CN102134168A - Microbial foliage synergist - Google Patents
Microbial foliage synergist Download PDFInfo
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- CN102134168A CN102134168A CN2010101031390A CN201010103139A CN102134168A CN 102134168 A CN102134168 A CN 102134168A CN 2010101031390 A CN2010101031390 A CN 2010101031390A CN 201010103139 A CN201010103139 A CN 201010103139A CN 102134168 A CN102134168 A CN 102134168A
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Abstract
The invention belongs to the technical field of microbial fertilizers, and particularly relates to a microbial foliage synergist; the preparation process flow of the invention is as follows: (1) the preparation of a bacillus laterosporus laubach WY9701 preparation; (2) the preparation of a streptomyces fradiae BD04002 metabolite; (3) the preparation of a microbial foliage synergist. The preparation process of the invention adopts an advanced continuous fermentation process of 'liquid submerged fermentation + closed solid fermentation', and uses a fully-closed self-controlled solid fermentation apparatus; the whole process of the solid fermentation is under fully-closed automatic monitoring; environment conditions such as the medium component, the formula, the temperature, the humidity, the ventilation, the pH value and the like are automatically detected, electronically displayed, and automatically controlled; not only the yield is greatly increased, but also the quality of the microbial foliage synergist is guaranteed.
Description
Technical field
The invention belongs to the microbial fertilizer technical field, specifically be meant microorganism blade face synergistic agent.
Background technology
The research and development and the application of China's microbial fertilizer start from the 1950's, because the reasearch funds scarcity, for a long time, microbial fertilizer industry integral level is not high, technological innovation is not enough, unstable product quality, utilize problems such as degree is lower.Existing in the world at present more than 70 countries and regions Application and Development microbial fertilizer, in the agriculture production of American-European developed country, the use of microbial fertilizer has reached more than 20% of fertilizer total amount, and ultimate production is with 10%～20% speed increase.And China's annual output microorganism fertilizer only accounts for 0.5% of fertilizer production.According to expert calculation, if this ratio reaches 3%, just can reduce by 500,000 tons of fertilizer amounts, produce more food 10,000,000,000 kilograms.
China is a large agricultural country, and crops planting area is wide.Agricultural is the basis of national economy, is " peace industry all over the world ".China and the world most of agricultural country is the same, is faced with all that the environmental pollution, the soil acidification that cause because of excessive use chemical fertilizer, agricultural chemicals, hormone harden, soil fertility fails, agricultural product quality descends at present, severe day by day problems such as toxic chemical substance residually exceeds standard, harm humans health.Especially the harm that brings to the mankind of food safety, pollution of dining table has caused international community's common concern.Therefore, develop eco-agriculture, produce green food, ensure people ' s health, extremely national governments pay attention to and support." green, environmental protection, health " has become the theme of current social development." solve the 21 century agricultural problem, crucial ", become countries in the world expert, political VIP's common understanding by biotechnology.Utilize biotechnology rehabilitating soil, degraded murder by poisoning, decontamination, the growth of promotion plant and animal, enhancing diseases prevention anti-adversity ability, raising output to improve quality, its unique remarkable effect is familiar with by the agriculturist and is accepted.Biotechnology is called " sunrise industry of 21 century ".The pollution-free food production data and the environmentfriendly products of Applied Biotechnology development have great demand and development space.The market outlook of China's microbial fertilizer are very wide.
Summary of the invention
The objective of the invention is to utilize microbial strains and advanced biotechnology development microorganism blade face synergistic agent.
The present invention is achieved in that
Technical process route of the present invention is as follows:
1,---------the seeding tank deep fermentation is cultivated, and---it is qualified that the closed type solid fermentation case is cultivated---work in-process---detection to shake a bottle spawn culture for the test tube slant spawn culture in the preparation of bacillus laterosporus preparation: bacterial classification (ampoul tube).
2,---------------fermentor tank continuously ferments, and---it is qualified that aftertreatment---work in-process---detects in cultivation for the seeding tank liquid fermentation and culture for shake-flask culture in the test tube slant for induction mutation of bacterium in the preparation of streptomyces fradiae metabolite: bacterial classification (ampoul tube).
3, the preparation of microorganism blade face synergy fertilizer:
The bacillus laterosporus preparation that ferments is determined viable count, and formulation content 50～10,000,000,000/gram bacillus laterosporus preparation and the streptomyces fradiae metabolite that ferments 2～5%: 95～98% ratios by weight percentage are mixed---detects---packing---finished product.
