CN103695340B - Subtilis ANSB0E1 and application thereof - Google Patents
Subtilis ANSB0E1 and application thereof Download PDFInfo
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Abstract
The invention provides a strain for the deposit number of degrading zearalenone, α-zearalenol and β-zearalenol is the subtilis ANSB0E1 of CGMCC No.8515 and cultural method thereof and application.Present invention also offers the method for fermented liquid degrading zearalenone, α-zearalenol and the β-zearalenol utilizing strains A NSB0E1, fermented liquid and zearalenone are reacted 72 hours, and degradation rate can reach more than 88%; Fermented liquid and α-zearalenol are reacted 72 hours, and degradation rate can reach more than 83%; Fermented liquid and β-zearalenol are reacted 72 hours, and degradation rate can reach more than 80%.Subtilis degrading activity of the present invention is high, high specificity, action effect gentle, can not destroy the nutritive ingredient in feed, is applicable to remove zearalenone, α-zearalenol and the β-zearalenol in feed.
Description
Technical field
The invention belongs to microbiological art, specifically, relate to a bacillus subtilis (Bacillus subtilis) ANSB0E1 and application thereof.
Background technology
Mycotoxin (Mycotoxin) is the secondary metabolite produced in mould-growth process, and have high toxicity, high mutagenicity and strong carinogenicity, growing of animal and human health in serious threat.Maximum mycotoxin is threatened to mainly contain aflatoxin (Aflatoxin to livestock industry and the mankind at present, AFT), zearalenone (Zearalenone, ZEN), Trichothecenes toxin (Trichothecenes, TRT), ochratoxin (Ochratoxin A, OTA) and FT (Fumonisins) etc.Zearalenone, also known as F-2 toxin, is by the mycetogenetic non-steroid mycotoxins of Fusarium, can combines with body inner estrogen acceptor, produces estrogen effect.α-zearalenol and β-zearalenol are the derivatives of zearalenone, also have the estrogen effect identical with zearalenone, and toxicity ratio zearalenone is stronger.Under high temperature low moisture environments, the cereal crop such as the corn gone mouldy, Chinese sorghum, wheat easily produce zearalenone, as the cereal of feedstuff raw material by after feed intake, can be deposited on through metabolism in the animal products such as meat, egg, milk, animal health and food safety in serious threat.Pig is the most responsive to zearalenone, and poisoning typical clinical symptom is that vaginal orifice enlargement, vagina and proctoptosis and mammary gland increase, and causes the breeding difficultys such as infertile, the miscarriage of sow and false heat.In addition, zearalenone also has hepatotoxicity, immunotoxicity, cytotoxicity and genotoxicity, also has certain influence to the generation of tumour.
The traditional physics and chemistry detoxicating method of mycotoxin exist effect instability, nutrient component damages large, affect feed palatability, and be difficult to the shortcomings such as large-scale production and can not be widely applied in actual production.Microorganism and biological enzyme removing toxic substances are because having high, the high specificity of efficiency that detoxifies, to the advantage such as feed and environmental nonpollution, having good application prospect, receive much concern.Research report, many fungies comprise Gliocladium roseum (Gliocladiumroseum), Rhizopus oryzae (R.oryzae), Rhizopus stolonifer (R.stolonifer) and Rhizopus microsporus (R.microsporus), Thamnidium elegans (Thamnidiumelegans), Beile can biological degradation corn zeranol by Mucor (Mucorbainieri), streptomyces rimosus (Streptomyces rimosus) and class Buddhist nun Cunninghamella sp (Cunninghamellabainieri) etc.But the report about degradation by bacteria zearalenone is less.
Up to now, not seeing can the relevant report of degrading zearalenone, α-zearalenol and β-zearalenol simultaneously about subtilis and metabolite thereof.
Summary of the invention
The object of this invention is to provide the subtilis of a strain for degrading zearalenone, α-zearalenol and β-zearalenol.
Another object of the present invention is to provide cultural method and the application thereof of above-mentioned subtilis.
