CN111820363A - Biodegradation method for gibberellin ketene toxin in DDGS - Google Patents
Biodegradation method for gibberellin ketene toxin in DDGS Download PDFInfo
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- CN111820363A CN111820363A CN201910310425.5A CN201910310425A CN111820363A CN 111820363 A CN111820363 A CN 111820363A CN 201910310425 A CN201910310425 A CN 201910310425A CN 111820363 A CN111820363 A CN 111820363A
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- 229930191978 Gibberellin Natural products 0.000 title claims abstract description 69
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 239000003448 gibberellin Substances 0.000 title claims abstract description 69
- CCGKOQOJPYTBIH-UHFFFAOYSA-N ethenone Chemical compound C=C=O CCGKOQOJPYTBIH-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 26
- 101710089042 Demethyl-4-deoxygadusol synthase Proteins 0.000 title claims abstract description 22
- 239000003053 toxin Substances 0.000 title claims abstract description 21
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- 238000006065 biodegradation reaction Methods 0.000 title claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 27
- 230000001580 bacterial effect Effects 0.000 claims abstract description 25
- 241000228245 Aspergillus niger Species 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 19
- 238000012258 culturing Methods 0.000 claims abstract description 17
- 230000015556 catabolic process Effects 0.000 claims abstract description 16
- 238000006731 degradation reaction Methods 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 15
- 239000012153 distilled water Substances 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 10
- 238000002474 experimental method Methods 0.000 claims abstract description 9
- 239000011248 coating agent Substances 0.000 claims abstract description 5
- 238000000576 coating method Methods 0.000 claims abstract description 5
- 239000003086 colorant Substances 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract description 5
- 239000002689 soil Substances 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 150000002576 ketones Chemical class 0.000 claims description 16
- 231100000033 toxigenic Toxicity 0.000 claims description 16
- 230000001551 toxigenic effect Effects 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000000047 product Substances 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 9
- 230000000593 degrading effect Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 9
- 241000233866 Fungi Species 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 241000228197 Aspergillus flavus Species 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 5
- 238000002965 ELISA Methods 0.000 claims description 5
- 229960001701 chloroform Drugs 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 230000005284 excitation Effects 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000002955 isolation Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 229920000858 Cyclodextrin Polymers 0.000 claims description 4
- 239000001116 FEMA 4028 Substances 0.000 claims description 4
- 231100000678 Mycotoxin Toxicity 0.000 claims description 4
- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims description 4
- 235000011175 beta-cyclodextrine Nutrition 0.000 claims description 4
- 229960004853 betadex Drugs 0.000 claims description 4
- 238000011081 inoculation Methods 0.000 claims description 4
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- 239000002636 mycotoxin Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000001965 potato dextrose agar Substances 0.000 claims 2
- 235000013555 soy sauce Nutrition 0.000 claims 2
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 230000009286 beneficial effect Effects 0.000 abstract description 4
- 239000002609 medium Substances 0.000 description 9
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 7
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 3
- 235000013339 cereals Nutrition 0.000 description 3
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- KHYSRSOCKZPZRY-UHFFFAOYSA-N 2,3-dihydroxybenzoic acid phenol Chemical compound C1(=CC=CC=C1)O.OC=1C(=C(C(=O)O)C=CC1)O KHYSRSOCKZPZRY-UHFFFAOYSA-N 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 244000075850 Avena orientalis Species 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 230000007674 genetic toxicity Effects 0.000 description 1
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- 239000003966 growth inhibitor Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000007688 immunotoxicity Effects 0.000 description 1
- 231100000386 immunotoxicity Toxicity 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 150000002596 lactones Chemical group 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 208000010484 vulvovaginitis Diseases 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of biodegradation, and discloses a biodegradation method for gibberellin ketene toxin in DDGS, which comprises the following steps: placing the collected soil sample in sterile distilled water in an ultraclean workbench, shaking for 15 minutes to prepare bacterial suspension, setting the rotating speed of a shaking table to be 200rpm, diluting the bacterial suspension with the sterile distilled water in a concentration gradient manner, coating the diluted bacterial suspension on an NA culture medium flat plate, culturing for 24 hours at the temperature of 30 ℃, enabling bacterial colonies to be distributed on the whole flat plate, picking bacterial strains with different shapes, sizes, colors and transparencies on the flat plate by using inoculating rings, carrying out flat plate scribing purification, and applying the bacterial strains subjected to spot grafting purification to a gibberellin ketene degradation experiment to obtain pseudomonas aeruginosa; the invention utilizes the characteristic that Aspergillus niger and Pseudomonas aeruginosa can degrade the gibberellin ketene toxin, so that the Aspergillus niger and the Pseudomonas aeruginosa are mutually matched to efficiently degrade the content of the gibberellin ketene toxin in the DDGS feed product, thereby improving the quality of the DDGS feed product, having high safety, causing no pollution and being beneficial to large-scale use.
