CN108624580A - A kind of preparation method with the trichoderma asperellum capsule and pill microbial inoculum for dropping heavy metal effect - Google Patents
A kind of preparation method with the trichoderma asperellum capsule and pill microbial inoculum for dropping heavy metal effect Download PDFInfo
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- CN108624580A CN108624580A CN201710186935.7A CN201710186935A CN108624580A CN 108624580 A CN108624580 A CN 108624580A CN 201710186935 A CN201710186935 A CN 201710186935A CN 108624580 A CN108624580 A CN 108624580A
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- 241001460073 Trichoderma asperellum Species 0.000 title claims abstract description 17
- 239000002775 capsule Substances 0.000 title claims abstract description 15
- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 15
- 239000006187 pill Substances 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 11
- 230000000694 effects Effects 0.000 title abstract description 16
- 241000894006 Bacteria Species 0.000 claims abstract description 23
- 239000000843 powder Substances 0.000 claims abstract description 19
- 239000000725 suspension Substances 0.000 claims abstract description 12
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 10
- 238000004108 freeze drying Methods 0.000 claims abstract description 9
- 241000233866 Fungi Species 0.000 claims abstract description 7
- 230000001580 bacterial effect Effects 0.000 claims abstract description 7
- 230000000813 microbial effect Effects 0.000 claims abstract description 7
- 238000009630 liquid culture Methods 0.000 claims abstract description 6
- 238000010521 absorption reaction Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims description 11
- 235000010413 sodium alginate Nutrition 0.000 claims description 10
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 9
- 239000000661 sodium alginate Substances 0.000 claims description 9
- 229940005550 sodium alginate Drugs 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 8
- 235000015097 nutrients Nutrition 0.000 claims description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 241000196324 Embryophyta Species 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 239000008103 glucose Substances 0.000 claims description 6
- 239000003223 protective agent Substances 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 244000061456 Solanum tuberosum Species 0.000 claims description 5
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 239000007836 KH2PO4 Substances 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 244000068988 Glycine max Species 0.000 claims description 2
- 235000010469 Glycine max Nutrition 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims 1
- 239000008188 pellet Substances 0.000 claims 1
- 239000002689 soil Substances 0.000 abstract description 8
- 238000000034 method Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000002045 lasting effect Effects 0.000 abstract description 2
- 230000002265 prevention Effects 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 238000000967 suction filtration Methods 0.000 abstract 1
- 241000223259 Trichoderma Species 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000266501 Ormosia ormondii Species 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 235000012015 potatoes Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000443 biocontrol Effects 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006835 compression Effects 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
Abstract
The present invention relates to microbial bacterial agent prevention and control fields, have the trichoderma asperellum strain for inhibiting plant absorption heavy metal in soil effect by using one plant filtered out early period, make a kind of capsule and pill microbial inoculum.The present invention is intended to provide a kind of preparation method using fungi production with the capsule and pill microbial inoculum for dropping heavy metal effect.Wherein method includes：The bacteria suspension of trichoderma asperellum is obtained by Liquid Culture, bacterium powder is made in freeze-drying after suction filtration, then bacterium powder is made as capsule and pill.The active height of this microbial inoculum, effect stability, lasting period length, environment influence advantage small, easy to use.
Description
Technical field
The present invention relates to microbial bacterial agent prevention and control fields, and in particular to a kind of trichoderma asperellum glue with drop heavy metal effect
The preparation method of ball microbial inoculum.
Background technology
The mode of heavy-metal contaminated soil usually has：Ore extraction activity causes, such as genesis near coal-mine surrounding soil
It is formed under the environmental factors long term such as water, air and heat；In waste mining rock stacking process caused by the reasons such as leaching.It is industrial dirty
The heavy metal pollution that water irrigated farmland is caused.
To administer heavy metal pollution of soil, various modifying agents are usually added to improve the physicochemical property of soil.Modifying agent
Itself the activity of heavy metal in the soil can be reduced, play an important role in plant stability, still, also have shortcoming, such as
It administers that area is small, low production efficiency, secondary pollution is easily caused in preparation process.
In recent years, carried out both at home and abroad and a large amount of eliminated heavy metal pollution of soil pair using the biology for capableing of enriching heavy metal
The research of ozone deplation.This method has many advantages, such as efficient, economic, environmental protection, becomes the hot spot in Environmental Studies.
