CN109020684A - A kind of sustained release bacillus subtilis microbial agent - Google Patents
A kind of sustained release bacillus subtilis microbial agent Download PDFInfo
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- CN109020684A CN109020684A CN201810905295.5A CN201810905295A CN109020684A CN 109020684 A CN109020684 A CN 109020684A CN 201810905295 A CN201810905295 A CN 201810905295A CN 109020684 A CN109020684 A CN 109020684A
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- 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
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- 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
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
- A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
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- 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
- A01N33/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic nitrogen compounds
- A01N33/02—Amines; Quaternary ammonium compounds
- A01N33/08—Amines; Quaternary ammonium compounds containing oxygen or sulfur
- A01N33/10—Amines; Quaternary ammonium compounds containing oxygen or sulfur having at least one oxygen or sulfur atom directly attached to an aromatic ring system
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05D3/00—Calcareous fertilisers
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/30—Layered or coated, e.g. dust-preventing coatings
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
- C05G5/00—Fertilisers characterised by their form
- C05G5/30—Layered or coated, e.g. dust-preventing coatings
- C05G5/37—Layered or coated, e.g. dust-preventing coatings layered or coated with a polymer
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Abstract
The present invention provides a kind of sustained release bacillus subtilis microbial agent, the microbial inoculum includes bacillus subtilis and/or its gemma, dopamine or its salt, the high-polymer molecule, sodium alginate and Ca2+.Then the present invention coats superabsorbent water the high-polymer molecule on dopamine surface, finally by sodium alginate and Ca by the way that dopamine is attached to bacillus subtilis and/or the surface of its gemma2+It is coated on the high-polymer molecule surface, so that a kind of sustained release bacillus subtilis microbial agent that can effectively keep soil moisture and slow release viable bacteria is prepared, to improve the utilization efficiency of soil moisture and bacillus subtilis.The present invention is sustained bacillus subtilis microbial agent, slowly discharges bacillus subtilis by continuing, and the sustainable nutritional condition and nutrient supply situation for improving crop inhibits harmful microbe activity, realize the efficient utilization of microbial inoculum.Sustained release bacillus subtilis microbial agent provided by the invention, preparation process is simple and environmentally-friendly, does not use organic solvent, is suitable for industrial mass production.
Description
Technical field
The invention belongs to bio-feritlizer development technique fields, and in particular to a kind of sustained release bacillus subtilis microbial agent.
Background technique
The use of agricultural micro organism microbial inoculum greatly reduces the dosage and its bring pollution of chemical fertilizer.Agricultural micro organism
Microbial inoculum can improve the nutritional condition of crop by the vital movement of wherein microorganism as a kind of bio-feritlizer and nutrient supplies
Situation is answered, accelerates absorption and conversion, inhibition harmful microbe activity of nutrient etc., in modern agricultural production, is largely made
With.
Bacillus subtilis is one kind of bacillus, is the bacillus of raw degeneration-resistant spore in one kind, is widely present
Such as soil, lake, ocean and the body surface of animals and plants in nature, itself is not pathogenic.The bacterium is as plant disease biological and ecological methods to prevent plant disease, pests, and erosion
One of bacterium has stronger prophylaxis effect, can be reduced the usage amount of fertilizer and pesticide, be a kind of micro- life of comparatively ideal biological and ecological methods to prevent plant disease, pests, and erosion
Object, therefore, how can be further improved its utilization efficiency is one of current urgent problem.
Summary of the invention
The present invention provides a kind of sustained release bacillus subtilis microbial agents.The present invention is by being attached to withered grass gemma for dopamine
Then the surface of bacillus coats superabsorbent water the high-polymer molecule on dopamine surface, finally by sodium alginate and Ca2+It is coated on high poly-
Object molecular surface, to prepare a kind of bacillus subtilis bacterium that can effectively keep soil moisture and continue slow release viable bacteria
Agent, to improve the utilization efficiency of soil moisture and bacillus subtilis.