One, raw material bacterium seed selection
By separation, screening to a large amount of microorganisms in the soil, with the bacillus laterosporus of diseases prevention, promotes growth, strong stress resistance as producing one of bacterial strain, this bacterial strain has carried out the bacterial classification virulence test through Nutrition and Food Safety Office of China Disease Prevention and control Centre, confirm that this bacterial strain is by food per os toxicity grading standard, true border non-toxic substance.China Committee for Culture Collection of Microorganisms's common micro-organisms center bacterial strain deposit number of this bacterial strain is: CGMCC NO.1755; From soil, isolate another strain bacterial strain streptomycete, it is remarkable that this bacterial strain is urged length, diseases prevention, disease-resistant effect, be accredited as streptomyces fradiae through Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms's common micro-organisms center bacterial strain deposit number of this bacterial strain is: CGMCC NO.2721.
The characteristic of one of raw material bacterial strain bacillus laterosporus:
1, strain improvement morphological characteristic
Shape and arrangement: thalline is shaft-like, hold thin slightly, single generate to, in heaps; The gemma ovalize, (1～1.2) * (2.5～3) μ m, being attached to gemma one side has the paddle body, and gemma is adnation, middle life in the fusiform sporangiocyst.
Motion and flagellum: can move.
2, physiological property
(1) gelatin stab culture: liquefaction: the positive; Liquefying speed: slow, a little less than.
(2) starch hydrolysis: feminine gender.
(3) with the relation of oxygen: aerobic, anaerobic all can be grown.
(4) casein hydrolysis: the positive.
(5) tyrosine hydrolysis: feminine gender.
(6) formation of Protosol: feminine gender.
3, cultivate characteristic: the culture medium culturing of choosing any one kind of them during cultivation
| Substratum | Culture condition | Cultivate and learn characteristic |
| Bacterium colony on the agar culture dish | Cultivated 1～3 day for 35 ℃～37 ℃ | Shape: circular surface: lubricious, gauffer, flat or margin of uplift slightly: neat or irregular slightly size: diameter 2-5mm color: white or light khaki color optical signature: dark, opaque |
| The agar inclined-plane | Cultivated 1～3 day for 35 ℃～37 ℃ | Growth form: streak culture, there is the pimple increment at the edge: middle generation color: no bacterium colony color: light khaki color smell: do not have obvious peculiar smell viscosity: not sticking |
| Nutrient solution: J-gravy | Cultivated 1～3 day for 35 ℃～37 ℃ | Opacity: middle smell: the free from extraneous odour thing that sinks: sticking, amount is few |
| The gelatin stab culture | Cultivated 8 days for 35 ℃～37 ℃ | Whether liquefy: positive liquefying speed: slow, a little less than |
4, with the relation of temperature
Substratum: J-gravy; PH value: 7.3～7.5
Top temperature: 47 ℃;
Optimum temperuture: 30～40 ℃;
Tolerable temperature: 80 ℃, 10 minutes.
5, the production technique of bacillus laterosporus preparation
Through continuous lab scale and pilot scale, we grope out cover effective (the closed fermentation of a liquid fermenting+solid) production technique scheme: the operational path production that a bottle spawn culture → seeding tank deep fermentation cultivation → closed type solid fermentation case is cultivated is cultivated → shaken to bacterial classification (ampoul tube) → slant strains.The continuous fermentation process of the employing advanced person that the project construction is initiative " deep fermentation+closed type solid fermentation ", developed totally-enclosed self-control type solid fermentation apparatus voluntarily, the whole process of solid fermentation all is under the totally enclosed automatic monitoring, to envrionment conditionss such as the medium component prescription in the fermenting process, temperature, humidity, ventilation, pH values, carry out automatization detection, electronics demonstration, control automatically, not only increase substantially output, and guaranteed the stability of quality product.
The strain characteristic of two streptomyces fradiaes of raw material bacterial strain:
1, strain improvement morphological characteristic
Mycelia becomes the unwrapping wire shape, and the branch filament has substrate mycelium and aerial hyphae.Substrate mycelium does not have tabula, does not rupture; Aerial hyphae is straight or slightly crooked, multi-branched; Form fibrillae of spores, the children is upright or ripple is bent during age, is 2-4 when ripe and encloses loose spirrillum circle, and 7 circles of discrete as many as form chain spore, spore circle or oval, smooth surface to certain period fibrillae of spores top.Bacterium colony surface opaque shape on synthetic medium, Se Bai or pink, the back side is apricot yellow or tiger fur is yellow.Lawn secretes light brown little dewdrop, and specific bacterium fragrance is arranged.