Description
Technical Field
The invention relates to the technical field of biodegradation, in particular to a biodegradation method for gibberellin ketene toxin in DDGS.
Background
Gibberellin, also known as F-2 toxin, is a lactone structure of phenol dihydroxy benzoic acid and has strong estrogenic activity. The toxin is thermally stable and is not destroyed by long term storage, baking or addition of propionic acid or mold growth inhibitors.
The accumulation of the gibberellin ketone in human and animal bodies can induce a series of estrogen effect symptoms, including the influence on the breast development of female mammals, vulvovaginitis, oestrus cycle disorder, pseudopregnancy, abortion, dead fetuses and teratocarcinoma, and in addition, researches prove that the gibberellin ketone also has genetic toxicity, cytotoxicity, immunotoxicity and tumor toxicity.
Zearalenone is present in a variety of cereal crops worldwide, such as: corn, barley, oats, wheat, rice, and sorghum. Production of zearalenone prior to harvest does not normally occur in significant amounts, but under appropriate environmental conditions is readily produced on corn and small grains in storage.
At present, a method for removing the gibberellin ketone in the DDGS feed can be divided into a chemical method and a physical method according to the action principle of the method, wherein the physical detoxification method can cause the loss of nutrient substances of the animal feed while removing the gibberellin ketone toxin; the chemical detoxification method can remove some grain feeds with serious mildew, but is time-consuming and labor-consuming, causes great damage to nutrient components, and can generate some chemical substances harmful to animal health.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a biodegradation method for the gibberellin ketene toxin in the DDGS, which has high safety, can specifically and efficiently degrade the gibberellin ketene toxin in the DDGS feed, and improves the quality of DDGS feed products.
(II) technical scheme
In order to achieve the purposes of convenient installation and good waterproofness, the invention provides the following technical scheme: a biodegradation method for a gibberellin ketene toxin in DDGS comprises the following steps:
the method comprises the following steps: isolation of bacteria
1) Placing the collected soil sample in sterile distilled water in an ultra-clean workbench, and oscillating for 15 minutes to prepare bacterial suspension, wherein the rotating speed of a shaking table is 200 rpm;
2) diluting the bacterial suspension with sterile distilled water in a concentration gradient manner, coating the diluted bacterial suspension on an NA culture medium plate, culturing for 24 hours at the temperature of 30 ℃, enabling bacterial colonies to be distributed on the whole plate, selecting strain plates with different shapes, sizes, colors and transparencies on the plate by using inoculating loops, carrying out streak purification, and applying the strain subjected to spot grafting purification to a gibberellic ketene degradation experiment to obtain pseudomonas aeruginosa;
3) qualitatively separating non-toxigenic filamentous fungi from the fermented paste for 4 days by using PDA culture medium containing beta-cyclodextrin, quantitatively analyzing the toxigenic capacity of the separated strain by ELISA method, and finally screening non-toxigenic fungi. And (3) carrying out re-screening on the aspergillus flavus toxigenic bacteria to screen out a bacterial strain aspergillus niger capable of preventing and controlling mycotoxin.