It is an object of the present invention to provide a kind of microbial inoculum, the microbial inoculum includes bacillus subtilis and/or its gemma, more
Bar amine or its salt, the high-polymer molecule, sodium alginate and Ca2+。
Specifically, the microbial inoculum further include it is following 1) and/or 2):
1) the high-polymer molecule includes superabsorbent water the high-polymer molecule；Specifically, the superabsorbent water the high-polymer molecule includes
The high-polymer molecule as made of acrylamide and acroleic acid polymerization；
2) dopamine or its salt are covered on the bacillus subtilis and/or the surface of its gemma, the high polymer
Molecule is coated on the dopamine or the surface of its salt；The sodium alginate and Ca2+It is coated on the high-polymer molecule surface.
Specifically, the microbial inoculum is prepared by the following method:
By careless bacillus and/or its gemma, in the Tris-HCl buffer of pH value 8~8.5, with Dopamine hydrochloride one
It rises and 2h or more is stirred at room temperature, after suction filtration, place the product in reaction vessel, add ultrapure water, lead to N2, high polymer is added into solution
Monomer, crosslinking agent, the catalyst of molecule are protected from light and continue logical N2, stir at room temperature, then reaction vessel be transferred in ice bath and is stirred
It mixes, initiator is added, is transferred in closed mold after stirring, polymerize at room temperature for 24 hours, product is crushed into obtain surface cladding superabsorbent
The careless gemma bacillus agent particle of water high molecular material；
Add after sodium alginate is mixed with the careless gemma bacillus agent particle that above-mentioned surface coats super Water-absorbing high polymer
Enter water, obtains mixed solution；Mixed solution is added drop-wise to CaCl2In aqueous solution；It filters and is rinsed with deionized water, it is dry to perseverance
Weight to obtain the final product.
Specifically, the described method includes:
It weighs 1.5g Tris to be placed in a beaker, 900mL deionized water about is added, with the HCl of 6M by Tris-HCl solution
PH is adjusted to 8~8.5；Bacillus subtilis (50% or more rate containing gemma) 50g is added in above-mentioned Tris-HCl solution, and will be molten
Liquid is settled to 1L；5g Dopamine hydrochloride is added, 2h or more is stirred at room temperature, filter surface package is attached with the withered grass bud of dopamine
Spore bacillus；
The surface of the above-mentioned preparation of 1-5g is taken to wrap up the bacillus subtilis for being attached with dopamine, the reaction for being placed in 100ml is held
In device, 50ml ultrapure water is added；Logical N20.5h removes dissolved oxygen therein, is added into solution by a certain amount of monomer propylene acyl
Amine (AM), acrylic acid (AA), crosslinking agent Al (OH)3NPs and catalyst TEMED, dosage are respectively AA 2.16g/100g H2O,
AM19.1g/100g H2O、Al(OH)3NPs5g/100gH28 μ E/100gH of O and TEMED2O, tinfoil cladding under be protected from light after
Continuous logical N2, quick magnetic agitation 15min at room temperature.Then reaction vessel is transferred in ice bath and is slowly stirred 5min, addition is drawn
Sending out agent KPS, (KPS dosage is 2g/100g H2O), it is transferred quickly in closed mold, polymerize at room temperature for 24 hours after stirring 30s；
Finally product is crushed, successively uses deionized water and ethanol washing, room temperature is dried to constant weight, and by 200 meshes, obtains surface packet
Cover the bacillus subtilis microbial agent particle of super Water-absorbing high polymer；
In mass ratio 1: 0.5~1: 2, sodium alginate and above-mentioned surface are coated to the withered grass gemma of super Water-absorbing high polymer
The mixing of bacillus microbial inoculum particle, takes 1-5g mixture to be added in 50ml water, obtains mixed solution；Mixed solution is slowly dropped to
The CaCl of 0.5~1wt%2In aqueous solution；The volume ratio of mixed solution and calcium chloride water is 1: 4；After a period of time, will
The filtering of calcium chloride crosslinker solution is simultaneously rinsed with deionized water to remove excessive calcium chloride, is then dried product extremely at 37 DEG C
Constant weight to obtain the final product.
