CN101406203A - Plant growth regulator and production thereof - Google Patents

Abstract

The invention relates to a plant growth regulator and a method for producing the same. The plant growth regulator is characterized by comprising raw materials according to the following weight ratio: 1 to 20 weight portions of copper sulfate, 1 to 50 weight portions of zinc sulfate, 1 to 30 weight portions of manganese sulfate, 1 to 40 weight portions of ferric sulfate, 1 to 25 weight portions of sodium molybdate, 0.1 to 1 weight portion of amino acid, 1 to 20 weight portions of nucleic acid protein, 0.001 to 0.002 weight portion of vitamin E, 2 to 6 weight portions of agricultural rare earth, 1 to 30 weight portions of silicic acid, 5 to 20 weight portions of sugar, and 1,000 to 2,000 weight portions of water, wherein each one hundred weight portions of the regulator contains 0.5 weight portion of potato polyphenol oxidase complex, 0.5 weight portion of rhodotorula glutinis L-phenylalanine ammonia-lyase complex, 0.5 weight portion of earthworm superoxide dismutase, 0.5 weight portion of seaweed bioactive substances, and 0.5 weight portion of anti-pest composite.

Description

Plant growth regulator and production method thereof

Technical field

The present invention relates to a plant growth regulators and production method thereof, the present invention relates to a kind of composition that promotes that plant increases.The present invention both can fixed nitrogen, phosphorus decomposing, potassium decomposing; Decomposition of cellulose and organic acid; Can decompose phenol, H again ₂S, nitroso; Secretion growth stimulant, antibiotic, multiple functional enzyme.It can satisfy plant nutrition, increases crop yield, improves quality, improves resistance, also can reduce the remaining toxin in the plant corpus simultaneously.

Technical background

In agricultural production, the main method that promotes the crops level of production to improve is to increase the needed fertilizer of plant growing, the application of single chemical fertilizer is played very big facilitation to modern agricultural development, but, use incorrect meeting to destroy soil structure, make edaphon very rare, organic matter decomposition is obstructed, nutritive element runs off, and has reduced the soil resource availability even has polluted well, river, lake because of loss.Especially partially nitrogen, and lack the cooperation of other elements, often make quality, resistance, the mouthfeel of crops all be subjected to bigger influence, nitrogen fertilizer application will be detrimental to health and environment partially.Crops nitrate enters in people's carcass carcinogenesis.Throughout the year a large amount of use chemical fertilizer easily causes hardening soil degree to improve constantly, cause grain, really, vegetable matter constantly descends, the shallow water resource is by pollution by chemical fertilizer, the content of organic matter in the soil is fewer and feweri, ecotope is damaged.In addition, agricultural products in China residue of pesticide problem is more serious.The producer uses in violation of rules and regulations high-toxic pesticide or excess to use the phenomenon ubiquity of agricultural chemicals on vegetable and fruit, causes that residue of pesticide exceed standard in many agricultural product.

There are more quality problems in the product of China's microbial manure production at present, and is low such as effective viable bacteria content, and assorted bacterium rate is higher, and the term of validity is short etc.Also have the formation of some products to show significantly unreasonable, for example in a kind of product, add tens kinds of microorganisms, this just exists a reproduction speed fast, slow, the problem in anti-adversity difference, contention nutrient, contention space, and how the bacterial classification that breeding amount is few works in soil.The bacterial classification difference, to making the effect difference of deposits yields, have to increase to have and subtract, the crop that has is effective, the crop that has is invalid substantially. the nutriment (comprising nitrogen, phosphorus, potassium and multiple mineral element) that bio-fertilizer can not directly provide crop to absorb, but make and assist crop absorption nutriment or produce the growth that somatotropin stimulates crop by the positive activity of microorganism in soil of a large amount of work.The chemical fertilizer or other additive that add underexercise in the bio-fertilizer that has make that nitrogen, phosphorus, the potassium content of chemical fertilizer in the fertilizer is too high to cause effective bacterium death, and lose the effect of bio-fertilizer.