Subtilis for degrading zearalenone, α-zearalenol and β-zearalenol provided by the invention (Bacillus subtilis) ANSB0E1, that contriver is separated and obtains from fish enteron aisle chyme, now be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, postcode 100101, deposit number CGMCC No.8515, preservation date on November 27th, 2013.
Form and the physicochemical characteristics of subtilis ANSB0E1 are as shown in table 1.
Table 1. cellular form and physicochemical characteristics
| Test subject | Result | Test subject | Result |
| Gramstaining | Positive | Carbohydrate is utilized to produce acid | + |
| Cell shape | Shaft-like | Glucose | ﹢ |
| Cell dia > 1 μm | ﹣ | Wood sugar | ﹢ |
| Form gemma | ﹢ | L-arabinose | ﹢ |
| Gemma expands | ﹣ | N.F,USP MANNITOL | ﹣ |
| Gemma is circular | + | Utilize glucose aerogenesis | ﹣ |
| Catalase | ﹢ | Utilize Citrate trianion | ﹢ |
| Oxydase | ﹢ | 50 DEG C of growths | ﹣ |
| Anaerobic growth | ﹣ | PH5.7 grows | ﹢ |
| VP tests | ﹢ | 7%NaCl grows | ﹢ |
| VP﹤pH6 | ﹢ | Starch Hydrolysis | ﹢ |
| VP﹥pH7 | ﹣ | Decompose casein | ﹢ |
| Methyl red test | ﹢ | Nitrate reduction | ﹢ |
The 16S rDNA sequencing result of subtilis ANSB0E1 is as shown in Seq ID No.1.
The present invention also provides the fermentation culture method of described subtilis ANSB0E1, and getting viable bacteria concentration is 10
9the subtilis ANSB0E1 seed liquor 1-4mL of CFU/mL, be inoculated in 50-100mL substratum and carry out shake flask fermentation cultivation, described substratum is made up of following component: Tryptones 7-12g, yeast extract 1.5-3g, glucose 2-5g, extractum carnis 2-5g, sodium-chlor 3-6g, Sodium phosphate dibasic 2.5-4g, magnesium sulfate heptahydrate 0.5-1.5g and distilled water 800-1200mL, and pH value is 6.8-7.4.
Preferably, described substratum is made up of following component: Tryptones 10g, yeast extract 2g, glucose 4g, extractum carnis 2g, sodium-chlor 6g, Sodium phosphate dibasic 2g, magnesium sulfate heptahydrate 1g and distilled water 1000mL, pH value 7.0.
The condition of shake flask fermentation is: 25-45 DEG C, fermentation 18-32h, pH value 6.8-7.4, rotating speed 150-225r/min.Preferably, the condition of shake flask fermentation is: 37 DEG C, fermentation 22h, pH value 7.0, rotating speed 200r/min.
In aforesaid fermentation cultural method, the substratum of seed liquor is identical with culture condition with the substratum of fermentation with culture condition.
The present invention also provides the complex micro organism fungicide prepared by described subtilis ANSB0E1.
The present invention also provides described subtilis ANSB0E1 or the application of its fermented liquid in degrading zearalenone, α-zearalenol and/or β-zearalenol.
The present invention also provides the method utilizing described subtilis ANSB0E1 degrading zearalenone, get the fermented liquid 700-900 μ L of described subtilis ANSB0E1, add 5000ppb zearalenone solution 100-300 μ L, pH value of reaction system is adjusted to 6.0-8.0, in 20-45 DEG C of reaction 2-72h.Preferably, the pH value of reaction system is adjusted to 7.2, in 37 DEG C of reaction 72h.
The present invention also provides the method utilizing described subtilis ANSB0E1 degraded α-zearalenol, get the fermented liquid 700-900 μ L of described subtilis ANSB0E1, add 5000ppb α-zearalenol solution 100-300 μ L, pH value of reaction system is adjusted to 6.0-8.0, in 20-45 DEG C of reaction 2-72h.Preferably, the pH value of reaction system is adjusted to 7.2, in 37 DEG C of reaction 72h.
