CN102960369B - Preparation method of granular paecilomyces lilacinus biological nematocide - Google Patents
Preparation method of granular paecilomyces lilacinus biological nematocide Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 49
- 241001465752 Purpureocillium lilacinum Species 0.000 title claims abstract description 27
- 230000001069 nematicidal effect Effects 0.000 title abstract description 9
- 239000005645 nematicide Substances 0.000 title abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 50
- 230000004151 fermentation Effects 0.000 claims abstract description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000012153 distilled water Substances 0.000 claims abstract description 26
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 23
- 229930006000 Sucrose Natural products 0.000 claims abstract description 23
- 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 abstract description 23
- 230000001580 bacterial effect Effects 0.000 claims description 33
- 239000005720 sucrose Substances 0.000 claims description 22
- 238000003756 stirring Methods 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 10
- 238000011068 loading method Methods 0.000 claims description 6
- 238000010899 nucleation Methods 0.000 claims description 6
- 238000012807 shake-flask culturing Methods 0.000 claims description 5
- 238000005469 granulation Methods 0.000 claims description 3
- 230000003179 granulation Effects 0.000 claims description 3
- 238000000034 method Methods 0.000 abstract description 18
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 abstract 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 abstract 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 abstract 1
- 235000019341 magnesium sulphate Nutrition 0.000 abstract 1
- 229960004793 sucrose Drugs 0.000 abstract 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 abstract 1
- 239000007788 liquid Substances 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 17
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- 230000001954 sterilising effect Effects 0.000 description 11
- 238000005138 cryopreservation Methods 0.000 description 10
- 238000011218 seed culture Methods 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 9
- 244000061456 Solanum tuberosum Species 0.000 description 9
- 235000002595 Solanum tuberosum Nutrition 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- 235000015097 nutrients Nutrition 0.000 description 9
- 241000196324 Embryophyta Species 0.000 description 8
- 241000243785 Meloidogyne javanica Species 0.000 description 7
- 241000244206 Nematoda Species 0.000 description 6
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- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
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- 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 description 4
- 241001143352 Meloidogyne Species 0.000 description 4
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Abstract
The invention provides a preparation method of granular paecilomyces lilacinus biological nematocide, and the method comprises the following steps of: preparing strain, producing strain, carrying out primary fermentation, carrying out secondary fermentation and pelleting. In the method, a culture medium comprises cane sugar, yeast, MgSO4, KCl, ZnSO4.7H2O and distilled water. By selecting various technological conditions in the preparation method of the paecilomyces lilacinus biological nematocide, the invention provides a method for producing the paecilomyces lilacinus biological nematocide on a large scale; and the prepared paecilomyces lilacinus biological nematocide is high in robe yield and low in production cost.
Description
Technical field
The present invention relates to a kind of preparation method of GR, the particularly preparation method of Paecilomyces lilacinus GR, belongs to Paecilomyces lilacinus GR preparation field.
Background technology
In the four large cause of diseases that cause plant infectious diseases, the harm of plant nematode exceedes bacterium and virus, is only second to fungal disease.Approximately 1,000 hundred million dollars of the losses that plant nematode causes world agriculture to produce every year, wherein root knot nematode (Meloidogyne spp.) is the maximum class plant nematode of harm, is often only the financial loss that important in the world cash crop are caused just up to tens billion of dollars.In Meloidogyne (Meloidogyne), root knot nematode disease generation with Meloidogyne incognita (M.incognita), peanut root-knot nematode (M.arenaria), javanese root knot nematode (M.javanica) and four kinds of northern root knot nematode (M.hapla) is the most general, all can cause heavy losses to most food crop, oil crops, fibre crops, tobacco, tealeaves, fruit tree, vegetables, medicinal material and flowers etc.