In the method, method after each compound is zoomed in or out together in proportion, also in protection model of the invention
Within enclosing.
It is a further object to provide a kind of preparation methods of microbial inoculum, which comprises by bacillus subtilis
2h or more is stirred at room temperature in the Tris-HCl buffer of pH value 8~8.5 in bacterium and/or its gemma together with Dopamine hydrochloride,
After suction filtration, place the product in reaction vessel, add ultrapure water, leads to N2, monomer, the crosslinking of the high-polymer molecule are added into solution
Agent, catalyst are protected from light and continue logical N2, it stirs at room temperature, then reaction vessel is transferred in ice bath and is stirred, initiator is added,
Be transferred in closed mold after stirring, polymerize at room temperature for 24 hours, by product crush surface coats super Water-absorbing high polymer
Careless gemma bacillus agent particle；
Add after sodium alginate is mixed with the careless gemma bacillus agent particle that above-mentioned surface coats super Water-absorbing high polymer
Enter water, obtains mixed solution；Mixed solution is added drop-wise to CaCl2In aqueous solution；It filters and is rinsed with deionized water, it is dry to perseverance
Weight to obtain the final product
Specifically, the method also includes: the high-polymer molecule includes superabsorbent water the high-polymer molecule；Specifically, described
Superabsorbent water the high-polymer molecule includes the high-polymer molecule as made of acrylamide and acroleic acid polymerization.
Specifically, the described method includes:
It weighs 1.5g Tris to be placed in a beaker, 900mL deionized water about is added, with the HCl of 6M by Tris-HCl solution
PH is adjusted to 8~8.5；Bacillus subtilis (50% or more rate containing gemma) 50g is added in above-mentioned Tris-HCl solution, and will be molten
Liquid is settled to 1L；5g Dopamine hydrochloride is added, 2h or more is stirred at room temperature, filter surface package is attached with the withered grass bud of dopamine
Spore bacillus；
The surface of the above-mentioned preparation of 1-5g is taken to wrap up the bacillus subtilis for being attached with dopamine, the reaction for being placed in 100ml is held
In device, 50ml ultrapure water is added；Logical N20.5h removes dissolved oxygen therein, is added into solution by a certain amount of monomer propylene acyl
Amine (AM), acrylic acid (AA), crosslinking agent Al (OH)3NPs and catalyst TEMED, dosage are respectively AA2.16g/100g H2O,
AM19.1g/100g H2O、Al(OH)3NPs5g/100gH28 μ L/100gH of O and TEMED2O, tinfoil cladding under be protected from light after
Continuous logical N2, quick magnetic agitation 15min at room temperature.Then reaction vessel is transferred in ice bath and is slowly stirred 5min, addition is drawn
Sending out agent KPS, (KPS dosage is 2g/100g H2O), it is transferred quickly in closed mold, polymerize at room temperature for 24 hours after stirring 30s；
Finally product is crushed, successively uses deionized water and ethanol washing, room temperature is dried to constant weight, and by 200 meshes, obtains surface packet
Cover the bacillus subtilis microbial agent particle of super Water-absorbing high polymer；
In mass ratio 1: 0.5~1: 2, sodium alginate and above-mentioned surface are coated to the withered grass gemma of super Water-absorbing high polymer
The mixing of bacillus microbial inoculum particle, takes 1-5g mixture to be added in 50ml water, obtains mixed solution；Mixed solution is slowly dropped to
The CaCl of 0.5~1wt%2In aqueous solution；The volume ratio of mixed solution and calcium chloride water is 1: 4；After a period of time, will
The filtering of calcium chloride crosslinker solution is simultaneously rinsed with deionized water to remove excessive calcium chloride, is then dried product extremely at 37 DEG C
Constant weight to obtain the final product；
In the method, method after each compound is zoomed in or out together in proportion, also in protection model of the invention
Within enclosing.
Of the invention a further object is provides the present invention microbial inoculum that any the method is prepared.
It is also another object of the present invention to provide the applications of any microbial inoculum of the present invention.