Existing fertilizer all is that the mankind apply extraneous strength to plant, and has ignored oneself's immunity of plant itself, self-protection, and self-anti-poor environment, therefore the ability of oneself's supply nutrition, needs to propose a plant growth regulators.

Summary of the invention

The object of the present invention is to provide a plant growth regulators and production method thereof, elements such as the potato PPO compound that contains among the present invention, rhodotorula glutinis L-phenylalnine ammonialyase compound, earthworm superoxide dismutase, seaweed bio active substance, disease-resistant worm compound, phytase, copper, zinc, manganese, iron are the indispensable most important elements of plant health, and the sterilization of preventing and curing diseases of plant is played special effect; The present invention with plant this as this, from natural zooblast, plurality of active ingredients in the plant cell that plant is required extracts respectively, prepares as required according to different crops then, real high efficiency, the specific aim that realizes plant health care product, the present invention can fixed nitrogen, phosphorus decomposing, potassium decomposing; Decomposition of cellulose and organic acid; Decompose phenol, H ₂S, nitroso; Secretion growth stimulant, antibiotic, multiple functional enzyme.It can satisfy plant nutrition, increases crop yield, improves quality, improves resistance, also can reduce the remaining toxin in the plant corpus simultaneously, the remains of pesticide on the crops of can effectively degrading.

The objective of the invention is to be realized by following technical proposals: a plant growth regulators comprises the following weight proportion raw material:

Copper sulphate 1-20

Zinc sulphate 1-50

Manganese sulphate 1-30

Iron sulfate 1-40

Molybdic acid is received 1-25

Amino acid 0.1-1

Nucleic acid-protein 1-20

Vitamin E 0.001-0.002

Agricultural rare-earth 2-6

Silicic acid 1-30

Sugar 5-20

Water 1000-2000;

Contain the potato PPO compound of 0.5 weight portion, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 weight portion, the earthworm superoxide dismutase of 0.5 weight portion, the seaweed bio active substance of 0.5 weight portion, the disease-resistant worm compound of 0.5 weight portion in the described conditioning agent of per 100 weight portions.

Another object of the present invention is realized by following technical proposals: the production method of a plant growth regulators, comprise the following weight proportion raw material, copper sulphate 1-20, zinc sulphate 1-50, manganese sulphate 1-30, iron sulfate 1-40, molybdic acid are received 1-25, amino acid 0.1-1, nucleic acid-protein 1-20, vitamin E 0.001-0.002, agricultural rare-earth 2-6, silicic acid 1-30, sugared 5-20, water 1000-2000; Contain the potato PPO compound of 0.5 weight portion, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 weight portion, the earthworm superoxide dismutase of 0.5 weight portion, the seaweed bio active substance of 0.5 weight portion, the disease-resistant worm compound of 0.5 weight portion in the described conditioning agent of per 100 weight portions;

The extraction step of I, rhodotorula glutinis L-phenylalnine ammonialyase compound:

A, preparation medium comprise following weight percentages in the medium: sucrose 1%, corn steep liquor 3%, earthworm slurry 2%, sodium chloride (NaCL) 0.5%, L-tyrosine 0.1%, dipotassium hydrogen phosphate (K ₂HPO ₄) 0.1%, defoamer 0.01%, all the other are clean waters, regulate the pH value 5.0～5.5 of medium;

B, medium is carried out sterilization processing, under 30 ℃ of temperature, inoculate bacterial classification then, under 30 ℃ of temperature, cultivated 12-15 hour, under 40 ℃ of temperature, cultivated 5-7 hour, generate zymotic fluid;

C, zymotic fluid is carried out centrifugal treating, collect wet cell;

D, working concentration are that 0.9% physiological saline carries out carrying out washing treatment to wet cell, add 1 gram wet cell in every ml physiological saline, then physiological saline are carried out centrifugal treating, collect wet cell;