2, cultural characteristic, physiological and biochemical property see the following form, the culture medium culturing of choosing any one kind of them during cultivation
The streptomyces fradiae physicochemical characteristics:
Physiological and biochemical property:
| Test subject | The result | Test subject | The result | Test subject | The result | Test subject | The result |
| Utilization of carbon source: inositol N.F,USP MANNITOL salicin raffinose rhamnosyl starch sorbyl alcohol sugar cane molasses two sugar,confectioner's seminoses | - + - + - + - - + + + | Utilization of carbon source (continuing) maltose synanthrin ribose glycerol-glucose acid sodium semi-lactosi Trisodium Citrate sodium succinate sodium malate cellobiose | + - + + + + + + + + | Utilization of carbon source (continuing) sodium malonate sorbose amygdaloside sodium tartrate D-glucose sodium hippurate trehalose wood sugar lactose melizitose | - - - - + - + + + + | Utilization of carbon source (continuing) L-arabinose galactitol tetrahydroxybutane sodium acetate Sodium Propionate casein amino acid enzyme amylase milk peptonizes the gelatine liquefication nitrate reduction | - - - + + + + + - + |
3, induction mutation of bacterium
(1) ultraviolet mutagenesis
Ultraviolet lamp (30 watts) preheating is after 30 minutes, filling the spore suspension (5.7 * 10 of 70ml with the physiological saline preparation
7Individual/ml) glass jar (diameter is 20 centimetres) places ultraviolet lamp tube 30 centimeters, places magnetic stirring apparatus below the glass jar, simultaneously aseptic rotor is put in the glass jar.Start agitator, the unlatching ultraviolet lamp also clocks, and places aseptic centrifuge tube standby at 0,5,10,15,20,30,40 minute timing sampling 10ml respectively.
(2) nitrosoguanidine mutagenesis
Take by weighing a certain amount of nitrosoguanidine, use the acetone hydrotropy, be mixed with the nitrosoguanidine mother liquor of 20mg/ml with the phosphate buffered saline buffer (the acetone volume is 1: 9 with the ratio of damping fluid volume) of pH6.0.
The spore suspension (5.7 * 10 that is prepared into the phosphate buffered saline buffer of pH6.0 at 70ml
7Individual/as to add 7ml nitrosoguanidine mother liquor in ml), put 200RPM, 28 ℃ of shaking tables are handled.Respectively effect after 0,5,10,15,20,25,30 minute timing sampling 10ml in aseptic centrifuge tube 3000RPM10 minute centrifugal, physiological saline washing three times suspends standby with 10ml physiological saline at last.
(3) ethyl sulfate mutagenesis
Get the 1ml ethyl sulfate with 1ml acetone hydrotropy, add the spore suspension (5.7 * 10 that 70ml is prepared into the phosphate buffered saline buffer of pH7.0
7Individual/as ml), to put 28 ℃ of shaking tables of 200RPM and handle.0,5,10,15,20,25,30 minutes timing sampling 10ml are in centrifuge tube, and each centrifuge tube adds the Sulfothiorine termination reaction of 0.6ml36%, and 3000RPM10 minute centrifugal, and physiological saline washing three times suspends standby with 10ml physiological saline at last.
By ultraviolet ray, nitrosoguanidine and ethyl sulfate mutagenic treatment, obtain the many strains of high yield bacterium of streptomyces microflavus fermentative production growth-promoting substance and antibacterial substance.The bacteriostatic activity of these high yield bacterium is respectively 2-5 a times of original strain.
4, the technological process of production: see shown in Figure 1.
Two, microorganism blade face synergy fertilizer product preparation
Will be through the bacillus laterosporus active bacteria formulation of seeding tank liquid fermenting and closed type solid fermentation and the streptomyces fradiae meta-bolites of producing through seeding tank liquid submerged fermentation and fermentor tank continuous fermentation process, reach the company standard requirement after being mixed according to detected result.
Compounding method: the bacillus laterosporus preparation that ferments is determined viable count, and formulation content 50～10,000,000,000/gram bacillus laterosporus work in-process and the streptomyces fradiae metabolite that ferments are in 2～5%: 98～95% ratios are mixed.
Three, product the key technical indexes of the present invention
Living bacteria count 〉=200,000,000/gram, assorted bacterium rate≤5%, pH value is 6.0-9.5, moisture≤10%, fineness (by the standard sieve of aperture 0.18mm) 〉=90% stimulates circle 〉=20mm, bacteriostasis rate 〉=50%.
