CN108059535A - A kind of production method of ecological fertilizer - Google Patents
A kind of production method of ecological fertilizer Download PDFInfo
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- CN108059535A CN108059535A CN201711482728.2A CN201711482728A CN108059535A CN 108059535 A CN108059535 A CN 108059535A CN 201711482728 A CN201711482728 A CN 201711482728A CN 108059535 A CN108059535 A CN 108059535A
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05B—PHOSPHATIC FERTILISERS
- C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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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
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- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
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Abstract
The present invention relates to a kind of production methods of ecological fertilizer, and ecological seaweed biostimulant or ecological seaweed liquid biostimulant are added among fertilizer to get ecological fertilizer.The present invention reaches stimulation plant growth, nurses one's health soil, the purpose of volume increase by the addition of ecological seaweed biostimulant.
Description
Technical field
The present invention relates to a kind of production methods of ecological fertilizer.
Background technology
At present since long-term fertilization is unreasonable, cause soil hardening, seriously polluted, soil pollution is directly related to food and is good for
Kang Wenti, market need ecological fertilizer, meet the needs of people's food security.
The content of the invention
The technical problem to be solved by the invention is to provide a kind of production method of ecological fertilizer, the present invention passes through ecology
The cooperation of seaweed bio stimulin and fertilizer achievees the purpose that volume increase and conditioning soil.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme：
A kind of production method of ecological fertilizer, it is characterised in that follow the steps below：
Ecological seaweed biostimulant or ecological seaweed liquid biostimulant are added among fertilizer to get ecology
Fertilizer；The fertilizer is simple substance fertilizer, compound fertilizer, Blending Fertilizer, composite fertilizer, organic fertilizer, organic and inorganic fertilizer, Water soluble fertilizer and biological organic
One kind in fertilizer；Ecological seaweed biostimulant or the mass ratio of ecological seaweed liquid biostimulant and fertilizer are 0.1~10:
90~99.9.
The ecology seaweed biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Seaweed after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, under the conditions of 60~120 DEG C sterilize 5~
40min is cooled to 27~45 DEG C of access enzymes, digests 30~60min, the mass ratio 5~30 of seaweed fragment, water and enzyme:70~
95:0.02~1, seaweed must be digested；
3) seaweed filtrate and seaweed slag, are prepared：Enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, residue obtained to be
Seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 50~70% to be adjusted to water content with water, at 70~150 DEG C
Under the conditions of sterilize 5~40min, be cooled to 27~36 DEG C, obtain mixed culture medium, access rhodotorula mucilaginosa, rhodotorula mucilaginosa and mixing are trained
The mass ratio for supporting base is 0.1~2:98~99.9, it is 27~36 DEG C in temperature, oxygen concentration is 8~21% in fermentation tank, fermentation
6~144h, sterilize 20~40min under the conditions of temperature is 70~150 DEG C, is cooled to 20~40 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 95~99.8:0.2~5, be 25~35 DEG C in temperature, oxygen content for 1~
It under the conditions of 8mg/L, ferments 1~6 day, gained filtrate is ecological seaweed liquid biostimulant after press filtration；Filter residue is seaweed
Residue；
6) ecological seaweed biostimulant, is prepared：Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant；
The seaweed is that one kind in Enteromorpha, kelp, sargassum, opotism and kelp or arbitrary proportion are two or more.
The preparation of thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：Thawing method is at least repeated once, and step is：The expansion that step 1) is obtained
It is further cultured for strain greatly to be put into -20 DEG C of refrigerator, freezes 4h, take out, dissolve at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, 20.0~25.0g of agar；
The production method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition, wherein lysozyme according to following steps into
Row：
(A) add the water of 5 times of weight to egg white or whole egg liquid, after stirring evenly add in homogenizer in, at least it is homogeneous once, it is even
Matter temperature is 30~60 DEG C, and homogeneous pressure is 2~40MPa, and the homogeneous time is 10~40min, is filtered after homogeneous, gained filtrate is
Dilution；
(B) the dilution molecular cut off for obtaining step (A) is the ultrafiltration membrane ultrafiltration of 15000~30000Da, is obtained
Thin liquid and dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
3~8, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.001~0.5mol/L,
Obtain eluent；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 2000~10000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E)；
The resin is that the resin is cation exchange resin or macroporous absorbent resin；The model of cation exchange resin
For 001*7,732, AmberliteIR-120, Dowex-50, Lewatit-100, Diaion SK-1, AllassionCS,
One or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II,
One or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
Step 3) the separation is included more than one or both of press filtration, filtering and centrifugation.