E, wet cell and buffer solution are mixed into cell suspending liquid, add 1 gram wet cell in per 4 milliliters of buffer solutions; In cell suspending liquid, add concentration then again and be 10% triton x-100 osmotic treated; The concentration of described buffer solution is 50mmol/L, pH value 8.8;

F, use processor for ultrasonic wave are handled described cell suspending liquid ultrasonic disruption, produce broken liquid;

G, to described broken liquid multilayer filtered through gauze, produce filtered fluid, filtered fluid is carried out centrifugal treating, get supernatant;

H, select molecular cut off 20,000 PS milipore filters to carry out ultrafiltration and concentration to supernatant to handle, obtain once concentration liquid;

The degree of saturation scale of I, the actual volume according to once concentration liquid, temperature and sulfate of ammoniac calculates and reaches 38% degree of saturation and should add sulfate of ammoniac consumption in the concentrate, prepares out solid sulphuric acid iron;

J, once concentration liquid is directly poured in the beaker, added solid sulphuric acid iron, then once concentration liquid is carried out refrigerated centrifuge and handle, obtain the secondary supernatant;

K, to secondary supernatant ultrafiltration and concentration, obtain secondary concentration liquid;

The degree of saturation scale of L, the actual volume according to secondary concentration liquid, temperature and sulfate of ammoniac calculates from the required sulfate of ammoniac consumption of 38%-75% degree of saturation, prepares out solid sulphuric acid iron;

M, secondary concentration liquid is directly poured in the beaker, added solid sulphuric acid iron, then secondary concentration liquid is carried out refrigerated centrifuge and handle, abandoning supernatant keeps sediment, obtains rhodotorula glutinis L-phenylalnine ammonialyase compound;

N, with the freeze-drying of rhodotorula glutinis L-phenylalnine ammonialyase compound, preserve down at-20 ℃ then;

II, potato PPO (PPO) compound extraction step:

A, with potato clean, section, carry out homogenized then; In the homogenized process, add the 0.03M phosphate buffer of 1 liter of parts by volume in the potato pulp of per 1 kilogram-mass part, mix, obtain potato pulp; The temperature of described 0.03M phosphate buffer is 1 ℃, pH value 5.5;

After B, the homogenized, use the multilayer gauze that potato pulp is filtered, then filtered fluid is carried out centrifugation and handle, obtain the polyphenol oxidase zyme extract;

C, in the polyphenol oxidase zyme extract, add solid ammonium sulfate and make it reach 40% degree of saturation, carry out centrifugation then and handle, obtain supernatant;

D, in supernatant, add solid ammonium sulfate and make it reach 70% degree of saturation, carry out centrifugation then and handle, collect thick enzyme sediment, obtain the potato PPO compound;

The extraction step of III, earthworm superoxide dismutase:

A, the earthworm that will live place light salt brine to clean, and the content of salt is no more than 0.3% in the light salt brine; The earthworm that will live is rubbed processing, and in the rubbing process, the lactose that adds about 0.5 vitamin C that restrains and 0.5 gram in per 1000 gram earthworms obtains earthworm juice as protectant;

B, earthworm juice is obtained earthworm freeze after freezing processing under-4 ℃～-1 ℃ the temperature;

C, earthworm frozen put into the phosphate buffer homogenized, the earthworm of per 1 kilogram-mass part is frozen the phosphate buffer of 3 liters of parts by volume of configuration, and the parameter of phosphate buffer is 0.05mol/L, pH value 7.8, and homogenized finishes, carry out centrifugation and handle, obtain one time supernatant;

D, supernatant is used the multilayer filtered through gauze, then filtered fluid is carried out centrifugation and handle, obtain the secondary supernatant;

E, will be in the above-mentioned clear liquid of secondary add the acetone mixed processing of 1.25 times of parts by volume, leave standstill processing after, the collecting precipitation thing;

F, described sediment joined in the phosphate buffer dissolve, obtain lysate; The phosphate buffer of 3 liters of parts by volume of sediment configuration of per 1 kilogram-mass part, the parameter of phosphate buffer is 0.05mol/L, pH value 7.8;

