CN108157043A - A kind of implantation methods in organic orchard - Google Patents

A kind of implantation methods in organic orchard Download PDF

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CN108157043A
CN108157043A CN201711482274.9A CN201711482274A CN108157043A CN 108157043 A CN108157043 A CN 108157043A CN 201711482274 A CN201711482274 A CN 201711482274A CN 108157043 A CN108157043 A CN 108157043A
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seaweed
organic
orchard
homogeneous
biostimulant
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郭肖宏
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
    • A01G17/005—Cultivation methods
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B7/00—Fertilisers based essentially on alkali or ammonium orthophosphates
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • 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
    • C05G5/20—Liquid fertilisers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

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  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
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  • General Engineering & Computer Science (AREA)
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  • Pest Control & Pesticides (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • General Chemical & Material Sciences (AREA)
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Abstract

The present invention relates to a kind of implantation methods in organic orchard, and by the plants interplanting with expelling parasite insecticidal effect in orchard, orchard is managed to get organic orchard according to organic planting requirement.The present invention can achieve the purpose that expelling parasite, reduce the usage amount of pesticide by interplanting the plant with expelling parasite insecticidal effect in orchard.

Description

A kind of implantation methods in organic orchard
Technical field
The present invention relates to a kind of implantation methods in organic orchard.
Background technology
Bread is the staff of life, strengthens food security work, the health and life security of relationship China more than 1,300,000,000, it is necessary to Grab tightly and tightly.These years, Party and government have descended very big strength to grab food security, and food security situation constantly improves, but deposits The problem of it is still many, common people still have many expectations, it is necessary to redouble one's efforts, work be run business into particular one reality, it is ensured that Ren Minqun Many " safety on the tip of the tongue ".
Organic plantation is the source of food security, and there has been no the organic plantation sides for reducing fertilizer and Pesticide use amount at present Method.
Invention content
The technical problem to be solved by the invention is to provide a kind of implantation methods in organic orchard, and the present invention is by fruit Plant of the interplanting with expelling parasite insecticidal effect, can achieve the purpose that expelling parasite and reduce Pesticide use amount in garden.
In order to solve the above-mentioned technical problem, the present invention uses following technical scheme：
A kind of implantation methods in organic orchard, it is characterised in that follow the steps below：
By the plants interplanting with expelling parasite insecticidal effect in orchard, orchard is managed according to organic planting requirement to get having Machine orchard；
The plant with expelling parasite insecticidal effect includes garlic, lavender, fish pelargonium, common nepenthes, shichongcao and folium artemisiae argyi In one kind or arbitrary proportion it is two or more.
Fertilizer used in organic orchard is one kind or arbitrary proportion in organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer It is two or more；
Ecological seaweed biostimulant is added in the organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer；
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 detached, 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 being 70~150 DEG C in temperature, 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.
Pesticide used in organic orchard is lime sulfur, Bordeaux mixture, mineral oil, trichoderma harzianum, quinolinone suspend Agent, ethyl alcohol, soda, China tree alkali, rotenone, Dalmatian chrysanthemum missible oil and one kind in matrine 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 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.
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, added in homogenizer after stirring evenly, it is at least homogeneous primary, 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) it adds 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, It is one or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II, It is one or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
Step 3) the separation includes one or more 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 being 120 DEG C in temperature.
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 CICC31192 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.
The mode that ecological seaweed biostimulant is added in fertilizer is：With the interior addition manner of fertilizer material mixing granulation With the outer addition manner for spraying to fertiliser granulates surface.
Invention has following advantageous effects：
1st, the present invention can reach expelling parasite and reduce pesticide by interplanting the plant with expelling parasite insecticidal effect in orchard The purpose of usage amount.