The present invention also provides the method utilizing described subtilis ANSB0E1 degraded β-zearalenol, get the fermented liquid 700-900 μ L of described subtilis ANSB0E1, add 5000ppb β-zearalenol solution 100-300 μ L, pH value of reaction system is adjusted to 6.0-8.0, in 20-45 DEG C of reaction 2-72h.Preferably, the pH value of reaction system is adjusted to 7.2, in 37 DEG C of reaction 72h.
The present invention further provides described subtilis ANSB0E1 or the application of complex micro organism fungicide prepared therefrom in feed additive field.
The invention has the advantages that:
(1) high, the high specificity of the efficiency of subtilis ANSB0E1 degrading zearalenone of the present invention, α-zearalenol and β-zearalenol, all can reach more than 80% to the degradation rate of zearalenone, α-zearalenol and β-zearalenol, and be irreversible degraded.
(2) the method detoxicating activity of degrading zearalenone of the present invention, α-zearalenol and β-zearalenol is high, act on single-minded, action effect is gentle, the nutritive ingredient in feed can not be destroyed, the organoleptic quality of feed is not affected, is applicable to remove zearalenone, α-zearalenol and the β-zearalenol in feed.
(3) method of degrading zearalenone of the present invention, α-zearalenol and β-zearalenol is simple to operate, to feed and environmental nonpollution, efficiently solves traditional detoxicating method Problems existing.
Accompanying drawing explanation
Fig. 1 is control group zearalenone collection of illustrative plates in the embodiment of the present invention 5.
Fig. 2 is subtilis ANSB0E1 treatment group zearalenone collection of illustrative plates in the embodiment of the present invention 5.
Fig. 3 is subtilis ANSB0E1 degrading zearalenone in the embodiment of the present invention 5, α-zearalenol and the time dependent collection of illustrative plates of β-zearalenol.
Fig. 4 is control group α-zearalenol collection of illustrative plates in the embodiment of the present invention 6.
Fig. 5 is subtilis ANSB0E1 treatment group α-zearalenol collection of illustrative plates in the embodiment of the present invention 6.
Fig. 6 is control group β-zearalenol collection of illustrative plates in the embodiment of the present invention 7.
Fig. 7 is subtilis ANSB0E1 treatment group β-zearalenol collection of illustrative plates in the embodiment of the present invention 7.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The cultural method of embodiment 1 subtilis ANSB0E1
Get subtilis Bacillus subtilis ANSB0E1(CGMCC No.8515) 1mL(viable bacteria concentration is 10
9cFU/mL), be inoculated in 50mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 37 DEG C, pH value 7.2, rotating speed 200r/min, fermentation time 24h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 10g, yeast extract 1.5g, glucose 5g, extractum carnis 3g, sodium-chlor 6g, Sodium phosphate dibasic 2.5g, magnesium sulfate heptahydrate 1g and distilled water 1000mL, and pH value is 7.0.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 2 subtilis ANSB0E1
Get subtilis Bacillus subtilis ANSB0E1(CGMCC No.8515) 1mL(viable bacteria concentration is 10
9cFU/mL), be inoculated in 80mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 25 DEG C, pH value 7.5, rotating speed 225r/min, fermentation time 28h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 12g, yeast extract 2g, glucose 4g, extractum carnis 2g, sodium-chlor 5g, Sodium phosphate dibasic 3g, magnesium sulfate heptahydrate 0.5g and distilled water 800mL, and pH value is 7.2.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 3 subtilis ANSB0E1
Get subtilis Bacillus subtilis ANSB0E1(CGMCC No.8515) 1mL(viable bacteria concentration is 10
9cFU/mL), be inoculated in 100mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 45 DEG C, pH value 6.8, rotating speed 185r/min, fermentation time 18h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 7g, yeast extract 2.5g, glucose 3g, extractum carnis 4g, sodium-chlor 4g, Sodium phosphate dibasic 3.5g, magnesium sulfate heptahydrate 1.25g and distilled water 1200mL, and pH value is 7.4.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
The cultural method of embodiment 4 subtilis ANSB0E1
Getting subtilis Bacillus subtilis ANSB0E1CGMCC No.85154mL(viable bacteria concentration is 10
9cFU/mL), be inoculated in 50mL substratum and carry out shake flask fermentation cultivation, leavening temperature is 35 DEG C, pH value 7.0, rotating speed 150r/min, fermentation time 32h.