G, to the water-bath heating 20 minutes under 65 ℃ of conditions of described lysate, left standstill 10 minutes, water-bath is heated to 65 ℃ again, be cooled to 4 ℃ then rapidly, lysate is carried out centrifugation handle, obtain three times supernatant, contain the earthworm superoxide dismutase in this supernatant;

H, the earthworm superoxide dismutase is preserved under-4 ℃ of conditions;

IV, seaweed bio active substance extraction step:

A, get 1 kilogram of clean marine alga, add 1 kilogram of wash clean earthworm, add one piece of raw egg and carry out homogenized, obtain mixed serum;

B, by per 1 kilogram of mixed serum add sucrose 40 grams, dusty yeast 20 grams, ammonium sulfate 1.0 grams, 1000 gram water stir the back, be warmed up to 40 ℃, fermentation process obtains zymotic fluid;

C, the 3mol/L phosphate buffer that adds 5 times of volumes in described zymotic fluid mix, and are heated to 100 ℃ of insulations 20-48 hour, obtain mixed solution;

D, the mixed solution temperature is cooled to normal temperature, the pH value of adjusting mixed solution with ammoniacal liquor reaches between the 6-7 then, and acquisition seaweed bio active substance extract is preserved under-4 ℃ of conditions;

V, disease-resistant worm compound extraction step:

A, balsam pear, ginger, garlic, rape flower, ginkgo skin, tree-of-heaven, fish Teng, green peel of walnut, castor bean leaf, bunge pricklyash leaf, tarragon are cleaned up with clear water, dry then;

B, above-mentioned raw materials is prepared burden by weight, mix then to pulverize and form crushed material, wherein, 10 parts in balsam pear, 3 parts in ginger, 7 parts in garlic, 10 parts of rape flowers, 10 parts of ginkgo skins, 10 parts of tree-of-heavens, 10 parts of fish Tengs, 10 parts of green peel of walnut, 10 parts of castor bean leaves, 10 parts of bunge pricklyash leafs, 10 parts of tarragons;

C, with the water that crushed material adds 5 times of parts by weight, add 1 ‰ phosphoric acid again, under 100 ℃ of conditions boiling 0.5-1 hour, soak then and placed 5-8 hour, obtain disease-resistant worm compound soup;

D, enantiopathy worm compound soup use the multilayer filtered through gauze, filtering liquid medicine is carried out centrifugation handle, and collect supernatant;

E, the pH value of using ammoniacal liquor to adjust supernatant reach 6.5-7, obtain disease-resistant worm compound, and disease-resistant worm compound is preserved under-4 ℃ of conditions.

Compared with the prior art the present invention has following advantage:

1, elements such as the copper that contains among the present invention, zinc, manganese, iron, rare earth, vitamin, carbohydrate are the indispensable most important elements of plant health, and the sterilization of preventing and curing diseases of plant is played special effect.

2, the superoxide dismutase that contains among the present invention (SOD) has synergistic effect to the nutritive element of plant absorbing, can improve resistance against diseases and the defence capability of crops greatly.

3, the superoxide dismutase that contains among the present invention, phytase, the agricultural rare-earth remains of pesticide on the crops of can effectively degrading.

Embodiment

Embodiment one:

One plant growth regulators, it comprises the following weight proportion raw material, is measurement unit with the gram:

Copper sulphate (in copper) 1

Zinc sulphate (in zinc) 1

Manganese sulphate (in manganese) 1

Iron sulfate (in iron) 1

Molybdic acid is received (in molybdenum) 1

Amino acid 0.1

Nucleic acid-protein 1

Vitamin E 0.001

Agricultural rare-earth 2

Silicic acid (in silicon) 1

Sugar (industrial granulated sugar) 5

Water (clean water) 1000;

Contain the potato PPO compound of 0.5 gram, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 gram, the earthworm superoxide dismutase of 0.5 gram, the seaweed bio active substance of 0.5 gram, the disease-resistant worm compound of 0.5 gram in the described conditioning agent of per one hectogram.