2nd, ecological seaweed biostimulant uses new fresh seaweed in the present invention, and life can largely be stimulated by containing in new fresh seaweed Long stimulin and resistance substance, activity is good, can achieve the purpose that reduce Fertilizer application amount.
3rd, ecological seaweed biostimulant is fermented under anaerobic using aerobic and anaerobism rhodotorula mucilaginosa in the present invention, The loss of seaweed nutritive ingredient can be prevented, and the alcohol that anaerobic fermentation generates is a supporting role seaweed extraction.
4th, ecological seaweed biostimulant is sent out under anoxic conditions using aerobic and anaerobism corynebacterium ammoniagenes in the present invention Ferment produces ATP, and ATP is a kind of high energy phosphate compound, and in cell, it can realize and energy storage and put with mutually converting for ADP Can, so as to ensure that the energy supply of cell items vital movement, promote plant growth.It is unreasonable due to long-term fertilization, it makes Into soil acidification, corynebacterium ammoniagenes can restore N03 -And urea, generating ammonia makes tunning that can improve acidification soil in alkalescent Earth problem.
5th, ecological seaweed biostimulant has heavy metal certain fixed function in the present invention.Contain in fermentate of the present invention There are polypeptide, alginic acid and other protides, heavy metal can make albumen, alginic acid and polypeptide inactivation, while heavy metal also can It is fixed by albumen, alginic acid and polypeptide, so as to prevent crop from absorbing.
6th, the present invention is using aerobic and oxygen bacterium, and soil hardening is serious at present, and the microorganism applied in ground is often in anoxic shape State is suitable for present soil environment using aerobic and oxygen bacterium.
7th, ecological seaweed biostimulant improves the permeability of corynebacterium ammoniagenes cell by thawing method in the present invention, can be with The ATP that corynebacterium ammoniagenes generate is made to be easier to penetrate into from cell membrane extracellularly, product effects are significantly better than with without place The product of the corynebacterium ammoniagenes of reason.
Specific embodiment
It is further illustrated the present invention with reference to specific example.
Embodiment 1
To have garlic and fish pelargonium interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is adds in ecological seaweed biostimulant organic fertilizer.
Pesticide used in organic orchard described above is Bordeaux mixture, mineral oil, trichoderma harzianum and quinolinone suspending agent.
Ecology seaweed biostimulant described above 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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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, It is filtered after homogeneous, 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) it adds 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
To have garlic and fish pelargonium interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is adds in ecological seaweed biostimulant organic fertilizer.
Pesticide used in organic orchard described above is Bordeaux mixture, mineral oil, trichoderma harzianum and quinolinone suspending agent.
Ecology seaweed biostimulant described above 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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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, It is filtered after homogeneous, 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) it adds 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
By shichongcao and folium artemisiae argyi interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is to add in the organic fertilizer of ecological seaweed liquid biostimulant and add in give birth to The biological organic fertilizer of state seaweed liquid biostimulant.
Pesticide used in organic orchard described above is rotenone, Dalmatian chrysanthemum missible oil and matrine according to mass ratio 1:1:1 Composition.
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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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, It is filtered after homogeneous, 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) it adds 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
By fish pelargonium and common nepenthes interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is to add in the organic fertilizer of ecological seaweed liquid biostimulant and add in give birth to The organic and inorganic fertilizer of state seaweed liquid biostimulant.
Pesticide used in organic orchard described above is soda, China tree alkali and rotenone.
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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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) it adds 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
By fish pelargonium and common nepenthes interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is the biological organic fertilizer for adding in seaweed residue.
Pesticide used in organic orchard described above is soda, China tree alkali and rotenone.
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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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) it adds 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