Step 4) the mixed culture medium to water content is 60%, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is 0.5:
99.5, it is 30 DEG C in temperature, oxygen concentration 12%, ferment 120h, and sterilize 20min under the conditions of temperature is 120 DEG C.
The mass ratio of corynebacterium ammoniagenes prepared by step 5) mixture and thawing method is 98:2, it is 28 DEG C in temperature, it is oxygen-containing
It measures under the conditions of 3mg/L, to ferment 5 days.
The rhodotorula mucilaginosa and corynebacterium ammoniagenes are purchased from Chinese industrial Microbiological Culture Collection administrative center, number difference
For CICC 31192 and CICC 10168.
Step A) described in it is homogeneous for homogeneous 2 times, homogeneous temperature is 40 DEG C, and the homogeneous time is 15min, wherein even for the first time
Matter pressure is 25MPa, and second of homogeneous pressure is 4MPa.
Invention has following advantageous effects：
1st, the present invention uses new fresh seaweed, and containing in new fresh seaweed largely can be with the stimulin of stimulating growth and resistance object
Matter, activity are good.
2nd, the present invention pre-processes seaweed using lysozyme and pectase, and lysozyme makes the insoluble glutinous polysaccharide of cell membrane
Soluble glycopeptide is resolved into, cell wall rupture content is caused to escape, facilitates subsequent extracted.
3rd, the present invention is fermented under anoxic conditions using aerobic and anaerobism corynebacterium ammoniagenes, produces ATP, and ATP is a kind of
High energy phosphate compound, in cell, it can realize energy storage and exoergic with mutually converting for ADP, so as to ensure that cell items
The energy supply of vital movement promotes plant growth.It is unreasonable due to long-term fertilization, cause soil acidification, corynebacterium ammoniagenes
N0 can be reduced3--And urea, generating ammonia makes tunning that can improve acidified soil problem in alkalescent.
4th, the present invention has heavy metal certain fixation.Contain polypeptide, alginic acid and other in fermentate of the present invention
Protide, heavy metal can make albumen, alginic acid and polypeptide inactivation, while heavy metal also can be by albumen, alginic acid and polypeptide
It is fixed, so as to prevent crop from absorbing.
5th, current soil hardening is serious, applies the microorganism in ground often in anaerobic condition, more can using aerobic and oxygen bacterium
Adapt to present soil environment.
6th, the permeability of corynebacterium ammoniagenes cell is improved by thawing method, the ATP that corynebacterium ammoniagenes generate can be made more to hold
It is easily penetrated into from cell membrane extracellularly, product effects are significantly better than the product with untreated corynebacterium ammoniagenes.
7th, the present invention combines fertilizer and ecological seaweed biostimulant, achievees the purpose that volume increase, improvement soil.
Specific embodiment
It is further illustrated the present invention with reference to specific example.
Embodiment 1
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 2
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 35 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 3
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 45 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 4
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 1 time, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 30min, and homogeneous pressure 25MPa is filtered after homogeneous, and gained filtrate is dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 5
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is crushed, obtains seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 3 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Third time homogeneous pressure is 4MPa, is filtered after homogeneous, and gained filtrate is dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 6
Ecological seaweed liquid biostimulant is sprayed onto to the surface of compound fertilizer granule, ecological seaweed liquid biostimulant and
The mass ratio of compound fertilizer granule is 0.4:99.6, obtain ecological compound fertilizer material after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is dried, dry kelp is crushed using pulverizer, is obtained
Seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 7
Ecological seaweed liquid biostimulant is added in urine slurry, the matter of ecological seaweed liquid biostimulant and urine slurry
Amount is than being 0.8:99.2, obtain Algae Ecology urea after dry.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is dried, dry kelp is crushed using pulverizer, is obtained
Seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 28000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
5, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.003mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 8000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
Embodiment 8
Ecological seaweed biostimulant is added in biological organic fertilizer raw material, ecological seaweed biostimulant and biology have
The mass ratio of machine fertilizer raw material is 1:99, by be granulated, dry, cool down, sieve after obtain Algae Ecology biological organic fertilizer.