Trichoderma filamentous fungi, septic strong, wide adaptability belong to more than 29 kind disease fungus and a variety of pathogenetic bacterias to 18
There is antagonism, it is considered to be one of most promising BIOLOGICAL CONTROL material.
At present in the world there are many with Trichoderma biological agent as main component, and significant effect.Trichoderma
Mechanism of action include by enhance the development compression resistance of root system and plant, the solubilising of inorganic salts and seal up for safekeeping, induction of resistance etc. it is complicated
Process.
One plant of Trichoderma-trichoderma asperellum ZZY with reduction plant absorption heavy metal in soil that this laboratory is isolated,
Depositary institution：China General Microbiological culture presevation administrative center；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Point
Class is named：Trichoderma asperellum Trichodema asperellum；Deposit number：CGMCC No.12071；Preservation date：2016
14 days 01 month.Through research, there is good effect in terms of reducing plant absorption heavy metal.
Invention content
It is a kind of biocontrol microorganisms with wide application prospect for Trichoderma asperellum.Present invention aims at provide a kind of work
Property height, effect stability, lasting period be long, environment influence it is small, easy to use, can inhibit the trichoderma asperellum glue that crops absorb heavy metal
Ball microbial inoculum.
The present invention provides a kind of trichoderma asperellum capsule and pill production method, and the wherein making of primary raw material bacterium powder needs following mistake
Journey,
(1) tablet culture：1. the fungi trichoderma asperellum ZZY that the present invention uses is seeded on PDA plate, it is permanent at 26 DEG C
Temperature culture 3d, covers with and bacteria suspension is made with sterile water after tablet, for use.
The PDA plate culture medium is potato culture, and specific preparation method is：Peeled potatoes 200g is cut into small
Block adds water 500mL to boil 30min, and glucose 20g is added in filtered through gauze in filtered juice, and agar 20g is simultaneously heated, and agar is complete
Moisture is supplied after dissolving to 1000mL, is dispensed, sterilizing, for use.
(2) Liquid Culture：The volume of fluid nutrient medium is 100mL, is fitted into the triangular flask of 500mL, ties mouth, is passed through
0.1Mp, 121 DEG C, 20min sterilizings.The bacteria suspension that tablet strain is accessed by 1-5% access amounts, shakes up, in 26 DEG C, 160r/min
3d is cultivated in constant-temperature shaking incubator.
The liquid medium is：Glucose 3%, peptone 0.25%, KH2PO4 0.3%, MgSO4 0.15% are yellow
Bean powder 0.25%, starting pH are nature pH.
(3) it is lyophilized：D- trehaloses 15% and sucrose 5% are mixed with cultured bacteria suspension, D- trehaloses and sucrose are made
For composite protectant, 200r/min shakes up 30min, filter, will filter out mycelium -20 DEG C of refrigerator freezings for 24 hours after,
Freeze-drying, grinding obtain bacterium powder.
(4) capsule and pill is made：Bacterium powder and sodium alginate mix according to 2: 5 ratio, the concentration of sodium alginate are adjusted to
2.5%, then instilled in the anhydrous calcium chloride solution of concentration 1.5% after being drawn with the needleless injector of 2mL, after standing 30min
It is put into -20 DEG C of refrigerators, for 24 hours, freeze-drying takes out after dry, seals up for safekeeping and finish, be put into drier for freezing.
Description of the drawings
The selection of Fig. 1 fluid nutrient medium carbon sources
The determination of Fig. 2 glucose optimised quantities
The selection of Fig. 3 fluid nutrient medium nitrogen sources
The determination of Fig. 4 peptone optimised quantities
The selection of Fig. 5 fluid nutrient medium trace elements
The selection of Fig. 6 Mg2+ optimised quantities
The selection of Fig. 7 K+ optimised quantities
The determination of Fig. 8 optimal pHs
The selection of Fig. 9 optimal mediums
The selection of Figure 10 analysis for soybean powder optimised quantities
Spore quantity under Figure 11-12 difference protective agents
Figure 13 different ratio capsule and pill sprouting situations
Figure 14 is that capsule and pill produced by the present invention is schemed in fact
Specific implementation mode
The technical solution further illustrated the present invention below by specific implementation mode.Those skilled in the art should be bright
, the embodiment, which is only to aid in, understands the present invention, should not be regarded as a specific limitation of the invention.