The polyphenol oxidase multienzyme complex that uses in the present embodiment, phenylalnine ammonialyase compound, superoxide dismutase, seaweed bio active substance, disease-resistant worm compound extracting method can use embodiment four disclosed methods, are not described in detail at this.

White sugar among the present invention is hydrocarbon, can promote the absorption to each trace elements of plant.Elements such as the copper that the present invention contains, zinc, manganese, iron, rare earth, vitamin, carbohydrate are the indispensable most important elements of plant health, and the sterilization of preventing and curing diseases of plant is played special effect.All plants that contain Ye Lvjia are all worked, crops and flue-cured tobacco, fruit tree, tealeaves etc. all have good effect of increasing production widely to vegetables, paddy rice, corn etc., it is improved about output 20%-50%, the piece root crop is increased production about 50%-80%, and low temperature cold current, overcast and rainy low light photograph and arid heat wave etc. are had functions such as tangible resistance, defence.

In the present embodiment, employed polyphenol oxidase (PPO), phenylalnine ammonialyase phenylalaninammo-nialyase (PAL), peroxidase (PO) belong to the defensive ferment of plant; Employed superoxide dismutase (Superoxide Dismutase is called for short SOD) is to remove the important enzyme .SOD of oxygen radical and the activity of CAT in the organism, reduce MDA content and membrane permeability, improving chlorophyll mass fraction and root system vigor has and is significantly just imitating .SOD and can improve crop anti-adversity, is playing a very important role aspect disease, freeze injury, aging, arid and the storage endurance.PAL is the constant speed enzyme of shikimic acid metabolic pathway, is the key enzyme in the antibacterial material building-up processes such as phenol, protective plant protecting agent, lignin.The effect synthetic and heteroauxin of PO and ethene is relevant, and two kinds of hormones can influence the great change of metabolism, and influences the metabolism of phenol with PO.Itself has disease resistance phenol, and its oxidation quinones sterilizing power is stronger, can the oxidation ectodesm and cause the pathogen expansion to be obstructed, cause the irritated resistance reaction of plant; The phenol material can also form the lignin precursor, makes the host cell lignification and disease-resistant.Even PPO itself also has inhibitory action to plant disease.

Occupation mode of the present invention is to use spraying apparatus to be sprayed directly on the blade face of crops.

Embodiment two:

Present embodiment is the preferred version on embodiment one basis, and it comprises the following weight proportion raw material, is measurement unit with the gram:

Copper sulphate 10

Zinc sulphate 25

Manganese sulphate 15

Iron sulfate 20

Molybdic acid receives 12

Amino acid 0.5

Nucleic acid-protein 10

Vitamin E 0.0015

Agricultural rare-earth 4

Silicic acid 20

Sugar 12

Water 1500.

Embodiment three:

Present embodiment is the preferred version on embodiment one basis, and it comprises the following weight proportion raw material, is measurement unit with the gram:

Copper sulphate 20

Zinc sulphate 50

Manganese sulphate 30

Iron sulfate 40

Molybdic acid receives 25

Amino acid/11

Nucleic acid-protein 20

Vitamin E 0.002

Agricultural rare-earth 6

Silicic acid 30

Sugar 20

Water 2000.

Embodiment four:

The production method of one plant growth regulators, comprise the following weight proportion raw material, copper sulphate 1-20, zinc sulphate 1-50, manganese sulphate 1-30, iron sulfate 1-40, molybdic acid are received 1-25, amino acid 0.1-1, nucleic acid-protein 1-20, vitamin E 0.001-0.002, agricultural rare-earth 2-6, silicic acid 1-30, sugared 5-20, water 1000-2000; Contain the potato PPO compound of 0.5 weight portion, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 weight portion, the earthworm superoxide dismutase of 0.5 weight portion, the seaweed bio active substance of 0.5 weight portion, the disease-resistant worm compound of 0.5 weight portion in the described conditioning agent of per 100 weight portions;