Step two: degradation of gibberellin by aspergillus niger
1) Inoculating the separated Aspergillus niger strain into 60ml sterile PDB culture medium at the inoculation amount of 2%, and culturing at 28 deg.C for 5 days with shaking table at 200 rpm. The fermentation broth was supplemented with gibberellin to a final concentration of 2ppm, and sterile PDB medium was supplemented with gibberellin as a blank.
Step three: pseudomonas aeruginosa for degrading gibberellic ketene
1) Inoculating pseudomonas aeruginosa in a liquid NB culture medium, carrying out shake culture for 24 hours at the temperature of 37 ℃ and the rotation speed of 200rpm (the rotation radius is 20mm), and collecting a culture product NS7 culture solution for a subsequent gibberellin ketene degradation experiment;
2) preparing gibberellin, namely dissolving 5mg of gibberellin standard substance in 25ml of chromatographic pure methanol to obtain a gibberellin solution with the concentration of 200 ppm;
3) degrading gibberellin ketone, experimental group: mu.l of 200ppm gibberellin solution was placed in a 10ml centrifuge tube and 975. mu.l of fresh MM medium was added to give a final concentration of 2 ppm. Adding 10 μ l NS7 culture solution, mixing well, shaking and culturing at 28 deg.C and 200rpm (rotation radius of 20mm) for 72h, centrifuging at 10000g for 10min to remove cells, and collecting supernatant.
Control group: mu.l of a solution of degraded zearalenone from Aspergillus niger was added to a volume of 1ml of MM medium containing 2ppm of zearalenone as a control.
Step four: chromatographic detection
1) Culturing the culture medium of the control group and the culture medium of the experimental group for 48 hours respectively, absorbing 2ml of liquid from the two groups of samples, adding 4ml of trichloromethane, extracting for 3 times respectively, drying by nitrogen, adding 1ml of mobile phase (acetonitrile: water =50:50 (v/v)) for redissolution, passing through an organic filter membrane of 0.22 mu m, and detecting by using High Performance Liquid Chromatography (HPLC);
the chromatographic detection conditions are as follows: a chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: water =50: 50; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 274 nm; emission wavelength: 440 nm; sample introduction amount: 20 mu L of the solution; the elution time is 20 min; the retention time of the gibberellin ketone is 6.5 + -0.5 min.
(III) advantageous effects
Compared with the prior art, the invention provides a biodegradation method for the gibberellin ketene toxin in DDGS, which has the following beneficial effects:
according to the method for biologically degrading the gibberellin ketone toxin in the DDGS, the aspergillus niger and the pseudomonas aeruginosa are matched with each other by utilizing the characteristic that the aspergillus niger and the pseudomonas aeruginosa can degrade the gibberellin ketone toxin, so that the content of the gibberellin ketone toxin in the DDGS feed product is efficiently degraded, the quality of the DDGS feed product is improved, the safety is high, the duration is long, no pollution is caused, and the DDGS feed product is beneficial to large-scale use.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example one
A biodegradation method for a gibberellin ketene toxin in DDGS comprises the following steps:
the method comprises the following steps: isolation of bacteria
1) Placing the collected soil sample in sterile distilled water in an ultra-clean workbench, and oscillating for 15 minutes to prepare bacterial suspension, wherein the rotating speed of a shaking table is 200 rpm;
2) diluting the bacterial suspension with sterile distilled water in a concentration gradient manner, coating the diluted bacterial suspension on an NA culture medium plate, culturing for 24 hours at the temperature of 30 ℃, enabling bacterial colonies to be distributed on the whole plate, selecting strain plates with different shapes, sizes, colors and transparencies on the plate by using inoculating loops, carrying out streak purification, and applying the strain subjected to spot grafting purification to a gibberellin ketene degradation experiment to obtain the pseudomonas aeruginosa.