Meta-bolites detected result: after testing, contain all kinds of endogenous hormones in the meta-bolites, what exist in a large number is zeatin (Z), ribosylzeatin (ZR), Plant hormones regulators,gibberellins (GA3) growth hormone (1AA), also can measure dormin (ABA), the about 35500ng/g of naphthylacetic acid (NAA) zeatin content sometimes; The about 350500ng of GA content; The about 21700ng/g of growth hormone content; The about 200ng/g of dormin content
2
The scope of application:
Various crop and edible fungis such as paddy and wheat, potato, corn, vegetables, rape, fruit tree, tea, sugarcane, cotton, mulberry, tobacco, flowers.
Generally at crops seedling stage, contain long-term, flowering fruit bearing stage and spray, main usage is as follows:
(1) general crop: every mu of 20-30g converts water 40-90kg and sprays.
(2) fruit tree: every mu of 40-60g converts water 150-300kg, sprays before and after SPRING WHEAT BEFORE AND AFTER FLOWERING, the phase of bearing fruit, expanding stage, harvesting.
(3) seed soaking: soaked seed after the dilution proportion or after planting watered and spread by 1: 1000.
Advantage of the present invention and positively effect are:
1, the continuous fermentation process of the initiative employing advanced person of the present invention " liquid submerged fermentation+closed type solid fermentation ", developed totally-enclosed self-control type solid fermentation apparatus voluntarily, the whole process of solid fermentation all is under the totally enclosed automatic monitoring, envrionment conditionss such as the medium component in the fermenting process, prescription, temperature, humidity, ventilation, pH value are carried out automatization detection, electronics demonstration, control automatically, not only increase substantially output, and guaranteed stable high-quality product.
A. the present invention has adopted novel process and equipment in commercial process, has realized deep fermentation and closed type solid fermentation continuous production, has realized totally-enclosed automatic control in the solid fermentation process.Novel in design, safe and reliable, efficient improves.
B. production process design advanced person of the present invention, the production unit dependable performance, technology is reasonable, and measuring means is complete, possesses the ability of batch process.Produce market is wide, and economic and social benefit is remarkable.
C. bacterial strain that this microbiobacterial agent adopts can be secreted the various active material through the dominant bacteria of research for many years and further screening reinforcement, not only can stimulate plant growth, and can suppress multiple pathogenic bacteria, the product strong stress resistance, reactivity is good, and reproduction speed is fast.On probation through the various crop test, for increase crop yield, improvement quality of agricultural product positive effect is arranged, help the environment protection development.
2, the function bacterial classification that a. successfully screens, purification, rejuvenation make new advances, inner called after WY97001 bacterium and BD04002 bacterium detect through Institute of Micro-biology of the Chinese Academy of Sciences and to be accredited as bacillus laterosporus and streptomyces fradiae.
The continuous fermentation process pattern of b. initiative application " liquid submerged fermentation+solid sealing fermentation " promotes fermentation production rate significantly.
C. develop totally-enclosed self-control type solid fermentation apparatus voluntarily, realize the computer monitor of solid fermentation link, improve the quality of products and stability processing parameters such as temperature, humidity, ventilation, pH values.
D. raw material is easy to get, inexpensive, zymotechnique is stable, and leavened prod living bacteria count height (the finished product viable count can reach more than 10,000,000,000/gram) is fit to large-scale industrial production, and production unit can be general with other microbiobacterial agent production units.
E. through experiment and demonstration and application proof, product stability of the present invention is good, and validity period is long, preserves at normal temperatures not lose efficacy in 2 years, and is easy to use; Harmless to crop, no specific peculiar smell, not skin irritation.And applicable crops is in extensive range, and effect is remarkable.
Product of the present invention to the main effect of crop is: 1. regulate enzymatic reaction, strengthen photosynthesis; 2. multiple nutrients is provided, promotes to grow; 3. improve crop resistance, reduce disease and take place; 4. secrete growth substance, promote root system development; 5. the harmful toxic matter of degrading improves output and improves quality.