In the method for such disease of control, due to root knot nematode conventionally survive in soil and roots of plants in, so general chemical pesticide is difficult to control its harm, the security that highly toxic pesticide is produced agricultural-food again constitutes a serious threat, and chemical nematocides can only be controlled nematode population in a short time, also have that cost is high, holding effect is short and the problem such as resistance simultaneously.In addition, what some were traditional prevents and treats method, though can play certain preventive and therapeutic effect as crop rotation, plantation disease-resistant variety etc., crop that can crop rotation in actual production is also few, and crop varieties root knot nematode to resistance is very limited.Therefore, traditional control method can not solve such disease.
Wireworm-killing biologic agricultural chemicals causes concern gradually due to its safety and efficiently, has boundless using value and market outlook.Wherein, Paecilomyces lilacinus (P.lilacinus) is one of the most promising natural enemy fungi preventing and treating at present plant nematode, has research and development widely and is worth.Paecilomyces lilacinus belongs to Mycophyta deuteromycetes Moniliales, ovum and female worm that can parasitic many plant pathogeny line insects, comprise the most serious root knot nematode of harm (Meloidogyne spp.) and Cyst nematode (Heterodera spp.), department of entomology doctor Jatala of International Potato Center reported first in 1979, Paecilomyces lilacinus to the percentage of egg parasitism of Meloidogyne incognita up to 60%-70%.Paecilomyces lilacinus bacterial strain can be grown surely at crop rhizosphere, and opposing rhizosphere harmful nematode infects, and crop rhizosphere soils microorganism species is produced to certain influence, is beneficial to growth and development of plants.
Although the cultural method of Paecilomyces lilacinus has had more research, as disclosed a kind of cultural method of Paecilomyces lilacinus in Chinese patent CN101845398A, a kind of liquid fermentation culturing method of Paecilomyces lilacinus is disclosed in Chinese patent CN102286421A, a kind of preparation method of paecilomyces lilacinus biological fertilizer is disclosed in Chinese patent CN101671210A, in CN101165016A, disclose a kind of biological organic fertilizer preparation method who prevents and treats eelworm harm, in CN1756482A, disclose a kind of manufacture method of granular nematocide.But these methods are limited to a small amount of manufacture, and large-scale production is restricted.
Summary of the invention
The present invention is for overcoming the deficiencies in the prior art, by the selection to various processing condition in Paecilomyces lilacinus GR preparation method, a kind of method of the Paecilomyces lilacinus GR that can be mass-produced is provided, and the robe output of the Paecilomyces lilacinus GR of acquisition is high, production cost is low.
The present invention is achieved through the following technical solutions:
A kind of preparation method of granular Paecilomyces lilacinus GR, it comprises the steps: to prepare bacterial strain, prepares bacterial classification, ferments for the first time, ferments for the second time and granulation, it is characterized in that, described prepare the substratum that adopts in bacterial classification consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water; Substratum in described fermentation for the first time consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water; Substratum in described fermentation for the second time consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water.
As mentioned above preparation method, describedly prepares that in bacterial classification, to adopt shake-flask culture, its shaking flask loading amount≤150ml/500ml bottle, shaking speed be that 150rpm, culture temperature are that 29～30 ° of C, time are 72h.
Preparation method as mentioned above, the inoculum size of the fermentor tank in described fermentation is for the first time 4% volume ratio, and the tinning coefficient of fermentor tank is that 0.6-0.7, temperature are that 27-29 ° of C, stirring velocity are 200-300rpm, and fermentation period is 45-54 hour.
Preparation method as mentioned above, the inoculum size of the fermentor tank in described fermentation is for the second time 4% volume ratio, and the tinning coefficient of fermentor tank is that 0.6-0.7, temperature are that 27-29 ° of C, stirring velocity are 200-300rpm, and fermentation period is 42-50 hour.
As mentioned above preparation method, described prepare substratum in bacterial classification consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg and distilled water 1000ml.The pH of described substratum is preferably 6.5.