Step two: pseudomonas aeruginosa for degrading gibberellic ketene
1) Inoculating pseudomonas aeruginosa in a liquid NB culture medium, carrying out shake culture for 24 hours at the temperature of 37 ℃ and the rotation speed of 200rpm (the rotation radius is 20mm), and collecting a culture product NS7 culture solution for a subsequent gibberellin ketene degradation experiment;
2) preparing gibberellin, namely dissolving 5mg of gibberellin standard substance in 25ml of chromatographic pure methanol to obtain a gibberellin solution with the concentration of 200 ppm;
3) degrading gibberellin ketone, experimental group: mu.l of 200ppm gibberellin solution was placed in a 10ml centrifuge tube and 975. mu.l of fresh MM medium was added to give a final concentration of 2 ppm. Adding 10 μ l NS7 culture solution, mixing well, shake culturing at 28 deg.C and 200rpm (rotation radius of 20mm) for 72h, centrifuging at 10000g for 10min to remove cells, and collecting supernatant;
control group: mu.l of non-inoculated NB medium was added to a volume of 1ml of MM medium containing 2ppm of gibberellin as a control.
Step three: chromatographic detection
1) Culturing the culture medium of the control group and the culture medium of the experimental group for 48 hours respectively, absorbing 2ml of liquid from the two groups of samples, adding 4ml of trichloromethane, extracting for 3 times respectively, drying by nitrogen, adding 1ml of mobile phase (acetonitrile: water =50:50 (v/v)) for redissolution, passing through an organic filter membrane of 0.22 mu m, and detecting by using High Performance Liquid Chromatography (HPLC);
the chromatographic detection conditions are as follows: a chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: water =50: 50; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 274 nm; emission wavelength: 440 nm; sample introduction amount: 20 mu L of the solution; the elution time is 20 min; the retention time of the gibberellin ketone is 6.5 + -0.5 min.
The result shows that the degradation rate of the gibberellin ketene reaches 75.34%.
Example two
A biodegradation method for a gibberellin ketene toxin in DDGS comprises the following steps:
the method comprises the following steps: isolation of bacteria
Qualitatively separating non-toxigenic filamentous fungi from the fermented paste for 4 days by using PDA culture medium containing beta-cyclodextrin, quantitatively analyzing the toxigenic capacity of the separated strain by ELISA method, and finally screening non-toxigenic fungi. And (3) carrying out re-screening on the aspergillus flavus toxigenic bacteria to screen out a bacterial strain aspergillus niger capable of preventing and controlling mycotoxin.
Step two: degradation of gibberellin by aspergillus niger
Inoculating the separated Aspergillus niger strain into 60ml sterile PDB culture medium at the inoculation amount of 2%, and culturing at 28 deg.C for 5 days with shaking table at 200 rpm.
Step three: chromatographic detection
After culturing for 48 hours, respectively absorbing 2ml of liquid from the two groups of samples, adding 4ml of trichloromethane, extracting for 3 times, drying by nitrogen, adding 1ml of mobile phase (acetonitrile: water =50:50 (v/v)) for redissolution, and detecting by using a High Performance Liquid Chromatography (HPLC) after passing through an organic filter membrane with the diameter of 0.22 mu m;
the chromatographic detection conditions are as follows: a chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: water =50: 50; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 274 nm; emission wavelength: 440 nm; sample introduction amount: 20 mu L of the solution; the elution time is 20 min; the retention time of the gibberellin ketone is 6.5 + -0.5 min.
The result shows that the degradation rate of the gibberellin ketene reaches 89.52%.
