CN102178128A - Application of bacillus subtilis ZDY1982 to degradation of mycotoxin deoxynivalenol - Google Patents
Application of bacillus subtilis ZDY1982 to degradation of mycotoxin deoxynivalenol Download PDFInfo
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- CN102178128A CN102178128A CN2011100723740A CN201110072374A CN102178128A CN 102178128 A CN102178128 A CN 102178128A CN 2011100723740 A CN2011100723740 A CN 2011100723740A CN 201110072374 A CN201110072374 A CN 201110072374A CN 102178128 A CN102178128 A CN 102178128A
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Abstract
The invention discloses application of bacillus subtilis ZDY1982 to degradation of mycotoxin deoxynivalenol. The bacillus subtilis ZDY1982 is cultured in a nutrient bouillon fluid nutrient medium by a table concentrator at the temperature of 37DEG C and at the speed of 180rpm for 12h; the deoxynivalenol mycotoxin in the liquid can be completely degraded and a degradation product is a nontoxic product. The invention has the advantages that the bacillus subtilis ZDY1982 can be used for completely degrading the polluted deoxynivalenol in food or feed by using biosynthesis and enzymology paths; and in addition, the defects that the degradation is incomplete and nutrients are damaged because the mycotoxin is treated by using a physical or chemical method can be avoided.
Description
Technical field
The present invention relates to a kind of degradative fungi toxin deoxynivalenol, relate in particular to the application of a kind of bacillus subtilis ZDY1982 in degradative fungi toxin deoxynivalenol.
Background technology
Along with the raising of people to the food security attention degree, many countries and regions are taked multiple quality of production management regulation one after another, as GAP (Good Agricultural Practices, GAP), good manufacture practice (Good Manufacture Practice, GMP) and hazard analysis and CCP (Hazard Analysis and Critical Control Point, HACCP) etc., guarantee is from the food security supply chain system of " rural area is to dining table ".China Department of Science and Technology has also started " 15 ", the great special project of Eleventh Five-Year Plan national science and technology " food security key technology " respectively in 2002,2006 years, emphasize to build and strengthen the support system of China's food security.Mycotoxin becomes the key problem of current tackling of key scientific and technical problems as a big hidden danger that influences food security.
The mode that is used to control food chain mycotoxin level at present has the method for physics, chemistry.Though physical methods such as heating, ultraviolet irradiation and ionizing radiation can destroy the mycotic spore activity, but can not control the level of mycotoxin in food or the feed completely effectively, though and chemical method can be destroyed mycotoxin, but also can cause great destruction, and cause the uncertainty of chemical agent residue health hazard to nutritional labeling.The focus of foreign study is that the alternative above-mentioned physico-chemical process of the ways and means of searching biotechnology is controlled the mycotoxin level in food and the feed.In recent years, adopt the mycotoxin in the method for microorganism control food chain to be paid close attention to by scientific circles.Many scholars attempt to reduce or eliminate by the method for microbial fermentation the pollution of deoxynivalenol in the cereal, and have obtained certain progress.
The present invention puts forth effort on the research of probio degradative fungi toxin, be based on the good physiological function of probio, with lactic acid bacteria, Bifidobacterium and bacillus subtilis is that the probio of representative can promote the growth of animal or human's body indigenous microorganisms flora and the host is produced wholesome effect, also have the growth that suppresses harmful bacterium, eliminate carcinogen, improve immunity of organisms, reduce important physiological functions such as cholesterol, and have safe in utilization, noresidue, advantage such as do not develop immunity to drugs.At present, the research report of seldom relevant both at home and abroad probio degraded or removing mycotoxin.
Summary of the invention
The object of the present invention is to provide the application of a kind of bacillus subtilis ZDY1982 in degradative fungi toxin deoxynivalenol, this bacillus subtilis strain has the ability of very strong degraded deoxynivalenol, safe in utilization, noresidue, does not develop immunity to drugs.
The present invention is achieved like this, bacillus subtilis ZDY1982 37 ℃, 180 rpm shaking tables in the nutrient broth fluid nutrient medium were cultivated 12 hours, deoxynivalenol toxin in the degradation liquid fully, catabolite is avirulent product, described bacillus subtilis ZDY1982 is deposited in Chinese typical culture collection center, June 22 2010 preservation time, deposit number: M2010153.Product can be prepared into enzyme preparation, active bacteria formulation, oral formulations or medicine.