Wherein, Medium of shaking flask fermentation is made up of following component: Tryptones 9g, yeast extract 3g, glucose 2g, extractum carnis 5g, sodium-chlor 3g, Sodium phosphate dibasic 4g, magnesium sulfate heptahydrate 1.5g and distilled water 1000mL, and pH value is 7.2.
After fermentation ends, fermented liquid is kept in 4 DEG C of refrigerators for subsequent use.
Embodiment 5 subtilis ANSB0E1 is used for degrading zearalenone
Get the fermentation of bacillus subtilis liquid of 900 μ L embodiment 1 preparations, react with 100 μ L zearalenones (5000ppb); Control group is add 100 μ L zearalenones (5000ppb) in 900 μ L PBS damping fluids; The pH value of reaction system is adjusted to 7.2, and treatment group and control group are all at 37 DEG C of reactions 2h, 12h, 24h, 48h and 72h.
By the centrifugal 5min of each sample obtained after reaction, with 0.45 μm of membrane filtration, high performance liquid chromatography is adopted to detect the concentration of zearalenone.
Testing conditions: chromatographic column: Diamonsil C18,150mm × 4.6mm × 5 μm; Moving phase is acetonitrile: water: methyl alcohol=46:46:8, flow velocity 1ml/min, excitation wavelength 374nm, emission wavelength 440nm, applied sample amount 20 μ L, the appearance time of zearalenone is respectively 11.228min(control group, Fig. 1) with 11.362min(treatment group, Fig. 2).
Detected result: the time dependent curve of the degradation rate of subtilis to zearalenone as shown in Figure 3.When 2h, 12h, 24h, 48h and 72h, the degradation rate of subtilis treatment group to zearalenone is respectively 14.21%, 33.11%, 56.54%, 76.18% and 88.61%.
Embodiment 6 subtilis ANSB0E1 is for α-zearalenol of degrading
Get the fermentation of bacillus subtilis liquid of 800 μ L embodiment 1 preparations, react with 100 μ L α-zearalenols (5000ppb); Control group is add 100 μ L α-zearalenols (5000ppb) in 900 μ L PBS damping fluids; The pH value of reaction system is adjusted to 7.2, and treatment group and control group are all at 37 DEG C of reaction 72h.
By the centrifugal 5min of each sample obtained after reaction, with 0.45 μm of membrane filtration, performance liquid chromatographic column is adopted to detect the concentration of α-zearalenol.
Testing conditions: chromatographic column: Diamonsil C18,150mm × 4.6mm × 5 μm; Moving phase is acetonitrile: water: methyl alcohol=50:43:7, flow velocity 0.8ml/min, ultraviolet detection wavelength: 265nm, applied sample amount 20 μ L, and the appearance time of α-zearalenol is respectively 10.068min(control group, Fig. 4) and 9.930min(treatment group, Fig. 5).
Detected result: compared with control group, the degradation rate of subtilis treatment group α-zearalenol is 84.25%.
Embodiment 7 subtilis ANSB0E1 is for β-zearalenol of degrading
Get the fermentation of bacillus subtilis liquid of 900 μ L embodiment 1 preparations, react with 100 μ L β-zearalenols (5000ppb); Control group is add 100 μ L β-zearalenols (5000ppb) in 900 μ L PBS damping fluids; The pH value of reaction system is adjusted to 7.2, and treatment group and control group are all at 37 DEG C of reaction 72h.
By the centrifugal 5min of each sample obtained after reaction, with 0.45 μm of membrane filtration, performance liquid chromatographic column is adopted to detect the concentration of β-zearalenol.