As mentioned above preparation method, the substratum in described fermentation for the first time consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg and distilled water 1000ml.The pH of described substratum is preferably 6.5.
As mentioned above preparation method, the substratum in described fermentation for the second time just consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg and distilled water 1000ml.The pH of described substratum is preferably 6.5.
Beneficial effect of the present invention: every batch of method of the present invention can obtain nematocides (microball preparation) more than 250kg, active constituent content>=10 in every gram of microball preparation
7cFU/g preparation colony number, microball preparation granular size scope is 4 ± 1mm, thousand microballoon weight ranges are 10-15 gram, and production cost is low, can produce on a large scale granular Paecilomyces lilacinus GR.
Embodiment
Preparation in accordance with the present invention, wherein bacterial strain is Paecilomyces lilacinus bacterial strain, is to purchase availablely, also can cultivate and obtain by the following method:
Adopt potato agar substratum (PDA), obtain by method known to those skilled in the art, parameter concrete in described cultivation is selected as follows: the temperature range of strain growth is 8-38 ° of C, and the best is 25-30 ° of C; The pH of strain growth is 2-11, and preferably pH value is 5-9.The preparation method of described substratum is: after peeling potatoes, be cut into piece, 200g boils 20min, after filtered through gauze, in filtrate, adds glucose 20g, and agar powder 15g is settled to 1000ml with distilled water, packing, and high pressure steam sterilization is for subsequent use after 20 minutes.
Described bacterial strain can be preserved by the following method: the bacterial strain of above-mentioned acquisition is adopted to common test tube PDA culture block method, cut and be grown in well-grown bacterium piece on PDA substratum and be placed in 1 milliliter of aseptic centrifuge tube, under low temperature, (4 ° of C) at least preserves 1 year.
Preparation in accordance with the present invention, the preparation method of described bacterial classification comprises:
(1) actication of culture
The strain transfer of cryopreservation, to PDA medium slant, is cultivated 3～4 days under 28～30 ° of C conditions, realized the abundant activation of bacterial classification.Bacterial classification after activation culture can be inoculated shaking flask and carry out seed culture.
(2) prepare shaking flask bacterial classification
Adopt liquid nutrient medium, it consists of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water.Described substratum can use following formula: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg, distilled water 1000ml, pH=6.5.
The parameter of shake-flask culture is as follows: shaking flask loading amount≤150ml/500ml bottle, and shaking speed is 150rpm, and culture temperature is 29～30 ° of C, and the time is about 72h.
When inoculation, adopt culture block directly to inoculate shaking flask, particularly, 4 to one shaking flasks of access diameter 0.5mm culture block.
The bacterial classification of gained color on PDA substratum is normal, and microscopy exists without other miscellaneous bacteria spores, and without impurity such as bacterium bacterial ooze, no acidic smell exists.
Preparation in accordance with the present invention, described fermentation for the first time (also referred to as seeding tank fermentation) is specially:
Adopt liquid nutrient medium, it consists of: sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water.Described substratum specifically can adopt following formula: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg, distilled water 1000ml, pH=6.5.
Fermentation is for the first time seeding tank fermentation, and its seeding tank sterilizing, inoculum size and inoculation temp are specially: 121 ° of C sterilizing 30min, and now pH scope is 6.0～6.5; Fermented liquid is pressed 4% volume ratio inoculation of first class seed pot loading amount, and inoculation temp is 30 ° below C.
Fermentation is controlled and fermentation time is specially: tinning coefficient 0.6-0.7, and 28 ± 1 ° of C, stirring velocity is 200-300rpm, sampling interval time is 4 hours; Fermentation time is 18-24 hour.Fermentation period 45～54 hours.
Gained seed liquor (being bacterium liquid) is khaki color to Vandyke brown, comparatively thickness, and spore content reaches 8 × 10
7more than/ml.
Preparation in accordance with the present invention, described fermentation for the second time (also referred to as producing tank fermentation) is specially:
Adopt liquid nutrient medium, it consists of: sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water.Described substratum specifically can adopt following formula: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg, distilled water 1000ml, pH=6.5.