The ecology seaweed liquid biostimulant is prepared according to following steps：
1) seaweed fragment, is prepared：Fresh Laminaria Japonica after cleaning is dried, dry kelp is crushed using pulverizer, is obtained
Seaweed fragment；
2) enzymolysis seaweed, is prepared：Seaweed fragment and water are fitted into agitator tank, sterilize 15min under the conditions of 100 DEG C,
30 DEG C of access enzymes are cooled to, digest 40min, the mass ratio 20 of seaweed fragment, water and enzyme:79.94:0.06, seaweed must be digested,
The enzyme is lysozyme and pectase according to mass ratio 5:1 composition；
3) seaweed filtrate and seaweed slag, are prepared：The press filtration of enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and gained is residual
Slag is seaweed slag；
4) fermentation of seaweed object, is prepared：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to
Mass ratio 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 60% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C
Bacterium 20min is cooled to 30 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is
0.5:99.5, it is 30 DEG C in temperature, oxygen concentration is 12% in fermentation tank, and ferment 120h, is sterilized under the conditions of temperature is 120 DEG C
20min is cooled to 35 DEG C, obtains fermentation of seaweed object；
5) ecological seaweed liquid biostimulant and seaweed residue, are prepared：The seaweed filtrate that step 3) obtains is added to
Stirred and evenly mixed in the fermentation of seaweed object that step 4) obtains, obtain mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and
The mass ratio of corynebacterium ammoniagenes prepared by thawing method is 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, fermentation 5
My god, press filtration, gained filtrate is ecological seaweed liquid biostimulant；Filter residue is seaweed residue；
6) ecological seaweed biostimulant, is prepared：Ecological seaweed liquid biostimulant it is concentrated and spray drying after, i.e.,
Obtain ecological seaweed biostimulant；
The preparation of wherein thawing method corynebacterium ammoniagenes follows the steps below：
A) expansion of strain is further cultured for：Using the thalline in exponential phase as seed, it is inoculated with 10% inoculum concentration
In fluid nutrient medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for
Strain；
B) thawing method improves the permeability of cell：In triplicate, step is thawing method：The expansion that step 1) is obtained is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved, be placed again into -20 DEG C of refrigerator at room temperature, is freezed
4h takes out, dissolves, be placed again into -20 DEG C of refrigerator at room temperature, freezes 4h, takes out, dissolves at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, agar 25.0g；
The preparation method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams of immersions
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
Wherein lysozyme follows the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, added in after stirring evenly in homogenizer, homogeneous 2 times, homogeneous temperature
Degree is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is 4MPa,
Filtering after homogeneous, gained filtrate are dilution；
(B) the ultrafiltration membrane ultrafiltration for being 26000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and
Dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:
6, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.02mol/L, is washed
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention of the ultrafiltration membrane are obtained
Molecular weight is 6000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E).
The advantageous effect further illustrated the present invention with reference to experimental data：
Experiment one
1st, material to be tested is tested
1 materials and methods：
1.1 test sites and experimental subjects：Yantai university chemistry analysis inspection center.
1.2 experiment detections：Survey the activity and the rate of recovery of lysozyme.
1.3 material to be tested：It is (even without homogenizer after the preparation method of lysozyme is except stirring evenly for common process
Matter, other production methods and embodiment 1 are consistent), embodiment 1, embodiment 2, embodiment 3, prepare in embodiment 4 and embodiment 5
Lysozyme do effect and compare.
1.4 experimental design：Using high performance liquid chromatography to sample treatment.
This experiment is consistent with other operations in addition to processing difference except testing.
2 results and analysis
Common process (homogeneous without homogenizer after stirring evenly, other production methods are consistent with embodiment 1) is implemented
Bacteriolyze enzymatic treatment prepared by example 1, embodiment 2, embodiment 3, embodiment 4 and embodiment 5 is compared, and data are shown in Table 1
Table 1
| The rate of recovery (%) | Enzymatic activity (U/mg) | |
| Common process | 72.7 | 14567 |
| Embodiment 1 | 91.7 | 21101 |
| Embodiment 2 | 84.7 | 19831 |
| Embodiment 3 | 88.3 | 20790 |
| Embodiment 4 | 91.7 | 20903 |
| Embodiment 5 | 89.3 | 20737 |
It is compared by 1 embodiment 1 of table with common process, the present invention is either in terms of the lysozyme rate of recovery or enzymatic activity
All it is significantly better than common process；Compared by embodiment 1 to embodiment 3 it can be seen that the rate of recovery and work of the homogeneous temperature to lysozyme
Property still have certain influence, wherein embodiment 1 is best using 40 DEG C of general effects；Compared by embodiment 1, embodiment 4 and embodiment 5
Relatively it can be seen that homogeneous pressure and number also have certain influence to the rate of recovery and activity of lysozyme, and non-pressure is bigger, the time
It is more long better, 1 best results of Integrated comparative embodiment.