1, eliminate fluid nutrient medium indirect competitive ELISA detected the method that deoxynivalenol disturbs:
When screening degradation bacteria strains on a large scale, the inventor finds MRS(Mann-Rogosa-Sharpe medium) reaction produces strong interference to culture medium to indirect competitive ELISA.For eliminating the interference of culture medium, the inventor has designed a series of MRS dilution factor experiments, is object of reference with the pure water, and experimental result shows that the interference of MRS culture medium can be ignored through 8 times of dilutions.
2, the strain culturing product prepares the method for enzyme preparation:
Enzyme preparation is one of additive of commercial Application, enzyme preparation has pulvis, tablet etc. at present, the enzyme preparation made from bacillus subtilis ZDY1982 culture be utilize biochemical means (comprising various splitters, preparative chromatography etc.) of separating with the bacterial strain metabolite in addition purifying obtain purer toxins degrading enzyme, make dry powder or make the tablet product by modes such as compressing tablets.
3, the method for preparing active bacteria formulation:
When pollutant was handled in large-scale industry, the live strain that utilizes biological method to obtain carries out liquid fermentation, and to handle the effect that reaches pollution abatement be the very interested application of present scientific circles.The bacterial strain that the present invention obtains can obtain the viable bacteria body of degraded deoxynivalenol in a large number through simple the cultivation, when being cultured to thalline logarithmic phase (12 to 16 hours), taking-up and addition portion divide nutrition to dress up the living bacterial liquid body preparation or the interpolation solid content is made the solid live bacteria preparation.
4, the oral formulations of preparation human body or animal body detoxifcation or the method for medicine:
The oral formulations of regulating enteron aisle is made with bacillus subtilis by present many producers, rather than is specifically designed to and gets rid of the deoxynivalenol of eating by mistake into human body or animal body.The important use that the present invention obtains bacterial strain is exactly that the thalline that Liquid Culture obtains was left the heart 10 minutes 4000, and with after the physiological saline washing 3 times, adds in the nutrient solution that is made into edible eggs white powder such as skimmed milk power and beef extract and makes bacteria suspension.
Advantage of the present invention is: bacillus subtilis ZDY1982 can be under the condition of gentleness, utilize biosynthesis and zymetology approach that the deoxynivalenol that pollutes in food or the feed is degraded fully, and can avoid handling the incomplete and ruined shortcoming of nutritional labeling of degraded that mycotoxin causes with physics or chemical method.
Description of drawings
Fig. 1 is a deoxynivalenol lab diagram in the strains for degrading fluid nutrient medium.
Fig. 2 is thalline and supernatant degraded deoxynivalenol toxin lab diagram.
Fig. 3 is a degraded deoxynivalenol lab diagram after the heat treatment of bacillus subtilis ZDY1982 supernatant.
The specific embodiment
Embodiment 1:
1. bacterial strain to be measured is connected to dull and stereotyped activation 2 times respectively from the freeze-drying pipe of preservation, cultivates for 37 ℃ and took out standby in 18 hours;
2. will activate good bacterial strain to be measured and insert MRS or nutrient broth (NB) fluid nutrient medium, and be modeled to the food pollution model of 1 μ g/mL according to document.Cultivated 12 hours at 37 ℃, anaerobism or 180 rpm shaking tables, nutrient solution centrifugal 15 minutes in 3000 rpm, supernatant is standby;
3. carry out 8 times of dilutions with the subalkaline ultra-pure water of furnishing in advance, and determine that its pH value is a neutrality;
4. detect with the nutrient solution of indirect competitive ELISA kit all bacterial strains to be measured;
5. experimental result as shown in Figure 1: abscissa is that (No. 1 is Bacillus subtillis ZDY1982 to the test strain numbering; No. 2 is Bacillus subtillis KM; No. 3 is bacillus licheniformis; No. 4 is Lactobacillus rhamnosus; No. 5 is lactobacillus bulgaricus; No. 6 is lactobacillus acidophilus 2c bacterial strain; No. 7 is lactobacillus cellobiosas; No. 8 is Lactobacillus crispatus; No. 9 is lactobacillus fermenti; No. 10 is lactobacillus acidophilus 124a bacterial strain; No. 11 is Lactobacillus delbrueckii; No. 12 is lactobacillus acidophilus 160b bacterial strain; No. 13 is Lactobacillus plantarum), establishing NB culture medium+deoxynivalenol simultaneously is No. 14, control group; MRS culture medium+deoxynivalenol is No. 15, control group.Ordinate is the residual concentration of deoxynivalenol in the specimen.ND represents not detect the residual concentration (ng/mL) of deoxynivalenol in the sample.The residual concentration of the deoxynivalenol among No. 1 Bacillus subtillis ZDY1982 is lower than detectability as can be seen from Figure 1, illustrates that it has strong degraded deoxynivalenol effect.