To have garlic and fish pelargonium interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is adds in ecological seaweed biostimulant organic fertilizer.
Pesticide used in organic orchard described above is Bordeaux mixture, mineral oil, trichoderma harzianum and quinolinone suspending agent.
Ecology seaweed biostimulant described above 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 detached, 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 being 120 DEG C in temperature 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 homogenizer after stirring evenly, 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, It is filtered after homogeneous, 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) it adds 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
To have garlic and fish pelargonium interplanting in orchard, orchard is managed to get organic orchard according to organic planting requirement；
Fertilizer used in organic orchard described above is adds in ecological seaweed biostimulant organic fertilizer.
Pesticide used in organic orchard described above is Bordeaux mixture, mineral oil, trichoderma harzianum and quinolinone suspending agent.
Ecology seaweed biostimulant described above 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 5min under the conditions of 120 DEG C, cold But to 30 DEG C of access enzymes, 30min, the mass ratio 15 of seaweed fragment, water and enzyme are digested:84.7:0.3, seaweed must be digested, it is described 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 detached, 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 65% to be adjusted to water content with water, is gone out under the conditions of 120 DEG C Bacterium 20min is cooled to 35 DEG C, obtains mixed culture medium, accesses rhodotorula mucilaginosa, and the mass ratio of rhodotorula mucilaginosa and mixed culture medium is 0.6:99.4, it is 30 DEG C in temperature, oxygen concentration is 15% in fermentation tank, and ferment 200h, is sterilized under the conditions of being 120 DEG C in temperature 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 99:1, it is 28 DEG C in temperature, under the conditions of oxygen content is 5mg/L, fermentation 4 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 homogenizer after stirring evenly, 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 10000Da with molecular cut off by the dilution that step (A) obtains, obtain thin liquid and Dope；
(C) it adds 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, the ultrafiltrate of lysozyme is obtained, it is described super The molecular cut off of filter membrane 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, and non-pressure is bigger to the rate of recovery and activity of lysozyme, the time It is more long better, 1 best results of Integrated comparative embodiment.
1st, material to be tested is tested
1 materials and methods：
1.1 test site：The U.S. early cherry orchard in Yantai Fushan District Dong Ting towns.
1.2 material to be tested：Common orchard (in addition to garlic and fish pelargonium is not interplanted, other production methods and embodiment 1 one Cause), comparison 1 (when preparing ecological seaweed liquid biostimulant, in addition to corynebacterium ammoniagenes are without thawing method processing directly use, Other production methods are consistent with embodiment 1), comparison 2 (be not added to when preparing lysozyme it is homogeneous in homogenizer in addition to, it is other Production method is consistent with embodiment 1), embodiment 6 and embodiment 1, compare.
This experiment is consistent with other management in addition to processing difference except testing.
1.3 experimental design：The cherry orchard for selecting 10 mu of age of trees consistent, 2 is parallel, and 1 mu is 1 processing unit.For common fruit Garden (in addition to garlic and fish pelargonium is not interplanted, other production methods are consistent with embodiment 1), comparison 1 (prepare ecological seaweed liquid to give birth to During object stimulin, in addition to corynebacterium ammoniagenes are without thawing method processing directly use, other production methods are consistent with embodiment 1), Comparison 2 (be not added to when preparing lysozyme homogeneous outer in homogenizer, other production methods are consistent with embodiment 1), implementation Example 6 and embodiment 1 compare, and observe orchard insect pest situation, count orchard cherry yield.
2 results and analysis
Cherry average product (kg) Orchard insect pest situation Soluble solid (%)
Common orchard 1379.7 Insect pest is serious 16.8
Comparison 1 1424.5 Substantially without worm 16.9
Comparison 2 1432.5 Substantially without worm 17.0
Embodiment 6 1456.6 Substantially without worm 17.2
Embodiment 1 1572.8 Substantially without worm 17.4
It is compared by common orchard with the present invention, it can be seen that the present invention has apparent anthelminthic effect, is capable of providing cherry Yield；Being compared by embodiment 1 and comparison 1 can show that corynebacterium ammoniagenes through thawing method high treating effect, can improve cherry yield； Compared by embodiment 1 and 2 data of comparison it is found that the effect through homogeneous processing is better than without homogeneous processing when prepared by lysozyme； Being compared by embodiment 1 and embodiment 6 can show that the effect using new fresh seaweed is better than using the seaweed effect dried, and can improve Orchard yield.
" managing orchard according to organic planting requirement " of the present invention refers to according to GB/T19630《Organic products》Mark Quasi- management orchard.