Experiment two
1st, material to be tested is tested
1 materials and methods：
1.1 test site：Linyi Luozhuang District Gao Dou towns.
1.2 material to be tested：Ordinary compound fertilizer (in addition to not ecological seaweed liquid biostimulant, other production methods with
Embodiment 1 is consistent), comparison 1 is (when preparing ecological seaweed liquid biostimulant, except corynebacterium ammoniagenes are straight without the processing of thawing method
Connect using outer, other production methods are consistent with embodiment 1), comparison 2 it is (even in homogenizer except not being added to when preparing lysozyme
Outside matter, other production methods are consistent with embodiment 1), 1 each 160kg of embodiment 6 and embodiment, do fertilizer efficiency experiment and compare；Rice
Kind teaches No. 5 for Henan, 40kg.
This experiment is consistent with other management in addition to processing difference except testing.
1.3 experimental design：The ground for selecting 20 mu of upper season crops the same, is divided into 5 groups, 4 is parallel.By ordinary compound fertilizer
(in addition to not ecological seaweed liquid biostimulant, other production methods are consistent with embodiment 1), comparison 1 (prepare ecological sea algae solution
During body biostimulant, except corynebacterium ammoniagenes are without the processing of thawing method directly in addition to use, other production methods with embodiment 1 one
Cause), comparison 2 (be not added to when preparing lysozyme homogeneous outer in homogenizer, other production methods with embodiment 1 unanimously),
It is average to apply 40kg processing fertilizer per acre in embodiment 6 and the soil that base application was turned over respectively of embodiment 1, it is broadcast using machinery
Kind, rice seed 5kg is sowed per acre, is counted after detection flag leaf width of rice, chlorophyll a, b content and harvest in yield and rice
Cadmium, chromium, lead, arsenic content, and survey soil pH.
In addition to different using soil fertility quality, other management are consistent for this experiment.
2 results and analysis
Be averaged sword-like leave width, chlorophyll a, b content of rice is shown in Table 1
1 rice of table is averaged sword-like leave width, chlorophyll a, b content
As can be seen from Table 1 under equal conditions, the present invention can substantially increase rice leaf width, improve chlorophyll a
The ratio of content and Chlorophyll-a Content/content of chlorophyll b improves Rice Photosynthesis.By comparison 1 and 1 data ratio of embodiment
Relatively it can be seen that addition uses thawing method to improve ecological seaweed liquid biostimulant prepared by the corynebacterium ammoniagenes of cytoactive,
Its ecological seaweed liquid biostimulant with obvious effects for making an addition to the corynebacterium ammoniagenes without the processing of thawing method well and preparing；By right
Than 2 and embodiment 1 compares it can be seen that the lysozyme bacteriolyze with obvious effects for being better than normal agitation for passing through the homogeneous preparation of homogenizer
Enzyme；It can be seen that by embodiment 1 and 6 data comparison of embodiment and be better than using the effect of new fresh seaweed using the effect for drying seaweed
Fruit.
Rice yield is shown in Table 2
Rice yield during table 2 is respectively handled
| Yield 1 (kg) | Yield 2 (kg) | Yield 3 (kg) | Yield 4 (kg) | Average product (kg) | |
| Common fertilizer | 439.6 | 438.7 | 435.3 | 434.8 | 437.1 |
| Embodiment 1 | 481.3 | 482.5 | 482.9 | 483.7 | 482.6 |
| Comparison 1 | 459.4 | 459.7 | 462.5 | 459.9 | 460.4 |
| Comparison 2 | 457.4 | 459.3 | 457.9 | 456.8 | 457.9 |
| Embodiment 6 | 467.2 | 463.4 | 465.6 | 468.4 | 466.2 |
As can be seen from Table 2 under equal conditions, the present invention can substantially increase crop yield.