Testing conditions: chromatographic column: Diamonsil C18,150mm × 4.6mm × 5 μm; Moving phase is acetonitrile: water: methyl alcohol=50:43:7, flow velocity 0.8mL/min, ultraviolet detection wavelength: 265nm, applied sample amount 20 μ L, and the appearance time of β-zearalenol is respectively 7.357min(control group, Fig. 6) and 7.287min(treatment group, Fig. 7).
Detected result: compared with control group, the degradation rate of subtilis treatment group β-zearalenol is 81.79%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1., for subtilis (Bacillus subtilis) ANSB0E1 of degrading zearalenone, α-zearalenol and β-zearalenol, its deposit number is CGMCCNo.8515.
2. the complex micro organism fungicide prepared by subtilis ANSB0E1 described in claim 1.
3. the fermentation culture method of subtilis ANSB0E1 described in claim 1, is characterized in that, getting viable bacteria concentration is 10
9the subtilis ANSB0E1 seed liquor 1-4mL of CFU/mL, be inoculated in 50-100mL substratum and carry out shake flask fermentation cultivation, described substratum is made up of following component: Tryptones 7-12g, yeast extract 1.5-3g, glucose 2-5g, extractum carnis 2-5g, sodium-chlor 3-6g, Sodium phosphate dibasic 2.5-4g, magnesium sulfate heptahydrate 0.5-1.5g and distilled water 800-1200mL, and pH value is 6.8-7.4.
4. fermentation culture method according to claim 3, is characterized in that, the condition of shake flask fermentation is: 25-45 DEG C, fermentation 18-32h, pH value 6.8-7.4, rotating speed 150-225r/min.
5. the subtilis ANSB0E1 fermented liquid obtained by method described in claim 3 or 4.
6. the application of fermented liquid in degrading zearalenone, α-zearalenol and/or β-zearalenol described in subtilis ANSB0E1 described in claim 1 or claim 5.
7. the method for a degrading zearalenone, it is characterized in that, get the fermented liquid 700-900 μ L of subtilis ANSB0E1 described in claim 5, add 5000ppb zearalenone solution 100-300 μ L, pH value of reaction system is adjusted to 6.0-8.0, in 20-45 DEG C of reaction 2-72h.
8. the method for α-zearalenol of degrading, it is characterized in that, get the fermented liquid 700-900 μ L of subtilis ANSB0E1 described in claim 5, add 5000ppb α-zearalenol solution 100-300 μ L, pH value of reaction system is adjusted to 6.0-8.0, in 20-45 DEG C of reaction 2-72h.
9. the method for β-zearalenol of degrading, it is characterized in that, get the fermented liquid 700-900 μ L of the ANSB0E1 of subtilis described in claim 5, add 5000ppb β-zearalenol solution 100-300 μ L, pH value of reaction system is adjusted to 6.5-8.5, in 20-45 DEG C of reaction 2-72h.
10. the application of complex micro organism fungicide in feed additive field described in subtilis ANSB0E1 described in claim 1 or claim 2.
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| CN109321485B (en) * | 2017-09-30 | 2020-05-01 | 吉林中粮生化有限公司 | Bacillus subtilis, microbial inoculum containing bacillus subtilis, kit containing bacillus subtilis, application of bacillus subtilis and kit and method for degrading zearalenone |
| CN108606235A (en) * | 2018-03-04 | 2018-10-02 | 河南亿万中元生物技术有限公司 | A kind of method of zearalenone in degrading maize DDGS |
| CN110172425B (en) * | 2019-05-30 | 2020-12-04 | 华中农业大学 | Screening and application of zearalenone virus-free probiotics |
| CN112063559B (en) * | 2020-09-20 | 2022-05-17 | 河南新汉博生物科技有限公司 | Zearalenone degrading strain and application thereof |
| CN113913340A (en) * | 2021-11-08 | 2022-01-11 | 中国农业科学院饲料研究所 | Bacillus subtilis and application thereof in degradation of zearalenone |
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