fermention medium as blank.
After testing, the fermented liquid that prepared by embodiment 1-3 is respectively 53.6%, 50.9% and 51.8% to the degradation rate of aflatoxin B1 in soy sauce.
Finally it should be noted that: above each embodiment, only in order to technical scheme of the present invention to be described, is not intended to limit; Although the present invention is had been described in detail with reference to aforementioned each embodiment, those of ordinary skill in the art is to be understood that: its technical scheme that still can record aforementioned each embodiment is modified, or some or all of technical characterictic is wherein equal to replacement; And these amendments or replacement do not make the essence of appropriate technical solution depart from the scope of various embodiments of the present invention technical scheme.
Claims (10)
1. a bacillus pumilus, is characterized in that, deposit number is CGMCC No.9086.
2. the application of bacillus pumilus claimed in claim 1 in aflatoxin degradation.
3. application according to claim 2, is characterized in that, described aflatoxin is aflatoxin B1 or aflatoxin M 1.
4. a method for aflatoxin degradation, is characterized in that, comprises the steps:
1) bacillus pumilus claimed in claim 1 is fermented, make fermented liquid;
2) described fermented liquid is contacted to for some time with the material that contains aflatoxin.
5. method according to claim 4, is characterized in that, adopts fermention medium to carry out described fermentation, and the formula of described fermention medium is: lactose 0.5-1.0%, yeast extract paste 0.5-1.0%, MgCl
20.05-0.15%, and regulate the pH value of described fermention medium to 7.0-7.5.
6. method according to claim 4, is characterized in that, the condition of described fermentation is: leavening temperature 35-39 DEG C, hunting speed 120-160r/min, fermentation time 48-96h.
7. according to the arbitrary described method of claim 4 to 6, it is characterized in that, described in contain aflatoxin the quality of material and the volume of described fermented liquid between proportioning be 1:(1.2-10).
8. according to the arbitrary described method of claim 4 to 6, it is characterized in that, described contact is carried out at the temperature of 35-39 DEG C, and described for some time is 48-96h.
9. according to the arbitrary described method of claim 4 to 6, it is characterized in that, described in contain aflatoxin material be grain, food or feed.
10. method according to claim 9, is characterized in that, described grain is corn, rice or wheat, and described food is soy sauce, and described feed is peanut meal or soybean meal.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039199A (en) * | 2015-06-10 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea Bacillus circulans and application thereof in suppression of aflatoxin |
| CN105349446A (en) * | 2015-04-20 | 2016-02-24 | 河北农业大学 | Degradation of aflatoxin M1Bacillus pumilus and active protein secreted by the same |
| CN108208537A (en) * | 2018-01-19 | 2018-06-29 | 河南工业大学 | A kind of method for releasing aflatoxin B1 |
| CN110452833A (en) * | 2019-07-05 | 2019-11-15 | 中国农业大学 | One plant degradation soybean trypsin inhibitor bacillus pumilus LZ013-2 and its application |
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| EP0537418A1 (en) * | 1991-07-01 | 1993-04-21 | Ahc Inc. | Novel bacillus bacterium and its usage |
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| EP0537418A1 (en) * | 1991-07-01 | 1993-04-21 | Ahc Inc. | Novel bacillus bacterium and its usage |
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| C. MUNIMBAZI 等: "Isolation and partial characterization of antifungal metabolites of Bacillus pumilus", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| CÉLESTIN MUNIMBAZI等: "Inhibition of aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus", 《MYCOPATHOLOGIA》 * |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349446A (en) * | 2015-04-20 | 2016-02-24 | 河北农业大学 | Degradation of aflatoxin M1Bacillus pumilus and active protein secreted by the same |
| CN105349446B (en) * | 2015-04-20 | 2019-09-10 | 河北农业大学 | Degradation of aflatoxin M1Bacillus pumilus and active protein secreted by the same |
| CN105039199A (en) * | 2015-06-10 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea Bacillus circulans and application thereof in suppression of aflatoxin |
| CN105039199B (en) * | 2015-06-10 | 2019-08-16 | 哈尔滨工业大学（威海） | One plant of deep-sea Bacillus circulans and its application for inhibiting aflatoxin |
| CN108208537A (en) * | 2018-01-19 | 2018-06-29 | 河南工业大学 | A kind of method for releasing aflatoxin B1 |
| CN110452833A (en) * | 2019-07-05 | 2019-11-15 | 中国农业大学 | One plant degradation soybean trypsin inhibitor bacillus pumilus LZ013-2 and its application |
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