EXAMPLE III
A biodegradation method for a gibberellin ketene toxin in DDGS comprises the following steps:
the method comprises the following steps: isolation of bacteria
1) Placing the collected soil sample in sterile distilled water in an ultra-clean workbench, and oscillating for 15 minutes to prepare bacterial suspension, wherein the rotating speed of a shaking table is 200 rpm;
2) diluting the bacterial suspension with sterile distilled water in a concentration gradient manner, coating the diluted bacterial suspension on an NA culture medium plate, culturing for 24 hours at the temperature of 30 ℃, enabling bacterial colonies to be distributed on the whole plate, selecting strain plates with different shapes, sizes, colors and transparencies on the plate by using inoculating loops, carrying out streak purification, and applying the strain subjected to spot grafting purification to a gibberellic ketene degradation experiment to obtain pseudomonas aeruginosa;
3) qualitatively separating non-toxigenic filamentous fungi from the fermented paste for 4 days by using PDA culture medium containing beta-cyclodextrin, quantitatively analyzing the toxigenic capacity of the separated strain by ELISA method, and finally screening non-toxigenic fungi. And (3) carrying out re-screening on the aspergillus flavus toxigenic bacteria to screen out a bacterial strain aspergillus niger capable of preventing and controlling mycotoxin.
Step two: degradation of gibberellin by aspergillus niger
Inoculating the separated Aspergillus niger strain into 60ml sterile PDB culture medium at the inoculation amount of 2%, and culturing at 28 deg.C for 5 days with shaking table at 200 rpm. The fermentation broth was supplemented with gibberellin to a final concentration of 2ppm, and sterile PDB medium was supplemented with gibberellin as a blank.
Step three: pseudomonas aeruginosa for degrading gibberellic ketene
1) Inoculating pseudomonas aeruginosa in a liquid NB culture medium, carrying out shake culture for 24 hours at the temperature of 37 ℃ and the rotation speed of 200rpm (the rotation radius is 20mm), and collecting a culture product NS7 culture solution for a subsequent gibberellin ketene degradation experiment;
2) preparing gibberellin, namely dissolving 5mg of gibberellin standard substance in 25ml of chromatographic pure methanol to obtain a gibberellin solution with the concentration of 200 ppm;
3) degrading gibberellin ketone, experimental group: mu.l of 200ppm gibberellin solution was placed in a 10ml centrifuge tube and 975. mu.l of fresh MM medium was added to give a final concentration of 2 ppm. Adding 10 μ l NS7 culture solution, mixing well, shaking and culturing at 28 deg.C and 200rpm (rotation radius of 20mm) for 72h, centrifuging at 10000g for 10min to remove cells, and collecting supernatant.
Control group: mu.l of a solution of degraded zearalenone from Aspergillus niger was added to a volume of 1ml of MM medium containing 2ppm of zearalenone as a control.
Step four: chromatographic detection
Culturing the culture medium of the control group and the culture medium of the experimental group for 48 hours respectively, absorbing 2ml of liquid from the two groups of samples, adding 4ml of trichloromethane, extracting for 3 times respectively, drying by nitrogen, adding 1ml of mobile phase (acetonitrile: water =50:50 (v/v)) for redissolution, passing through an organic filter membrane of 0.22 mu m, and detecting by using High Performance Liquid Chromatography (HPLC);
the chromatographic detection conditions are as follows: a chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: water =50: 50; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 274 nm; emission wavelength: 440 nm; sample introduction amount: 20 mu L of the solution; the elution time is 20 min; the retention time of the gibberellin ketone is 6.5 + -0.5 min.
The result shows that the degradation rate of the gibberellin ketene reaches 99.13%.