Fermentation for the second time produces tank fermentation, and it is produced tank sterilizing, inoculum size and inoculation temp and is specially: nutrient solution is through 121 ° of C sterilizing 30min, inoculum size 4% volume ratio, and inoculation temp is 30 ° below C.
Fermentation is controlled and fermentation time is specially: tinning coefficient 0.6-0.7, and 28 ± 1 ° of C of leavening temperature, stirring velocity is 200-300rpm, does not need to regulate pH value; Sampling interval time is 3 hours; Fermentation period is 42～50 hours.
Gained bacterium liquid is khaki color to Vandyke brown, comparatively thickness, and in fermented liquid, the raw spore content of liquid is 4 × 10
8more than/ml, dry cell weight is more than 1.5%.
Preparation in accordance with the present invention, described granulation is specially:
Paecilomyces lilacinus, after above-mentioned production tank has fermented, through fully stirring, disperses mycelia and spore in fermentor tank, is prepared into bacteria suspension for subsequent use.Bacteria suspension in the sodium alginate soln fully having dissolved and tank, according to 1:1 ratio thorough stirring and evenly mixing in stirred pot, is added to aseptic diatomite immediately, mix and become ore deposit soil solution.This ore deposit soil solution is added in ready microballoon producer, and drop oozes from aperture, forms sodium alginate micro ball.Microballoon aseptic water washing, dries in the shade under room temperature condition, is dried to after microball preparation water content is down to below 2% and packs and use.
Active constituent content>=10 in every gram of microball preparation
7cFU/g preparation colony number, microball preparation granular size scope is 4 ± 1mm, thousand microballoon weight ranges are 10-15 gram.
Preparation in accordance with the present invention, described preparation is packed, is stored by following steps:
Adopt plastics bag to vacuumize preservation, this preparation is deposited after 6 months at ambient temperature, and preparation effective constituent still maintains vigour.
Beneficial effect of the present invention: every batch of method of the present invention can obtain nematocides (microball preparation) more than 250kg, preferably at 280-360kg, active constituent content>=10 in every gram of microball preparation
7cFU/g preparation colony number, microball preparation granular size scope is 4 ± 1mm, thousand microballoon weight ranges are 10-15 gram, and production cost is low, can produce on a large scale granular Paecilomyces lilacinus GR.
Below in conjunction with embodiment, the invention will be further described, but protection scope of the present invention is not limited to embodiment.
One, substratum and formula thereof
1, potato agar substratum (PDA)
After peeling potatoes, be cut into piece, 200g boils 20min, after filtered through gauze, in filtrate, adds glucose 20g, and agar powder 15g is settled to 1000ml with distilled water, packing, and high pressure steam sterilization is for subsequent use after 20 minutes.
2, liquid nutrient medium
Sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o 10mg, distilled water 1000ml, pH=6.5.
Two, the cultivation of bacterial strain and preservation
1, potato agar substratum (PDA)
After peeling potatoes, be cut into piece, 200g boils 20min, after filtered through gauze, in filtrate, adds glucose 20g, and agar powder 15g is settled to 1000ml with distilled water, packing, and high pressure steam sterilization is for subsequent use after 20 minutes.
2, strain culturing
The temperature range of this strain growth is 8-38 ° of C, and the best is 25-30 ° of C.This bacterial strain all can be grown between pH 2-11, and appropriate pH value is 5-9.
(1), in above-mentioned potato agar substratum, the concrete culture condition of bacterial strain is: 25 ° of C of temperature, and pH=6.5, obtained strains is numbered A1.
(2), in above-mentioned potato agar substratum, the concrete culture condition of bacterial strain is: 30 ° of C of temperature, and pH=7, obtained strains is numbered A2.