Embodiment 2:
With bacillus respectively at 37 ℃, 180 rpm shaken cultivation, 12 h, centrifugal 10 min separation of supernatant and the thalline of 4000 rpm.
2. supernatant is collected filtered fluid with 0.22 μ m micro-filtrate membrane filtration;
3. thalline physiological saline washed twice to remove residual supernatant, suspends with physiological saline at last, makes that cell concentration is 10
10CFU/mL;
4. get the supernatant and the bacteria suspension 980 μ L of filtration sterilization respectively, and add the deoxynivalenol aqueous solution 20 μ L of 50 μ g/mL respectively, make that the deoxynivalenol final concentration is 1 μ g/mL, 37 ℃ of static incubation 3 h and 14 h sampling;
5. use the residual concentration of deoxynivalenol in the indirect competitive ELISA kit test sample;
6, experimental result as shown in Figure 2: abscissa represents that (No. 1 is Bacillus subtillis ZDY1982 to the test strain numbering; No. 2 is bacillus licheniformis; No. 3 is bacillus cereus; Represent supernatant, represent thalline), (water+deoxynivalenol is a control group 4 to establish three control groups simultaneously; The NB culture medium is a control group 5; NB culture medium+deoxynivalenol is a control group 6).Ordinate is the residual concentration (ng/mL) of deoxynivalenol in the specimen.ND represents not detect the residual concentration of deoxynivalenol in the sample.As can be seen from Figure 2, the deoxynivalenol residual concentration is lower than the LDL (20ng/mL) of ELISA detection kit in the supernatant () of bacillus subtilis ZDY1982, and the thalline () of bacillus subtilis ZDY1982 does not have the ability of degraded toxin deoxynivalenol substantially.
Embodiment 3:
1. the supernatant of pressing the method collection bacillus subtilis ZDY1982 among the embodiment 2 is standby;
2. supernatant is heated 10 min down at 65,75,85,95,100 ℃ respectively, add deoxynivalenol (final concentration is 1000 ng/mL) then to cultivate altogether 3 hours;
3, all samples detects the residual concentration of deoxynivalenol with indirect competitive ELISA;
4, experimental result is as shown in Figure 3: abscissa represent heat treated temperature (℃), ordinate is the residual concentration (ng/mL) of deoxynivalenol in the specimen.ND represents not detect the residual concentration of deoxynivalenol in the sample.As can be seen from Figure 3 the effective degradable component in the supernatant progressively loses activity with the rising of heat treatment temperature, between 65-75 ℃, and the loss of activity large percentage.
Embodiment 4:
1. bacillus subtilis ZDY1982 is cultivated in a large number, its supernatant is cooked following processing: (1) concentrates through vacuum, and low temperature drying obtains crude enzyme preparation; (2) separate the enzyme material that purifying procedure obtains degraded deoxynivalenol toxin through classification ultrafiltration, sour fractional precipitation, anion exchange etc., make the high-purity enzyme preparation.
2. crude enzyme preparation or high-purity enzyme preparation are dissolved in the low amounts of water, mode by spraying is dispersed in the grain trough that mycotoxin pollutes in batches it, feed is through repeatedly tedding accumulation, its internal temperature is controlled at 25-37 ℃, after enzyme effect a period of time, measure the reduction pollution level of deoxynivalenol.
3. after the cereal that takes a morsel is used the immersion of methyl alcohol: water=1:1 successively, centrifugally abandons precipitation, crossed solid-phase extraction column, immune affinity column etc. handled, detect wherein the toxin remains at 218 nm with HPLC.
Claims (2)
1. the application of bacillus subtilis ZDY1982 in degradative fungi toxin deoxynivalenol, it is characterized in that bacillus subtilis ZDY1982 37 ℃, 180 rpm shaking tables in the nutrient broth fluid nutrient medium cultivated 12 hours, deoxynivalenol toxin in the degradation liquid fully, catabolite is avirulent product, described bacillus subtilis ZDY1982 is deposited in Chinese typical culture collection center, June 22 2010 preservation time, deposit number: M2010153.