Specifically, the application includes following 1) -3) described at least one:
1) as bacillus subtilis and/or the sustained release or controlled release product of its gemma；
2) product as useful plants growth；
3) as Ecological Distribution of Soil Microorganisms or the adjusting product of nutrition condition.
The plant includes crops；
It is also another object of the present invention to provide the applications of any the method for the present invention.
Specifically, the application includes following 1) -3) described at least one:
1) answering in the product or its Related product that preparation has sustained release or controlled release bacillus subtilis and/or its gemma
With；
2) application in the product or its Related product that preparation has useful plants growth；
3) there is the application in the product or its Related product for adjusting Ecological Distribution of Soil Microorganisms or nutrition condition in preparation.
The plant includes crops；
One kind provided by the invention, has at least the following advantages:
(1) sustained release bacillus subtilis microbial agent provided by the invention is slowly discharged by continuing beneficial to crop growth
Bacillus subtilis, carry out the sustainable nutritional condition and nutrient supply situation for improving crop, inhibit harmful microbe living
It is dynamic, thus the disease-resistant anti-adversity ability for improving the growth rate of crop, enhancing crop, the improvement soil fertility of sustainability.
(2) sustained release bacillus subtilis microbial agent provided by the invention, preparation process is simple and environmentally-friendly, without using organic molten
Agent is suitable for industrial mass production application.
(3) in a specific embodiment, high polymer of the present invention has selected super absorbent polymer, super absorbent polymer
The water greater than tens times to thousands of times of its weight can be absorbed.The sustained release bacillus subtilis prepared using super absorbent polymer
Microbial inoculum can absorb the moisture saved in soil, and the moisture saved can promote its internal bacillus subtilis wrapped up again
Growth, breeding, and gemma can be promoted to switch to bacillus subtilis, slowing down soil moisture evaporation rate simultaneously, can persistently delay
Slow discharges viable bacteria into soil, improves soil moisture, the utilization efficiency beneficial to viable bacteria and nutrient.
(4) in a specific embodiment, the super absorbent polymer that the present invention selects is shown with the raising of temperature
The enhancing of water absorbing capacity, thus under slowing down 35 DEG C or more of hot environment, nutrient in soil and fertilizer, moisture rapid evaporation and
Release, improves the utilization rate of liquid manure.In addition, super absorbent polymer shows the decline of water holding capacity with the raising of temperature, this
Performance is conducive under hot environment, and the present invention is sustained bacillus subtilis microbial agent intelligent control itself and is sustained a certain amount of moisture,
The temperature of crop ambient enviroment is reduced, crop is helped to resist hot environment.
(5) sodium alginate and Ca2+It is cross-linked to form three-dimensional netted " eggshell " structure, wraps up coating superabsorbent water polymerization therein
The viable bacteria kernel of object achievees the purpose that be sustained viable bacteria using the variation of inside and outside osmotic pressure.With super absorbent polymer absorb water swelling,
" eggshell " inside water pressure is also gradually increased, interior to be inhaled when " eggshell " inside water pressure is greater than the hydraulic pressure of external soil or environment
The viable bacteria kernel of the water dissolution of receipts is discharged into outside with water, and the portion of water of release is utilized by soil absorption, external soil
Hydraulic pressure in earth increases, high polymer again by soil water and the absorption of viable bacteria kernel wrap up into sodium alginate and Ca2+It is cross-linked to form
In three-dimensional netted " eggshell " structure, repeatedly, achieve the purpose that slow release water and viable bacteria.
(6) sustained release bacillus subtilis microbial agent provided by the invention, selected bacillus subtilis is in extreme condition
Under, it can induce and generate the very strong endogenous gemma of resistance, heat-resisting, cold-resistant, the anti-organic solvent of gemma energy, chemical proof etc.,
Both it is easy to industrial production, carries out formulation, and be easy to survive, colonize and breed, Storage period is long, and the present invention is prepared into controlling
Microbial inoculum is released, the stability of preparation is good with the compatibility of chemicals, in a specific embodiment, the experimental results showed that,
Controlled release bacillus subtilis microbial agent provided by the invention, when placing 3 months, shape that the burst size of viable bacteria continues to increase
State.