3, the strain fermentation product mainly contains four class active substances:
Zeatin, growth hormone, Plant hormones regulators,gibberellins, dormin.Each parahormone interacts, the collaborative adjusting, to the growth of plant, bloom, maturation and dormancy as a result carry out comprehensive regulation, and the yield and quality of crop is played an important role, its main effect is:
1. promote cell fission, strengthen photosynthesis.Contained growth hormone, zeatin, Plant hormones regulators,gibberellins (GA) in the synergistic agent of microorganism blade face, lead multiple natural phant growth substances such as pentenyl VITAMIN B4 (ZIP), indolylacetic acid, dormin, after the plant absorbing utilization, the short reaction of regulatable enzyme, strengthen the activity of enzyme, improve the crop disease prevention anti-adversity ability, effectively regulate the plant growth level; Promote cell fission, increase chlorophyll content, improve photosynthesis.Can significantly improve crop quality, promote the crop precocity;
2. nutrition is provided, promotes plant growth.The VITAMIN that contains in the synergistic agent of microorganism blade face, amino acid, nutritive substances such as polysaccharide can promote crop growth after directly being absorbed by crop; Make blade increase, thicken, cane increases slightly, increases, and percentage of fertile fruit improves, the growth potential of performance crop.Product is rich in trace element in the solubility, can increase trace elements absorbed in the succession crop.
3. disease resistance improves crop resistance.The beneficial microorganism of microorganism blade face synergistic agent and excretory various active material thereof can suppress or directly kill multiple pathogenic micro-organism, make that plant growth is sane, resistance strengthens.Microorganism blade face synergistic agent also can make because of some disease cause dormant crop recover and recover the growth (as the recovery of the early stage mosaic disease of tobacco).Show according to authority's experiment: this microorganism blade face synergistic agent has obvious restraining effect to multiple germ viruses such as tobacco mosaic virus, aspergillus niger, cotton seedling blight, banded sclerotial blight, anthrax, and inhibiting rate can reach about 80%.
4. breaking dormancy promotes to sprout.This microorganism blade face synergistic agent seed soaking can be broken seed dormancy, promotes seed germination, improves seedling rate, makes Miao Qimiao strong.
5. this product enters in the soil and can effectively improve the soil micro-ecosystem environment, increases the humus content of the soil, plays the effect of culture fertility.
Description of drawings
Fig. 1 is technological process of production figure of the present invention
Embodiment
1. the preparation of bacillus laterosporus:
A. strain improvement
(ampoul tube) shape and arrangement: thalline is shaft-like, hold thin slightly, single generate to, in heaps; The gemma ovalize, (1～1.2) * (2.5～3) μ m, being attached to gemma one side has the paddle body, and gemma is adnation, middle life in the fusiform sporangiocyst.
Motion and flagellum: can move.
B. breeding strain
Slant strains is cultivated: test tube slant substratum: extractum carnis-protein culture medium; Culture temperature: 32 ± 1 ℃; Incubation time: 72～96 hours.
Shake-flask culture: culture temperature: 32 ± 1 ℃ of incubation times: 30～32 hours
C. preparation production
Seeding tank liquid fermentation and culture: culture temperature: 32 ± 1 ℃ of incubation times: 18～20 hours aeration condition 1:0.3～0.5V/V
The closed type solid fermentation incubator is cultivated:
28～35 ℃ of incubation times of culture temperature: 96～120 hours cultivation humidity: 60%～90%
The bacillus laterosporus preparation---it is qualified to detect
Work in-process bacillus laterosporus preparation test rating: effective viable bacteria 〉=5,000,000,000/gram; Assorted bacterium rate≤5%; Moisture≤10%.
2. the preparation of streptomyces fradiae:
(1) strain improvement
(ampoul tube) mycelia becomes the unwrapping wire shape, and the branch filament has substrate mycelium and aerial hyphae.Substrate mycelium does not have tabula, does not rupture; Aerial hyphae is straight or slightly crooked, multi-branched; Form fibrillae of spores, the children is upright or ripple is bent during age, is 2-4 when ripe and encloses loose spirrillum circle, and 7 circles of discrete as many as form chain spore, spore circle or oval, smooth surface to certain period fibrillae of spores top.Bacterium colony surface opaque shape on synthetic medium, Se Bai or pink, the back side is apricot yellow or tiger fur is yellow.Lawn secretes light brown little dewdrop, and specific bacterium fragrance is arranged.
(2) induction mutation of bacterium
A. ultraviolet mutagenesis
Ultraviolet lamp (30 watts) preheating is after 30 minutes, filling the spore suspension (5.7 * 10 of 70ml with the physiological saline preparation
7Individual/ml) glass jar (diameter is 20 centimetres) places ultraviolet lamp tube 30 centimeters, places magnetic stirring apparatus below the glass jar, simultaneously aseptic rotor is put in the glass jar.Start agitator, the unlatching ultraviolet lamp also clocks, and places aseptic centrifuge tube standby at 0,5,10,15,20,30,40 minute timing sampling 10ml respectively.