2. the application of bacillus subtilis ZDY1982 according to claim 1 in degradative fungi toxin deoxynivalenol is characterized in that being prepared into enzyme preparation, active bacteria formulation, oral formulations or medicine.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816745A (en) * | 2012-09-11 | 2012-12-12 | 国家粮食局科学研究院 | Deoxynivalenol toxin degrading enzyme as well as encoding gene and application thereof |
| CN103243047A (en) * | 2013-05-09 | 2013-08-14 | 中国农业大学 | Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis |
| CN107348323A (en) * | 2017-06-27 | 2017-11-17 | 湖北华大瑞尔科技有限公司 | A kind of bacillus subtilis, de- mould dose and de- mould dose application |
| CN108251323A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | One bacillus licheniformis, the microbial inoculum containing the bacterium and its application, the method and kit of vomitoxin of degrading |
| CN108251324A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | One bacillus subtilis, the microbial inoculum containing the bacterium and its application, the method and kit of vomitoxin of degrading |
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| CN109730245A (en) * | 2019-03-20 | 2019-05-10 | 盱眙大地运输有限公司 | Vomitoxin removal methods in a kind of wheat |
| CN111315235A (en) * | 2017-10-31 | 2020-06-19 | 诺力益维公司 | Antifungal toxin composition |
| CN114874932A (en) * | 2022-04-22 | 2022-08-09 | 南京农业大学 | Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412976A (en) * | 2008-07-02 | 2009-04-22 | 南昌大学 | Use of bacillus subtilis in degradation of deoxynivalenol |
-
2011
- 2011-03-24 CN CN2011100723740A patent/CN102178128A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101412976A (en) * | 2008-07-02 | 2009-04-22 | 南昌大学 | Use of bacillus subtilis in degradation of deoxynivalenol |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816745B (en) * | 2012-09-11 | 2014-02-05 | 国家粮食局科学研究院 | Deoxynivalenol toxin degrading enzyme as well as encoding gene and application thereof |
| CN102816745A (en) * | 2012-09-11 | 2012-12-12 | 国家粮食局科学研究院 | Deoxynivalenol toxin degrading enzyme as well as encoding gene and application thereof |
| CN103243047A (en) * | 2013-05-09 | 2013-08-14 | 中国农业大学 | Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis |
| CN103243047B (en) * | 2013-05-09 | 2015-04-08 | 中国农业大学 | Bacillus subtilis capable of effectively degrading vomitoxin and application of bacillus subtilis |
| CN108251324B (en) * | 2016-12-29 | 2021-06-11 | 中粮营养健康研究院有限公司 | Bacillus subtilis, microbial inoculum containing bacillus subtilis, application of microbial inoculum, method for degrading vomitoxin and kit |
| CN108251323A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | One bacillus licheniformis, the microbial inoculum containing the bacterium and its application, the method and kit of vomitoxin of degrading |
| CN108251324A (en) * | 2016-12-29 | 2018-07-06 | 中粮营养健康研究院有限公司 | One bacillus subtilis, the microbial inoculum containing the bacterium and its application, the method and kit of vomitoxin of degrading |
| CN108251323B (en) * | 2016-12-29 | 2021-06-11 | 中粮营养健康研究院有限公司 | Bacillus licheniformis, microbial inoculum containing bacillus licheniformis, application of microbial inoculum, method for degrading vomitoxin and kit |
| CN107348323A (en) * | 2017-06-27 | 2017-11-17 | 湖北华大瑞尔科技有限公司 | A kind of bacillus subtilis, de- mould dose and de- mould dose application |
| CN109251933B (en) * | 2017-07-13 | 2020-12-08 | 华中农业大学 | Fusarium toxin and toxic aldehyde detoxification related gene AKR18A1 and application thereof |
| CN109251933A (en) * | 2017-07-13 | 2019-01-22 | 华中农业大学 | A kind of and fusarium toxin and toxic aldehydes detoxification related gene AKR18A1 and its application |
| CN109593665A (en) * | 2017-09-30 | 2019-04-09 | 中粮营养健康研究院有限公司 | The method of bacillus subtilis, the microbial inoculum containing the bacterium and kit and their application and degradation vomitoxin |
| CN109593665B (en) * | 2017-09-30 | 2022-07-29 | 中粮营养健康研究院有限公司 | Bacillus subtilis, microbial inoculum containing bacillus subtilis, kit containing bacillus subtilis, application of bacillus subtilis and kit and method for degrading vomitoxin |
| CN111315235A (en) * | 2017-10-31 | 2020-06-19 | 诺力益维公司 | Antifungal toxin composition |
| CN109730245A (en) * | 2019-03-20 | 2019-05-10 | 盱眙大地运输有限公司 | Vomitoxin removal methods in a kind of wheat |
| CN114874932A (en) * | 2022-04-22 | 2022-08-09 | 南京农业大学 | Lactobacillus johnsonii and application thereof in degrading deoxynivalenol and inhibiting pathogenic bacteria |
| CN114874932B (en) * | 2022-04-22 | 2023-05-19 | 南京农业大学 | Lactobacillus johnsonii and application thereof in degradation of deoxynivalenol and inhibition of pathogenic bacteria |
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Application publication date: 20110914 |