Cadmium content is shown in Table 3 in rice
Table 3 respectively cadmium, chromium, lead, arsenic content in rice in processing
| Cadmium (%) | Chromium (%) | Lead (%) | Arsenic (%) | |
| Common fertilizer | 0.00003 | 0 | 0.00001 | 0 |
| Embodiment 1 | 0.00001 | 0 | 0 | 0 |
| Comparison 1 | 0.00002 | 0 | 0 | 0 |
| Comparison 2 | 0.00002 | 0 | 0 | 0 |
| Embodiment 6 | 0.00001 | 0 | 0 | 0 |
As can be seen from Table 3, the present invention can inhibit absorption of the rice to heavy metal, be played centainly to improving crop quality
Effect.
Each processing soil pH is shown in Table 4
| Handle 1pH | Handle 2pH | Handle 3pH | Handle 4pH | Average pH | |
| Common fertilizer | 5.82 | 5.84 | 5.85 | 5.84 | 5.85 |
| Embodiment 1 | 5.89 | 5.91 | 5.87 | 5.89 | 5.90 |
| Comparison 1 | 5.89 | 5.90 | 5.86 | 5.89 | 5.91 |
| Comparison 2 | 5.88 | 5.90 | 5.87 | 5.89 | 5.89 |
| Embodiment 6 | 5.89 | 5.91 | 5.88 | 5.88 | 5.90 |
There is table 4 as can be seen that the present invention can improve soil acidification problem compared with common fertilizer；It is differed with other processing
Less.
It can be drawn by testing above, the present invention can improve flag leaf width of rice, blade in terms of plant growth is promoted
Photosynthesis improves rice yield；Corynebacterium ammoniagenes are better after the processing of thawing method；Lysozyme is homogeneous using homogenizer
Afterwards, it is with obvious effects to be better than the uniform product of simple agitation.
The present invention can fix heavy metal in terms of soil is nursed one's health, and prevent crop from absorbing and improving the work of acidified soil
With.
Claims (9)
1. a kind of production method of ecological fertilizer, it is characterised in that follow the steps below：
Ecological seaweed biostimulant or ecological seaweed liquid biostimulant are added among fertilizer to get ecological fertilizer；
The fertilizer is in simple substance fertilizer, compound fertilizer, Blending Fertilizer, composite fertilizer, organic fertilizer, organic and inorganic fertilizer, Water soluble fertilizer and biological organic fertilizer
One kind；Ecological seaweed biostimulant or the mass ratio of ecological seaweed liquid biostimulant and fertilizer are 0.1~10:90~
99.9。
2. the production method of ecological fertilizer as described in claim 1, it is characterised in that：It is described ecology seaweed biostimulant according to
Following steps are prepared：
1）, prepare seaweed fragment：Seaweed after cleaning is crushed, obtains seaweed fragment；
2）, prepare enzymolysis seaweed：Seaweed fragment and water are fitted into agitator tank, under the conditions of 60~120 DEG C sterilize 5~
40min is cooled to 27~45 DEG C of access enzymes, digests 30~60min, the mass ratio 5~30 of seaweed fragment, water and enzyme:70~
95:0.02~1, seaweed must be digested；
3）, prepare seaweed filtrate and seaweed slag：Enzymolysis seaweed is separated, gained filtrate is seaweed filtrate, and residue obtained is seaweed
Slag；
4）, prepare fermentation of seaweed object：By seaweed slag, urea, magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and glucose according to quality
Than 95.5:1:0.5:1:1:1 is added to mixing in fermentation tank, and it is 50~70% to be adjusted to water content with water, in 70~150 DEG C of conditions
5~40min of lower sterilizing, is cooled to 27~36 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, rhodotorula mucilaginosa and mixed culture medium
Mass ratio be 0.1~2:98~99.9, it is 27~36 DEG C in temperature, oxygen concentration is 8~21% in fermentation tank, fermentation 6~
144h, sterilize 20~40min under the conditions of temperature is 70~150 DEG C, is cooled to 20~40 DEG C, obtains fermentation of seaweed object；
5）, prepare ecological seaweed liquid biostimulant and seaweed residue：By step 3）Obtained seaweed filtrate is added to step
4）It is stirred and evenly mixed in obtained fermentation of seaweed object, obtains mixture, corynebacterium ammoniagenes prepared by access thawing method, mixture and thawing
The mass ratio of corynebacterium ammoniagenes prepared by method is 95~99.8:0.2~5, it is 25~35 DEG C in temperature, oxygen content is 1~8mg/L
Under the conditions of, it ferments 1~6 day, gained filtrate is ecological seaweed liquid biostimulant after press filtration；Filter residue is seaweed residue；
6）, prepare ecological seaweed biostimulant：Ecological seaweed liquid biostimulant it is concentrated and spray drying after to get life
State seaweed bio stimulin；
The seaweed is that one kind in Enteromorpha, kelp, sargassum, opotism and kelp or arbitrary proportion are two or more.
3. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：The preparation of thawing method corynebacterium ammoniagenes is pressed
It is carried out according to following steps：
a）The expansion of strain is further cultured for：Using the thalline in exponential phase as seed, liquid is inoculated in 10% inoculum concentration
In culture medium, 48h is cultivated in 30 DEG C of shaking table 160r/min, after 8000r/min centrifuges 10min, expansion is obtained and is further cultured for strain；
b）Thawing method improves the permeability of cell：Thawing method is at least repeated once, and step is：By step 1）Obtained expansion is again
Culture strain is put into -20 DEG C of refrigerator, freezes 4h, is taken out, is dissolved at room temperature；
PH7.2~7.4 of the fluid nutrient medium, and be made into according to following mass ratio：Beef infusion broth 1.0L, peptone
10.0g, NaCl 5.0g, 20.0~25.0g of agar;
The production method of wherein beef infusion broth follows the steps below：Lean beef is cleaned, and chopping weighs 550 grams and is soaked in
It in 1375ml water, impregnates a night, filtering, 0.6kg/cm after filtrate packing2Sterilize 40min.
4. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：The enzyme be lysozyme and pectase by
According to mass ratio 5:1 composition, wherein lysozyme follow the steps below：
(A) add the water of 5 times of weight to egg white or whole egg liquid, after stirring evenly add in homogenizer in, at least it is homogeneous once, homogeneous temperature
It spends for 30~60 DEG C, homogeneous pressure is 2~40MPa, and the homogeneous time is 10~40min, is filtered after homogeneous, and gained filtrate is dilution
Liquid；
(B) the dilution molecular cut off for obtaining step (A) is the ultrafiltration membrane ultrafiltration of 15000~30000Da, obtains thin liquid
And dope；
(C) add in resin in the thin liquid obtained in step (B) to be adsorbed, the weight ratio of the thin liquid and resin is 1:3~
8, the resin after being adsorbed；
(D) resin after the absorption for obtaining step (C) is eluted with the NaCl aqueous solutions of 0.001~0.5mol/L, is obtained
It washes
De- liquid；
(E) by the eluent that step (D) obtains ultrafiltration membrane ultrafiltration desalination, ultrafiltrate, the retention molecule of the ultrafiltration membrane are obtained
It measures as 2000~10000Da；
(F) to get to lysozyme after the ultrafiltrate drying obtained step (E)；
The resin is that the resin is cation exchange resin or macroporous absorbent resin；The model of cation exchange resin
001*7、732、AmberliteIR-120、Dowex-50、Lewatit-100、Diaion SK-1、AllassionCS、
One or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II,
One or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
5. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：Step 3）It is described separation include press filtration,
It is more than one or both of filtering and centrifugation.
6. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：Step 4）The mixed culture medium is to containing
Water is 60%, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is 0.5:99.5, it is 30 DEG C in temperature, oxygen concentration 12%,
Ferment 120h, and sterilize 20min under the conditions of temperature is 120 DEG C.
7. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：Step 5）It is prepared by mixture and thawing method
Corynebacterium ammoniagenes mass ratio be 98:2, it is 28 DEG C in temperature, under the conditions of oxygen content is 3mg/L, ferments 5 days.
8. the production method of ecological fertilizer as claimed in claim 2, it is characterised in that：The rhodotorula mucilaginosa and corynebacterium ammoniagenes
Chinese industrial Microbiological Culture Collection administrative center is purchased from, number is respectively CICC 31192 and CICC 10168.
9. the production method of ecological fertilizer as claimed in claim 4, it is characterised in that：Step A）Described in it is homogeneous be homogeneous 2
Secondary, homogeneous temperature is 40 DEG C, and the homogeneous time is 15min, and wherein first time homogeneous pressure is 25MPa, and second of homogeneous pressure is
4MPa。
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Application publication date: 20180522 |