Claims (10)

1. a kind of implantation methods in organic orchard, it is characterised in that follow the steps below：
By the plants interplanting with expelling parasite insecticidal effect in orchard, orchard is managed to get organic fruit according to organic planting requirement Garden；
The plant with expelling parasite insecticidal effect is included in garlic, lavender, fish pelargonium, common nepenthes, shichongcao and folium artemisiae argyi A kind of or arbitrary proportion is two or more；
Fertilizer used in organic orchard is two kinds of one kind or arbitrary proportion in organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer More than;
Ecological seaweed biostimulant is added in the organic fertilizer, organic and inorganic fertilizer and biological organic fertilizer;
The ecology seaweed biostimulant is prepared according to following steps：
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 detached, 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 being 70~150 DEG C in temperature, 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.
2. the implantation methods in organic orchard as described in claim 1, it is characterised in that：Pesticide used in organic orchard is stone sulphur Mixture, Bordeaux mixture, mineral oil, trichoderma harzianum, quinolinone suspending agent, ethyl alcohol, soda, China tree alkali, rotenone, Dalmatian chrysanthemum breast One kind or arbitrary proportion in oil and matrine is two or more.
3. the implantation methods in organic orchard as described in claim 1, it is characterised in that：The preparation of thawing method corynebacterium ammoniagenes according to Following steps carry out：
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 preparation 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 implantation methods in organic orchard as described in claim 1, 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, added in homogenizer after stirring evenly, at least homogeneous primary, homogeneous temperature It is 30~60 DEG C to spend, and 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) it adds 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、 It is one or more in Duolite C-20, SDB-3, macroporous absorbent resin AB-8, D101, D3520, X-5, NKA-II, It is one or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
5. the implantation methods in organic orchard as described in claim 1, it is characterised in that：Step 3）It is described separation include press filtration, One or more of filtering and centrifugation.
6. the implantation methods in organic orchard as described in claim 1, 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 being 120 DEG C in temperature.
7. the implantation methods in organic orchard as described in claim 1, 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 implantation methods in organic orchard as described in claim 1, 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 implantation methods in organic orchard as claimed in claim 3, 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。
10. the implantation methods in organic orchard as described in claim 1, it is characterised in that：Ecological seaweed biostimulant adds in Mode into fertilizer is：With the interior addition manner of fertilizer material mixing granulation and spray to the outer addition side of fertiliser granulates surface Formula.
CN201711482274.9A 2017-12-29 2017-12-29 A kind of implantation methods in organic orchard Withdrawn CN108157043A (en)

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CN103814742B (en) * 2014-03-10 2016-04-13 北京农学院 The method of organic orchard mixing intercropping plant
CN107384671A (en) * 2017-09-14 2017-11-24 吴毅 A kind of production method of organic wine
CN107382554A (en) * 2017-09-14 2017-11-24 杨丽丽 A kind of production method of Algae Ecology fertilizer
CN107418809A (en) * 2017-09-14 2017-12-01 吴毅 A kind of production method for not adding sulfur dioxide organic wine
CN107409928A (en) * 2017-09-14 2017-12-01 吴毅 A kind of implantation methods in organic orchard

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103814743A (en) * 2014-03-10 2014-05-28 北京农学院 Method for improving soil nutrition of organic orchard
CN103814742B (en) * 2014-03-10 2016-04-13 北京农学院 The method of organic orchard mixing intercropping plant
CN107384671A (en) * 2017-09-14 2017-11-24 吴毅 A kind of production method of organic wine
CN107382554A (en) * 2017-09-14 2017-11-24 杨丽丽 A kind of production method of Algae Ecology fertilizer
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Application publication date: 20180615