Specific embodiment
Made in following embodiments, experimental method is conventional method unless otherwise specified.
Material as used in the following examples etc., is commercially available unless otherwise specified.
Following embodiments and its illustrate for explanation and understanding the present invention, do not constitute to improper limit of the invention
It is fixed.
Embodiment 1, the preparation for being sustained bacillus subtilis microbial agent
(1) bacillus subtilis surface package attachment dopamine
It weighs 1.5g Tris to be placed in a beaker, 900mL deionized water about is added, with the HCl of 6M by Tris-HCl solution
PH is adjusted to 8~8.5；Bacillus subtilis (50% or more rate containing gemma) 50g is added in above-mentioned Tris-HCl solution, and will be molten
Liquid is settled to 1L；5g Dopamine hydrochloride is added, 2h or more is stirred at room temperature, filter surface package is attached with the withered grass bud of dopamine
Spore bacillus.
(2) cladding of superabsorbent water the high-polymer molecule materials A A-co-AM
The surface of the above-mentioned preparation of 1-5g is taken to wrap up the bacillus subtilis for being attached with dopamine, the reaction for being placed in 100ml is held
In device, 50ml ultrapure water is added；Logical N20.5h removes dissolved oxygen therein, is added into solution by a certain amount of monomer propylene acyl
Amine (AM), acrylic acid (AA), crosslinking agent Al (OH)3NPs and catalyst TEMED, dosage are respectively AA 2.16g/100g H2O,
AM19.1g/100g H2O、Al(OH)3NPs5g/100gH28 μ L/100gH of O and TEMED2O, tinfoil cladding under be protected from light after
Continuous logical N2, quick magnetic agitation 15min at room temperature.Then reaction vessel is transferred in ice bath and is slowly stirred 5min, addition is drawn
Sending out agent KPS, (KPS dosage is 2g/100g H2O), it is transferred quickly in closed mold, polymerize at room temperature for 24 hours after stirring 30s.
Finally product is crushed, successively uses deionized water and ethanol washing, room temperature is dried to constant weight, and by 200 meshes, obtains surface packet
Cover the bacillus subtilis microbial agent particle of super Water-absorbing high polymer.
(3) it is sustained the preparation of bacillus subtilis microbial agent
In mass ratio 1: 0.5~1: 2, sodium alginate and above-mentioned surface are coated to the withered grass gemma of super Water-absorbing high polymer
The mixing of bacillus microbial inoculum particle, takes 1-5g mixture to be added in 50ml water, obtains mixed solution；Mixed solution is slowly dropped to
The CaCl of 0.5~1wt%2In aqueous solution.The volume ratio of mixed solution and calcium chloride water is 1: 4.After a period of time, will
The filtering of calcium chloride crosslinker solution is simultaneously rinsed with deionized water to remove excessive calcium chloride, is then dried product extremely at 37 DEG C
Constant weight to obtain the final product.
Embodiment 2, the performance for being sustained bacillus subtilis microbial agent
(1) water suction and water retention property test of bacillus subtilis microbial agent are sustained
The sustained release bacillus subtilis microbial agent prepared according to 1 the method for embodiment is taken, tests 15 DEG C, 25 DEG C, 35 respectively
DEG C when, the sustained release bacillus subtilis microbial agent is to the water conservation after the absorbability (g/g) and 12h of 0.9wt%NaCl aqueous solution
Rate (wt%), test result is as shown in table 1.
Table 1
Table 1 the result shows that, the sustained release water suction of bacillus subtilis microbial agent of 1 the method for embodiment preparation, water retention property
It is excellent, and as the temperature rises, water absorbing capacity is gradually increasing, and therefore, which can effectively keep away
Exempt from the moisture excessive evaporation in soil, especially at temperature higher summer and noon, so, the sustained release bacillus subtilis bacterium
Agent is suitble to the application of early rice, winter wheat and the biggish crop planting area of the morning and evening temperature difference, and in noon summer of high temperature, this hair
The sustained release bacillus subtilis microbial agent of bright offer can also automatically save the withered grass bud of water and its package in soil to a certain extent
Spore bacillus is not lost, the utilization efficiency for keeping soil liquid manure, improving bacillus subtilis.