The making of 1 microbial inoculum primary raw material of embodiment-freeze-dried powder
In the present embodiment, fungi strain trichoderma asperellum ZZY is cultivated by the following method, is included the following steps：
(a tablet cultures：Trichoderma asperellum ZZY is seeded on PDA plate, the constant temperature incubation 3d at 25 DEG C, after ripe, is used
Spore suspension is made in sterile water, for use.
The PDA plate culture medium is potato culture, and specific preparation method is：Peeled potatoes 200g is cut into small
Block adds water 500mL to boil 30min, after four layers of filtered through gauze, glucose 20g is added in filtered juice, agar 20g is simultaneously heated, fine jade
Fat supplies moisture to 1000mL after being completely dissolved, packing, 121 DEG C of high pressure sterilization 20min, spare.
(b) Liquid Culture：The volume of fluid nutrient medium is 100mL, is fitted into the triangular flask of 250mL, ties mouth, is passed through
0.1Mp, 121 DEG C, 20min sterilizings.The spore suspension that tablet strain is accessed by 2-4% amounts, shakes up, in 26 DEG C, 160r/min
3d is cultivated in constant-temperature shaking incubator.
The liquid medium is：Glucose 3%, peptone 0.25%, KH2PO4 0.3%, MgSO4 0.15% are yellow
Bean powder 0.25%, starting pH are nature pH.
The selection of the Liquid Culture based component and concentration, can be seen that from Fig. 1-10, when mentioned component matches, measure it
The biomass of culture is maximum, therefore uses the proportioning.
(c) it is lyophilized：Cultured bacteria suspension is added into 10% D- trehaloses and 5% sucrose as protective agent, is being shaken
30min is shaken up on bed, is then filtered, bacteria cake out will be filtered and frozen in -20 DEG C of refrigerators, freezing is for 24 hours；Bacteria cake is placed on again
Freeze dryer is lyophilized, and grinding obtains required bacterium powder.
The effect of different freeze drying protectants can be seen that from Figure 11-12.The spore amount that the freeze drying protectant obtains after being added
As shown above, in single protective agent, D- trehaloses and sucrose effect are best, and 10% D- trehaloses and 5% sucrose are answered
When conjunction, reach best, therefore it is compound as freeze drying protectant to select the ratio to carry out.
The making of 2 capsule and pill microbial inoculum of embodiment
Bacterium powder and sodium alginate mix according to 2: 5 ratio, the concentration of sodium alginate are adjusted to 2.5%, then with 2mL's
Needleless injector instills after drawing in the anhydrous calcium chloride solution of concentration 1.5%, is put into -20 DEG C of refrigerators after standing 30min,
For 24 hours, freeze-drying takes out after dry, seals up for safekeeping and finish, be put into drier for freezing.
The determination of bacterium powder and the ratio of sodium alginate：It is matched by 1: 5,2: 5,3: 5,4: 5 ratio, while to viscosity
Be adjusted, with it is smooth drip and it is only dilute be advisable, final proportioning is set to 1g sodium alginates and 40mL water is about added.Curing agent is selected
Anhydrous calcium chloride solution, when concentration is excessive, the capsule and pill to drip floats on solution, easily forms condensation, finally schedules concentration
1.5%, and do not influence solidification effect.
Capsule and pill is sprouted with same plating medium, sprouts effect as can be seen from Figure 13, the sprouting effect of No. 2 tablets
Fruit is more satisfactory, and bacterium powder ratio used is less, so bacterium powder and sodium alginate (SA) is selected to make capsule and pill with 2: 5 ratio.
Claims (10)
1. a kind of microbial bacterial agent, which is characterized in that the microbial bacterial agent includes fungi and microbial inoculum protective agent, makes plastic
Ball.The wherein described fungi is trichoderma asperellum (Trichoderma asperellum) ZZY, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center (CGMCC), deposit number are CGMCC No.12071.
2. microbial inoculum according to claim 1, which is characterized in that the fungi protective agent includes D- trehaloses 10% and sucrose
5%.