(3), in above-mentioned potato agar substratum, the concrete culture condition of bacterial strain is: 28 ° of C of temperature, and pH=9, obtained strains is numbered A3.
3, bacterial classification is preserved
The bacterial strain of above-mentioned acquisition is adopted to common test tube PDA culture block method (cut be grown in well-grown bacterium piece on PDA substratum be placed in 1 milliliter of aseptic centrifuge tube), and under low temperature, (4 ° of C) preserves.
Three, prepare bacterial classification
1, activated spawn
(1) strains A of above-mentioned cryopreservation 1 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A11).
(2) strains A of above-mentioned cryopreservation 1 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 4 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A12).
(3) strains A of above-mentioned cryopreservation 1 is forwarded in PDA medium slant, under 30 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A13).
(4) strains A of above-mentioned cryopreservation 2 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A21).
(5) strains A of above-mentioned cryopreservation 2 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 4 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A22).
(6) strains A of above-mentioned cryopreservation 2 is forwarded in PDA medium slant, under 30 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A23).
(7) strains A of above-mentioned cryopreservation 3 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A31).
(8) strains A of above-mentioned cryopreservation 3 is forwarded in PDA medium slant, under 28 DEG C of conditions, cultivates 4 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A32).
(9) strains A of above-mentioned cryopreservation 3 is forwarded in PDA medium slant, under 30 DEG C of conditions, cultivates 3 days, can be used for inoculating shaking flask and carry out seed culture (being designated as A33).
2, preparation shaking flask bacterial classification
(1) shake-flask culture base
Liquid nutrient medium: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o10mg, distilled water 1000ml, pH=6.5.
(2) shake-flask culture
Control shaking flask loading amount≤150ml/500ml bottle, shaking speed is 150rpm, 29～30 ° of C of culture temperature, and the time is about 72h.
(3) inoculation
Adopt culture block directly to inoculate shaking flask, because this bacterium can produce a large amount of conidiums, therefore access 4 to one shaking flasks of diameter 0.5mm culture block.
(4) obtain bacterial classification
The bacterial classification color on PDA substratum more than obtaining is normal, and microscopy exists without other miscellaneous bacteria spores, and without impurity such as bacterium bacterial ooze, no acidic smell exists.
Four, fermentation (seeding tank fermentation) for the first time
1, seed tank culture base
Liquid nutrient medium: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o10mg, distilled water 1000ml, pH=6.5.
2, seeding tank sterilizing, inoculum size and inoculation temp
121 ° of C sterilizing 30min, now pH scope is 6.0～6.5.Fermented liquid is pressed 4% volume ratio inoculation of first class seed pot loading amount, and inoculation temp is 30 ° below C.
3, fermentation is controlled and fermentation time
Tinning coefficient 0.6-0.7,28 ± 1 ° of C, stirring velocity is 200-300rpm, sampling interval time is 4 hours.Fermentation time is 18-24 hour.Fermentation period 45～54 hours.
4, obtain seed liquor
The seed liquor (being bacterium liquid) obtaining is khaki color to Vandyke brown, comparatively thickness, and spore content reaches 8 × 10
7more than/ml.
Five, fermentation for the second time (producing tank fermentation)
1, produce tank substratum
Liquid nutrient medium: sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl 0.5g, ZnSO
47H
2o10mg, distilled water 1000ml, pH=6.5.
2, produce tank sterilizing, inoculum size and inoculation temp
Nutrient solution is through 121 ° of C sterilizing 30min, inoculum size 4% volume ratio, and inoculation temp is 30 ° below C.
3, fermentation is controlled and fermentation time
Tinning coefficient 0.6-0.7,28 ± 1 ° of C of temperature, stirring velocity is 200～300rpm, does not need to regulate pH value.Sampling interval time is 3 hours, and fermentation period is 42～50 hours.
4, obtain bacterium liquid
The bacterium liquid obtaining is khaki color to Vandyke brown, comparatively thickness, and in fermented liquid, the raw spore content of liquid is 4 × 10
8more than/ml, dry cell weight is more than 1.5%.