In addition, as the temperature rises, the water absorbing capacity of the sustained release bacillus subtilis microbial agent of 1 the method for embodiment preparation
While power is in rising trend, water holding capacity is on a declining curve, and water absorbing capacity rises the evaporation waste for advantageously reducing moisture,
Meanwhile it being sustained the decline of bacillus subtilis microbial agent water holding capacity, be conducive to sustained release bacillus subtilis microbial agent and delay into soil
Partial moisture is released, the temperature on crop periphery is effectively reduced, is conducive to plant resistant hot environment.
(2) viable bacteria release experiment
The multi-functional sustained release bacillus subtilis microbial agent 6g prepared according to 1 the method for embodiment is taken, is equally divided into 3
Part, it is placed 10 days respectively in 15 DEG C, 25 DEG C, 35 DEG C of environment (humidity keeps 40% or less), then detects different temperatures not
With the viable bacteria burst size under standing time, being sustained bacillus subtilis microbial agent.
Plate detection method detects viable bacteria and discharges quantity measuring method:
1) in 1.2 micron membrane filter of suction funnel middle berth, the sample after placement is placed on filter membrane, is filtered, side is taken out when suction filtration
Vacuum side is into funnel plus sterile water, the total volume of sterile water are 10 times of sample quality；The step does not discharge viable bacteria for filtering off
Surface package be attached with the sustained release bacillus subtilis microbial agent of sodium alginate and the high-polymer molecule；
2) collect filtrate, be added bead, stand 20min, on rotary shaker 200r/min vibrate 30min to get
Mother liquor bacteria suspension；
3) it draws the above-mentioned mother liquor bacteria suspension of 5.0ml respectively with Sterile pipette to be added in 45ml sterile water, is by volume 1
: 10 ratio is serially diluted, and a series of dilution bacteria suspensions of different extension rates are respectively obtained；
4) each sample takes 3 dilutions for being continuously suitable for, and draws different dilution bacteria suspensions respectively with Sterile pipette
0.1ml is added on solid agar medium, and the bacteria suspension of different dilutions is uniformly applied to solid with sterile glass scraper respectively
On agar medium；Each dilution is repeated 3 times, while making blank control with sterile water, is trained in 37 DEG C of constant incubator
Support 12-36h；
5) bacterium colony count and sample in viable count purpose calculate: according to national standards the method in GB20287-2006 into
Row.
Testing result is as shown in table 2.Table 2 the result shows that, the sustained release withered grass bud being prepared according to 1 the method for embodiment
Spore bacillus microbial inoculum can effectively discharge viable bacteria, and as the temperature rises, the burst size of viable bacteria also gradually increases, but discharge speed
Degree slows down.This is the experimental results showed that sustained release bacillus subtilis microbial agent provided by the invention is very suitable to early rice and the winter is small
The fertilising of wheat.And in the summer of high temperature, sustained release bacillus subtilis microbial agent provided by the invention can slow down its package to a certain degree
Viable bacteria release.Because of crop itself slow growth at this time, excessive fertilizer, sustained release withered grass gemma provided by the invention are not needed
Bacillus microbial inoculum viable bacteria rate of release slows down, and is to a certain extent exactly the utilization efficiency for improving viable bacteria.
Table 2
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as, as long as skill obtained in the form of equivalent substitutions or equivalent transformations
Art scheme should all be fallen within the scope and spirit of the invention.