B. nitrosoguanidine mutagenesis
Take by weighing a certain amount of nitrosoguanidine, use the acetone hydrotropy, be mixed with the nitrosoguanidine mother liquor of 20mg/ml with the phosphate buffered saline buffer (the acetone volume is 1: 9 with the ratio of damping fluid volume) of pH6.0.
The spore suspension (5.7 * 10 that is prepared into the phosphate buffered saline buffer of pH6.0 at 70ml
7Individual/as to add 7ml nitrosoguanidine mother liquor in ml), put 200RPM, 28 ℃ of shaking tables are handled.Respectively effect after 0,5,10,15,20,25,30 minute timing sampling 10ml in aseptic centrifuge tube 3000RPM10 minute centrifugal, physiological saline washing three times suspends standby with 10ml physiological saline at last.
C. ethyl sulfate mutagenesis
Get the 1ml ethyl sulfate with 1ml acetone hydrotropy, add the spore suspension (5.7 * 10 that 70ml is prepared into the phosphate buffered saline buffer of pH7.0
7Individual/as ml), to put 28 ℃ of shaking tables of 200RPM and handle.0,5,10,15,20,25,30 minutes timing sampling 10ml are in centrifuge tube, and each centrifuge tube adds the Sulfothiorine termination reaction of 0.6ml 36%, and 3000RPM10 minute centrifugal, and physiological saline washing three times suspends standby with 10ml physiological saline at last.
By ultraviolet ray, nitrosoguanidine and ethyl sulfate mutagenic treatment, obtain to protect the many strains of high yield bacterium of streptomyces microflavus fermentative production growth-promoting substance and antibacterial substance.The bacteriostatic activity of these high yield bacterium is respectively 2-5 a times of original strain.
(3) breeding strain
Slant strains is cultivated: test tube slant substratum: potato sucrose substratum; Culture temperature: 28 ± 1 ℃; Incubation time: 96～120 hours.
Shake-flask culture: culture temperature: 28 ± 1 ℃ of incubation times: 20～24 hours
(4) work in-process production
The seeding tank liquid fermentation and culture
Culture temperature: 28 ± 1 ℃ of incubation times: 1: 0.5～0.8V/V of 20～24 hours aeration conditions
The fermentor tank cultivation of continuously fermenting
Culture temperature: 28 ± 1 ℃ of incubation times: 1: 0.8～1.2V/V of 40～48 hours aeration conditions
(5) aftertreatment---last handling process is that fermented liquid passes through solid-liquid separation, and solid part adopts the flash method drying, and liquid portion adopts the spray method drying, and merging dry powder is the streptomyces fradiae metabolite.
Streptomyces fradiae metabolite work in-process---it is qualified to detect
Inspection of semifinished product index: bacteriostasis rate 〉=70%; Stimulate circle 〉=25mm; Moisture≤10%.
2. the preparation of microorganism blade face synergy fertilizer:
Compounding method: the bacillus laterosporus preparation that ferments is determined viable count, and formulation content 50～10,000,000,000/gram bacillus laterosporus preparation and the streptomyces fradiae metabolite that ferments are in 2～5%: 95～98% ratios are mixed.
Above content is a better embodiment of the present invention only, and for those of ordinary skill in the art, according to thought of the present invention, the part that all can change in specific embodiments and applications, this description should not be construed as limitation of the present invention.
Claims (9)
1. microorganism blade face synergistic agent, it is characterized in that: its preparation technology's flow line is as follows:
(1)---------the seeding tank deep fermentation is cultivated, and---the closed type solid fermentation case is cultivated---bacillus laterosporus preparation work in-process---, and it is qualified to detect to shake a bottle spawn culture for the test tube slant spawn culture in the preparation of bacillus laterosporus preparation: bacterial classification;
(2)---------------fermentor tank continuously ferments, and---it is qualified to detect in aftertreatment---streptomyces fradiae metabolite work in-process---in cultivation for the seeding tank liquid fermentation and culture to shake a bottle spawn culture for the test tube slant spawn culture for induction mutation of bacterium in the preparation of streptomyces fradiae metabolite: bacterial classification;
(3) preparation of microorganism blade face synergy fertilizer:
The bacillus laterosporus preparation that ferments is determined viable count, formulation content 50～10,000,000,000/gram bacillus laterosporus preparation work in-process and the streptomyces fradiae metabolite that ferments are mixed by weight 2～5%: 98～95% ratios, detect, pack, make finished product then.