3. the preparation method of microbial bacterial agent according to claim 1 or 2, which is characterized in that include the following steps：
(1) tablet culture：The strain trichoderma asperellum ZZY that the present invention uses is seeded on PDA plate, spore suspension is made；
(2) Liquid Culture：By in the spore suspension access fluid nutrient medium of step (1), cultivated in constant-temperature shaking incubator；
(3) it is lyophilized：Cultured fluid nutrient medium is filtered to obtain mycelium, is lyophilized, obtains bacterium powder；
(4) capsule and pill is made：Pellet is made in bacterium powder, is lyophilized, is sealed up for safekeeping and finish, be put into drier.
4. preparation method according to claim 3, which is characterized in that the tablet of step (1) the fungi trichoderma asperellum ZZY
Incubation step is：Trichoderma asperellum ZZY is aseptically inoculated on PDA, the constant temperature incubation 3d at 26 DEG C, after covering with tablet
Spore suspension is made with sterile water.
5. preparation method according to claim 3, which is characterized in that step (2) the Liquid Culture step is：Liquid is trained
The volume for supporting base is 100mL, is fitted into the triangular flask of 250mL, is sealed, and through 0.1Mp, 121 DEG C, 20min sterilizings are connect by 1-5%
Kind amount access step (1) spore suspension, shakes up, 3d is cultivated in 26 DEG C, 160r/min constant-temperature shaking incubators.
6. preparation method according to claim 3, which is characterized in that step (3) described step of freeze drying is：By D- trehaloses
15% and sucrose 5% mixed with cultured bacteria suspension, as protective agent, 200r/min shakes up 30min, filter, will filter out
Come mycelium -20 DEG C of refrigerator freezings for 24 hours after, freeze-drying, grinding obtain bacterium powder.
7. preparation method according to claim 3, which is characterized in that step (4) the making capsule and pill step is：Bacterium powder and
Sodium alginate is mixed according to 2: 5 ratio, the concentration of sodium alginate is adjusted to 2.5%, then inhaled with the needleless injector of 2mL
It instilling in the anhydrous calcium chloride solution of concentration 1.5% after taking, is put into -20 DEG C of refrigerators after standing 30min, freezing for 24 hours, is lyophilized,
It is taken out after drying, seals up for safekeeping and finish, be put into drier.
8. preparation method according to claim 4, which is characterized in that the PDA culture medium making includes the following steps：It goes
Skin potato 200g is cut into small pieces, and water 500mL is added to boil 30min, and after four layers of filtered through gauze, glucose 20g is added in filtered juice,
Agar 20g is simultaneously heated, and agar supplies moisture to 1000mL after being completely dissolved, dispense, sterilizing.
9. preparation method according to claim 5, which is characterized in that the fluid nutrient medium includes the following steps：Grape
Sugar 3%, peptone 0.25%, KH2PO4 0.3%, MgSO4 0.15%, analysis for soybean powder 0.25%, starting pH are nature pH.
10. the microbial bacterial agent according to claim 1-2 or the preparation method according to any one of claim 3-9
Being prepared reduces the microbial bacterial agent of plant absorption heavy metal.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022286A (en) * | 2017-06-12 | 2018-12-18 | 天津科技大学 | A kind of preparation method of the fermenting agent for reducing grain fruits and vegetables content of beary metal |
| CN112795557A (en) * | 2020-12-30 | 2021-05-14 | 天津开发区坤禾生物技术有限公司 | Nutrition-rich immobilized freeze-dried microbial agent and preparation method and application thereof |
| CN114515555A (en) * | 2022-03-16 | 2022-05-20 | 中南大学 | Preparation method and application of sulfate reducing microorganism capsule |
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|---|---|---|---|---|
| US4668512A (en) * | 1985-03-20 | 1987-05-26 | The United States Of America As Represented By The Secretary Of Agriculture | Preparation of pellets containing fungi and nutrient for control of soilborne plant pathogens |
| US4724147A (en) * | 1983-06-22 | 1988-02-09 | The United States Of America As Represented By The Secretary Of Agriculture | Preparation of pellets containing fungi for control of soilborne diseases |
| US4767441A (en) * | 1985-09-06 | 1988-08-30 | The United States Of America As Represented By The Secretary Of Agriculture | Method for the preparation of mycoherbicide-containing pellets |
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| CN112795557A (en) * | 2020-12-30 | 2021-05-14 | 天津开发区坤禾生物技术有限公司 | Nutrition-rich immobilized freeze-dried microbial agent and preparation method and application thereof |
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