Claims (10)
1. a kind of microbial inoculum, which is characterized in that the microbial inoculum includes bacillus subtilis and/or its gemma, dopamine or its salt, height
Polymers molecule, sodium alginate and Ca2+。
2. microbial inoculum according to claim 1, which is characterized in that the microbial inoculum further include it is following 1) and/or 2):
1) the high-polymer molecule includes superabsorbent water the high-polymer molecule；Specifically, the superabsorbent water the high-polymer molecule includes by third
The high-polymer molecule made of acrylamide and acroleic acid polymerization；
2) dopamine or its salt are covered on the bacillus subtilis and/or the surface of its gemma, the high-polymer molecule
It is coated on the dopamine or the surface of its salt；The sodium alginate and Ca2+It is coated on the high-polymer molecule surface.
3. according to claim 1 and/or microbial inoculum described in 2, which is characterized in that the microbial inoculum is prepared by the following method:
By careless bacillus and/or its gemma, in the Tris-HCl buffer of pH value 8~8.5, the room together with Dopamine hydrochloride
Temperature stirring 2h or more, after suction filtration, place the product in reaction vessel, adds ultrapure water, leads to N2, the high-polymer molecule is added into solution
Monomer, crosslinking agent, catalyst, be protected from light and continue logical N2, it stirs at room temperature, then reaction vessel is transferred in ice bath and is stirred,
Initiator is added, is transferred in closed mold after stirring, polymerize at room temperature for 24 hours, product is crushed into obtain surface cladding superabsorbent water
The careless gemma bacillus agent particle of high molecular material；
Water is added after sodium alginate is mixed with the careless gemma bacillus agent particle that above-mentioned surface coats super Water-absorbing high polymer,
Obtain mixed solution；Mixed solution is added drop-wise to CaCl2In aqueous solution；It filters and is rinsed with deionized water, drying to constant weight is
?.
4. a kind of preparation method of microbial inoculum, which is characterized in that the described method includes: by bacillus subtilis and/or its gemma, in
In the Tris-HCl buffer of pH value 8~8.5, it is stirred at room temperature 2h or more together with Dopamine hydrochloride, after suction filtration, place the product in
In reaction vessel, add ultrapure water, leads to N2, monomer, crosslinking agent, the catalyst of the high-polymer molecule are added into solution, is protected from light continuation
Logical N2, stir at room temperature, then reaction vessel be transferred in ice bath and is stirred, initiator is added, is transferred to closed die after stirring
In tool, polymerize at room temperature for 24 hours, by product crush surface coats the careless gemma bacillus agent of super Water-absorbing high polymer
Grain；
Water is added after sodium alginate is mixed with the careless gemma bacillus agent particle that above-mentioned surface coats super Water-absorbing high polymer,
Obtain mixed solution；Mixed solution is added drop-wise to CaCl2In aqueous solution；It filters and is rinsed with deionized water, drying to constant weight is
?.
5. according to the method described in claim 4, it is characterized in that, including super the method also includes: the high-polymer molecule
Water sucting superpolymer molecule；Specifically, the superabsorbent water the high-polymer molecule includes the height as made of acrylamide and acroleic acid polymerization
Polymers molecule.
6. the microbial inoculum being prepared by claim 4 and/or 5 the methods.
7. the application of microbial inoculum described in any microbial inoculum of claim 1-3 and/or claim 6.
8. application according to claim 7, which is characterized in that the application includes following 1) -3) described at least one
Kind:
1) as bacillus subtilis and/or the sustained release or controlled release product of its gemma；
2) product as useful plants growth；
3) as Ecological Distribution of Soil Microorganisms or the adjusting product of nutrition condition.
9. the application of claim 4 and/or 5 the methods.
10. application according to claim 9, which is characterized in that the application includes following 1) -3) described at least one
Kind:
1) application in the product or its Related product that preparation has sustained release or controlled release bacillus subtilis and/or its gemma；
2) application in the product or its Related product that preparation has useful plants growth；
3) there is the application in the product or its Related product for adjusting Ecological Distribution of Soil Microorganisms or nutrition condition in preparation.
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| CN114736077A (en) * | 2022-03-30 | 2022-07-12 | 中诚国联（河南）生物科技有限公司 | Compound microbial agent for acid soil remediation and preparation method and application thereof |
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