2. microorganism according to claim 1 blade face synergistic agent, it is characterized in that: the substratum of described bacillus laterosporus and training method are chosen any one kind of them below being: agar medium is cultivated, the agar slant medium is cultivated, J-gravy nutrient solution is cultivated, the gelatin stab culture.
3. microorganism according to claim 1 blade face synergistic agent, it is characterized in that: the production technique of described bacillus laterosporus preparation is: bacterial classification, and---------the seeding tank deep fermentation is cultivated, and---the closed type solid fermentation case is cultivated---bacillus laterosporus preparation work in-process---, and it is qualified to detect to shake a bottle spawn culture in the slant strains cultivation, wherein, the temperature in the seeding tank fermenting process is that 32 ± 1 ℃, 1: 0.3～0.5V/V of aeration condition, fermentation time are 18～20 hours; Closed type solid fermentation case culture condition is: humidity 60%～90%, 28～35 ℃ of temperature, fermentation time are 96～120 hours.
4. microorganism according to claim 1 blade face synergistic agent is characterized in that: the preparation technology of described streptomyces fradiae metabolite: bacterial classification, and---------------fermentor tank continuously ferments, and---it is qualified to detect in aftertreatment---streptomyces fradiae metabolite work in-process---in cultivation for the seeding tank liquid fermentation and culture for shake-flask culture in the test tube slant cultivation for induction mutation of bacterium; Wherein, the leavening temperature in the seeding tank liquid fermentation and culture is that 28 ± 1 ℃, 1: 0.5～0.8V/V of aeration condition, fermentation time are 20～24 hours; The fermentor tank culture condition of continuously fermenting is, leavening temperature is that 28 ± 1 ℃, 1: 0.8～1.2V/V of aeration condition, fermentation time are 40～48 hours.
5. microorganism according to claim 1 blade face synergistic agent, it is characterized in that: the slant medium of streptomyces fradiae is chosen any one kind of them below being: czapek's solution, glucose asparagine substratum, glycerine asparagine substratum, inorganic salt starch culture-medium, ISP-2 substratum, oatmeal substratum, Gause I substratum, mulberry Ta Shi substratum.
6. microorganism according to claim 1 blade face synergistic agent is characterized in that: in the preparation of described technology (2) streptomyces fradiae metabolite, the induction mutation of bacterium method is as follows:
(1) ultraviolet mutagenesis
30 watts ultraviolet lamp preheating was 5.7 * 10 filling 70ml with the index that physiological saline prepares after 30 minutes
7The glass jar of the spore suspension of individual/ml places ultraviolet lamp tube 30 centimeters, places magnetic stirring apparatus below the glass jar, simultaneously aseptic rotor is put in the glass jar; Start agitator, the unlatching ultraviolet lamp also clocks, and places aseptic centrifuge tube standby at 0,5,10,15,20,30,40 minute timing sampling 10ml respectively;
(2) nitrosoguanidine mutagenesis
Take by weighing a certain amount of nitrosoguanidine, use the acetone hydrotropy, be mixed with the nitrosoguanidine mother liquor of 20mg/ml with the phosphate buffered saline buffer of pH6.0, the acetone volume is 1: 9 with the ratio of damping fluid volume;
Being prepared into spore count at 70ml with the phosphate buffered saline buffer of pH6.0 is 5.7 * 10
7The spore suspension of individual/ml adds 7ml nitrosoguanidine mother liquor in spore suspension, put 200RPM, 28 ℃ of shaking tables processing; Respectively effect after 0,5,10,15,20,25,30 minute timing sampling 10ml in aseptic centrifuge tube 3000RPM10 minute centrifugal, physiological saline washing three times suspends standby with 10ml physiological saline at last;
(3) ethyl sulfate mutagenesis
Get the 1ml ethyl sulfate with 1ml acetone hydrotropy, adding 70ml is 5.7 * 10 with the spore count that the phosphate buffered saline buffer of pH7.0 is prepared into
7The spore suspension of individual/ml is put 28 ℃ of shaking tables of 200RPM and is handled; 0,5,10,15,20,25,30 minutes timing sampling 10ml are in centrifuge tube, each centrifuge tube adds the Sulfothiorine termination reaction of 0.6ml36%, 3000RPM10 minute centrifugal, physiological saline washing three times is at last with the suspend many strains of high yield bacterium of standby and antibacterial substance of 10ml physiological saline; The bacteriostatic activity of these high yield bacterium is respectively 2-5 a times of original strain.