Six, preparation and storage
1, the aftertreatment of fermented liquid
Paecilomyces lilacinus after producing tank fermentation through fully stirring, disperses mycelia and spore in fermentor tank, is prepared into bacteria suspension for subsequent use.
2, mixture
Bacteria suspension in the sodium alginate soln fully having dissolved and tank, according to 1:1 ratio thorough stirring and evenly mixing in stirred pot, is added to aseptic diatomite immediately, mix and become ore deposit soil solution.This ore deposit soil solution is added in ready microballoon producer, and drop oozes from aperture, forms sodium alginate micro ball.
3, prepare finished product
Microballoon aseptic water washing, dries in the shade under room temperature condition, is dried to after microball preparation water content is down to below 2% and packs and use.
4, the quality of finished product
In thus obtained microsphere, active constituent content>=10 in every gram of microball preparation
7cFU/g preparation colony number, microball preparation granular size scope is 4 ± 1mm, thousand microballoon weight ranges are 10-15 gram.
5, the storage of product
Adopt plastics bag to vacuumize preservation, this preparation is deposited after 6 months at ambient temperature, and preparation effective constituent still maintains vigour.
Concrete operating parameters and product parameters are in table 1.
Table 1 operating parameters and product parameters
Claims (1)
1. the preparation method of a granular Paecilomyces lilacinus GR, it comprises the steps: to prepare bacterial strain, prepares bacterial classification, ferments for the first time, ferments for the second time and granulation, described fermentation for the first time also claims seeding tank fermentation, described fermentation for the second time also claims to produce tank fermentation, it is characterized in that, described prepare the substratum that adopts in bacterial classification consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water; Substratum in described fermentation for the first time consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water; Substratum in described fermentation for the second time consist of sucrose, yeast, MgSO
4, KCl, ZnSO
47H
2o and distilled water;
Describedly prepare that in bacterial classification, to adopt shake-flask culture, its shaking flask loading amount≤150ml/500ml bottle, shaking speed be that 150rpm, culture temperature are that 29～30 DEG C, time are 72h;
The inoculum size of fermentor tank in described fermentation is for the first time 4% volume ratio, and the tinning coefficient of fermentor tank is that 0.6-0.7, temperature are that 27-29 DEG C, stirring velocity are 200-300rpm, and fermentation period is 45-54 hour;
The inoculum size of fermentor tank in described fermentation is for the second time 4% volume ratio, and the tinning coefficient of fermentor tank is that 0.6-0.7, temperature are that 27-29 DEG C, stirring velocity are 200-300rpm, and fermentation period is 42-50 hour;
Described prepare substratum in bacterial classification consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl0.5g, ZnSO
47H
2o10mg and distilled water 1000ml; PH=6.5;
Substratum in described fermentation for the first time consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl0.5g, ZnSO
47H
2o10mg and distilled water 1000ml; PH=6.5;
Substratum in described fermentation for the second time consist of sucrose 15g, yeast 6.12g, MgSO
40.5g, KCl0.5g, ZnSO
47H
2o10mg and distilled water 1000ml; PH=6.5.
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| CN105766900A (en) * | 2016-04-01 | 2016-07-20 | 广西南宁益土生物科技有限责任公司 | Nematode granula for promoting slow seedling of crops |
| CN106376602A (en) * | 2016-08-31 | 2017-02-08 | 江西天人生态股份有限公司 | Paecilomyces lilacinus granule and preparation method thereof |
| CN107410366A (en) * | 2017-06-12 | 2017-12-01 | 北京中农富源生物工程技术有限公司 | A kind of long-acting biological kills the preparation method of nematode combination agent |
| CN110878257B (en) * | 2019-10-09 | 2021-05-11 | 浙江初农生物科技有限公司 | Paecilomyces lilacinus culture method and application |
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