The invention utilizes the characteristic that Aspergillus niger and pseudomonas aeruginosa can degrade the gibberellin ketene toxin to mutually match the Aspergillus niger and the pseudomonas aeruginosa to efficiently degrade the content of the gibberellin ketene toxin in the DDGS feed product, thereby improving the quality of the DDGS feed product, having high safety and long duration, causing no pollution and being beneficial to large-scale use.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A biodegradation method for a gibberellin ketene toxin in DDGS is characterized by comprising the following steps:
the method comprises the following steps: isolation of bacteria
Placing the collected soil sample in sterile distilled water in an ultra-clean workbench, and oscillating for 15 minutes to prepare bacterial suspension, wherein the rotating speed of a shaking table is 200 rpm;
diluting the bacterial suspension with sterile distilled water in a concentration gradient manner, coating the diluted bacterial suspension on an NA culture medium plate, culturing for 24 hours at the temperature of 30 ℃, enabling bacterial colonies to be distributed on the whole plate, selecting strain plates with different shapes, sizes, colors and transparencies on the plate by using inoculating loops, carrying out streak purification, and applying the strain subjected to spot grafting purification to a gibberellic ketene degradation experiment to obtain pseudomonas aeruginosa;
qualitatively separating non-toxigenic filamentous fungi in soy sauce mash from soy sauce fermented for 4 days by using a PDA (potato dextrose agar) culture medium containing beta-cyclodextrin, then quantitatively analyzing the toxigenic capacity of the separated strains by using an ELISA (enzyme-linked immunosorbent assay) method, finally screening non-toxigenic fungi, re-screening aspergillus flavus toxigenic bacteria for inhibiting aspergillus flavus, and screening a strain aspergillus niger capable of preventing and controlling mycotoxin;
step two: degradation of gibberellin by aspergillus niger
1) Inoculating the separated aspergillus niger strain into 60ml of sterile PDB culture medium according to the inoculation amount of 2%, setting the temperature to be 28 ℃, and carrying out shake culture for 5 days at the rotation speed of 200rpm of a shaker;
step three: pseudomonas aeruginosa for degrading gibberellic ketene
1) Inoculating pseudomonas aeruginosa in a liquid NB culture medium, carrying out shake culture for 24 hours at the temperature of 37 ℃ and the rotation speed of 200rpm (the rotation radius is 20mm), and collecting a culture product NS7 culture solution for a subsequent gibberellin ketene degradation experiment;
2) preparing gibberellin, namely dissolving 5mg of gibberellin standard substance in 25ml of chromatographic pure methanol to obtain a gibberellin solution with the concentration of 200 ppm;
3) degrading gibberellin ketone, experimental group: putting 10 μ l of 200ppm gibberellin ketene solution into a 10ml centrifuge tube, adding 975 μ l of fresh MM culture medium to make the final concentration 2ppm, adding 10 μ l of the obtained NS7 culture solution, mixing well, shaking and culturing at 28 deg.C and 200rpm (rotation radius 20MM) for 72h, centrifuging at 10000g for 10min to remove cells, and collecting supernatant;
control group: adding 10 μ l of degraded gibberellin solution of Aspergillus niger into 1ml MM culture medium containing 2ppm gibberellin as control group;
step four: chromatographic detection
1) Culturing the culture medium of the control group and the culture medium of the experimental group for 48 hours respectively, absorbing 2ml of liquid from the two groups of samples, adding 4ml of trichloromethane, extracting for 3 times respectively, drying by nitrogen, adding 1ml of mobile phase (acetonitrile: water =50:50 (v/v)) for redissolution, passing through an organic filter membrane of 0.22 mu m, and detecting by using High Performance Liquid Chromatography (HPLC);
the chromatographic detection conditions are as follows: a chromatographic column: c18 column 6mm × 150mm × 5 um; mobile phase: acetonitrile: water =50: 50; detecting the temperature: 25 ℃; flow rate: 1 ml/min; detection wavelength: excitation wavelength: 274 nm; emission wavelength: 440 nm; sample introduction amount: 20 mu L of the solution; the elution time is 20 min; the retention time of the gibberellin ketone is 6.5 + -0.5 min.
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| CN107502566A (en) * | 2017-07-10 | 2017-12-22 | 中国农业科学院饲料研究所 | One plant of lysine bacillus and its application in degrading zearalenone |
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| CN113984930A (en) * | 2021-10-25 | 2022-01-28 | 西安市食品药品检验所(西安市药品不良反应监测中心) | Method for detecting 7-protein assimilation preparation type stimulant in animal feed |
| CN113984930B (en) * | 2021-10-25 | 2023-08-18 | 西安市食品药品检验所(西安市药品不良反应监测中心) | Method for detecting 7 protein assimilation preparation type stimulants in animal feed |
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