7. microorganism according to claim 1 blade face synergistic agent, it is characterized in that: in the preparation of described preparation technology (2) streptomyces fradiae metabolite, its last handling process is that fermented liquid passes through solid-liquid separation, solid part adopts the flash method drying, liquid portion adopts the spray method drying, and merging dry powder is the streptomyces fradiae metabolite.
8. microorganism according to claim 1 blade face synergistic agent is characterized in that: the preparation of described preparation technology (3) microorganism blade face synergy fertilizer: will be that 2～5%: 95～98% ratios are mixed by weight percentage through bacillus laterosporus active bacteria formulation formulation content 50～10,000,000,000/gram bacillus laterosporus work in-process of seeding tank liquid fermenting and closed type solid fermentation and the streptomyces fradiae meta-bolites of process seeding tank liquid submerged fermentation and the production of fermentor tank continuous fermentation process.
9. microorganism according to claim 1 blade face synergistic agent, it is characterized in that: described technical target of the product is: living bacteria count 〉=200,000,000/gram, assorted bacterium rate≤5%, the pH value is 6.0-9.5, moisture≤10%, fineness are the standard sieves by aperture 0.18mm, percent of pass 〉=90%, stimulate circle 〉=20mm, bacteriostasis rate 〉=50%; Contain zeatin, ribosylzeatin, Plant hormones regulators,gibberellins, growth hormone in the streptomyces fradiae meta-bolites, and dormin, naphthylacetic acid; Zeatin content 28000～41000ng/g; GA content 300500～420000ng/g; Growth hormone content 15700～32500ng/g; Dormin content 140～220ng/g.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102432352A (en) * | 2011-10-12 | 2012-05-02 | 四川宏塔生物有机肥有限公司 | Biological synergist |
| CN102442865A (en) * | 2011-09-29 | 2012-05-09 | 北京可力美施生物科技有限公司 | Functional bacterial manure and preparation method thereof |
| CN103875893A (en) * | 2014-03-28 | 2014-06-25 | 东莞市天盛生物科技有限公司 | Multi-strain composite microorganism feed additive and preparation method thereof |
| CN104446694A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Frankia super bacterial manure fermenting technology |
| CN105349449A (en) * | 2015-09-22 | 2016-02-24 | 黑龙江棵乐农业有限公司 | ECMA (ecols composite microbial agents) preparation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106834179A (en) * | 2017-02-23 | 2017-06-13 | 麻林涛 | A kind of Novel culture method of bacillus laterosporus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055198C (en) * | 1992-11-13 | 2000-08-09 | 北京大学 | Process for selectively breeding and producing high-efficiency crop disease prevention and output increasing strain |
| CN100451105C (en) * | 2006-08-04 | 2009-01-14 | 东莞市保得生物工程有限公司 | Bacillus laterosporus and soil inoculation agent prepared from the strain |
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2010
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102442865A (en) * | 2011-09-29 | 2012-05-09 | 北京可力美施生物科技有限公司 | Functional bacterial manure and preparation method thereof |
| CN102442865B (en) * | 2011-09-29 | 2013-06-05 | 北京可力美施生物科技有限公司 | Functional bacterial manure and preparation method thereof |
| CN102432352A (en) * | 2011-10-12 | 2012-05-02 | 四川宏塔生物有机肥有限公司 | Biological synergist |
| CN102432352B (en) * | 2011-10-12 | 2014-04-16 | 四川宏塔生物有机肥有限公司 | Biological synergist |
| CN103875893A (en) * | 2014-03-28 | 2014-06-25 | 东莞市天盛生物科技有限公司 | Multi-strain composite microorganism feed additive and preparation method thereof |
| CN103875893B (en) * | 2014-03-28 | 2016-05-04 | 东莞市天盛生物科技有限公司 | Many bacterial classifications composite microbial feed additive and preparation method thereof |
| CN104446694A (en) * | 2014-11-25 | 2015-03-25 | 张吉科 | Frankia super bacterial manure fermenting technology |
| CN105349449A (en) * | 2015-09-22 | 2016-02-24 | 黑龙江棵乐农业有限公司 | ECMA (ecols composite microbial agents) preparation method |
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| CN103725626A (en) | 2014-04-16 |
| CN103725626B (en) | 2016-04-27 |
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