The extraction step of I, rhodotorula glutinis L-phenylalnine ammonialyase compound:

A, preparation medium comprise following weight percentages in the medium: sucrose 1%, corn steep liquor 3%, earthworm slurry 2%, sodium chloride (NaCL) 0.5%, L-tyrosine 0.1%, dipotassium hydrogen phosphate (K ₂HPO ₄) 0.1%, defoamer 0.01%, all the other are clean waters, regulate the pH value 5.0～5.5 of medium;

B, medium is carried out sterilization processing, under 30 ℃ of temperature, inoculate bacterial classification then, under 30 ℃ of temperature, cultivated 15 hours, under 40 ℃ of temperature, cultivated 7 hours, generate zymotic fluid; Conventional equipment is used in sterilization;

C, zymotic fluid was carried out centrifugal treating 7 minutes, collect wet cell, the centrifugal separator rotating speed is 5000rpm;

D, working concentration are that 0.9% physiological saline carries out carrying out washing treatment to wet cell, add 1 gram wet cell in every ml physiological saline, then physiological saline are carried out centrifugal treating 7 minutes, collect wet cell, and the centrifugal separator rotating speed is 5000rpm;

E, wet cell and buffer solution are mixed into cell suspending liquid, add 1 gram wet cell in per 4 milliliters of buffer solutions; In cell suspending liquid, added concentration (quality volumetric concentration) then again and be 10% triton x-100 (TrionX-100) osmotic treated 7 hours; The concentration of described buffer solution is 50mmol/L, pH value 8.8; Described buffer solution is Pehanorm-hydrochloride buffer (Tris-HCL);

F, use processor for ultrasonic wave were handled 25 minutes described cell suspending liquid ultrasonic disruption under 850W power, produced broken liquid;

G, to described broken liquid with three layers of filtered through gauze, produce filtered fluid, filtered fluid was carried out centrifugal treating 15 minutes, get supernatant (containing rhodotorula glutinis L-phenylalnine ammonialyase compound in the supernatant), the centrifugal separator rotating speed is 5000rpm;

H, select molecular cut off 20,000 PS milipore filters to carry out ultrafiltration and concentration to supernatant to handle, obtain once concentration liquid;

The degree of saturation scale of I, the actual volume according to once concentration liquid, temperature and sulfate of ammoniac calculates and reaches 38% degree of saturation and should add sulfate of ammoniac consumption in the concentrate, prepares out solid sulphuric acid iron;

J, once concentration liquid is directly poured in the beaker, the limit is slowly stirred the limit and is slowly added the solid sulphuric acid iron that weighs up, and after treating all to add dissolving, slowly stirs 10 minutes again; Then once concentration liquid is carried out refrigerated centrifuge and handled 10 minutes, obtain the secondary supernatant, centrifugal rotational speed is 5000rpm, and temperature condition is-4 ℃;

K, to secondary supernatant ultrafiltration and concentration, obtain secondary concentration liquid;

The degree of saturation scale of L, the actual volume according to secondary concentration liquid, temperature and sulfate of ammoniac calculates from the required sulfate of ammoniac consumption of 38%-75% degree of saturation, prepares out solid sulphuric acid iron;

M, secondary concentration liquid is directly poured in the beaker, the limit is slowly stirred the limit and is slowly added the solid sulphuric acid iron that weighs up, and after treating all to add dissolving, slowly stirs 10 minutes again; Then secondary concentration liquid is carried out refrigerated centrifuge and handled 10 minutes, abandoning supernatant keeps sediment, obtains rhodotorula glutinis L-phenylalnine ammonialyase compound, and centrifugal rotational speed is 5000rpm, and the temperature condition of refrigerated centrifuge is-4 ℃;

N, with the freeze-drying of rhodotorula glutinis L-phenylalnine ammonialyase compound, preserve down at-20 ℃ then;

II, potato PPO (PPO) compound extraction step:

A, with potato clean, section, use the plant tissue refiner to carry out homogenized then; In the homogenized process, add the 0.03M phosphate buffer of 1 liter of parts by volume in the potato pulp of per 1 kilogram-mass part, mix, obtain potato pulp; The temperature of described 0.03M phosphate buffer is 1 ℃, pH value 5.5, comprises EDTA, 5% glycerine, 1% polyvinylpyrrolidone of 0.02M mercaptoethanol, 0.001M in the described 0.03M phosphate buffer;

After B, the homogenized, use 4 layers of gauze that potato pulp is filtered, then filtered fluid is carried out centrifugation and handle, obtain the polyphenol oxidase zyme extract; The centrifugal separator rotating speed is 6000rpm, centrifugal treating 10 minutes;

C, in the polyphenol oxidase zyme extract, add solid ammonium sulfate and make it reach 40% degree of saturation, stir while adding and reach abundant dissolving, carry out centrifugation then and handle, obtain supernatant; The centrifugal separator rotating speed is 6000rpm, centrifugal treating 20 minutes;

D, add solid ammonium sulfate make it reach 70% degree of saturation in supernatant, carry out centrifugation then and handle, the reject supernatant is collected thick enzyme sediment, obtains the potato PPO compound; The centrifugal separator rotating speed is 6000rpm, centrifugal treating 20 minutes;

The extraction step of III, earthworm superoxide dismutase:

A, the earthworm that will live place light salt brine to clean three times, and the content of salt is no more than 0.3% in the light salt brine; The earthworm that will live is rubbed processing, and in the rubbing process, the lactose that adds about 0.5 vitamin C that restrains and 0.5 gram in per 1000 gram earthworms obtains earthworm juice as protectant;

B, earthworm juice is obtained earthworm freeze after freezing processing under-4 ℃～-1 ℃ the temperature;

C, earthworm frozen put into the phosphate buffer homogenized, the earthworm of per 1 kilogram-mass part is frozen the phosphate buffer of 3 liters of parts by volume of configuration, the parameter of phosphate buffer is 0.05mol/L, pH value 7.8, continuous stirring 1～2 hour, homogenized finishes, and carries out centrifugation and handles, and obtains one time supernatant, the centrifugal separator rotating speed is 15000rpm, centrifugal treating 30 minutes;

D, supernatant is used 4 layers of filtered through gauze, then filtered fluid is carried out centrifugation and handle, obtain the secondary supernatant, the centrifugal separator rotating speed is 6000rpm, centrifugal treating 20 minutes;

E, with adding the acetone mixed processing of 1.25 times of parts by volume in the above-mentioned clear liquid of secondary, left standstill the collecting precipitation thing 1 hour;

F, described sediment joined in the phosphate buffer dissolve, obtain lysate; The phosphate buffer of 3 liters of parts by volume of sediment configuration of per 1 kilogram-mass part, the parameter of phosphate buffer is 0.05mol/L, pH value 7.8;

G, to the water-bath heating 20 minutes under 65 ℃ of conditions of described lysate, left standstill 10 minutes, water-bath is heated to 65 ℃ again, be cooled to 4 ℃ then rapidly, lysate is carried out centrifugation to be handled, obtain three times supernatant, contain earthworm superoxide dismutase (earthworm superoxide dismutase-peroxidase complex enzyme) in this supernatant; The centrifugal separator rotating speed is 6000rpm, centrifugal treating 20 minutes;

H, the earthworm superoxide dismutase is preserved under-4 ℃ of conditions;

IV, seaweed bio active substance extraction step:

A, get 1 kilogram of clean marine alga, add 1 kilogram of wash clean earthworm, add one piece of raw egg and carry out homogenized, obtain mixed serum;

B, by per 1 kilogram of mixed serum add sucrose 40 grams, dusty yeast 20 grams, ammonium sulfate 1.0 grams, 1000 gram water stir the back, be warmed up to 40 ℃, fermentation time 84 hours obtains zymotic fluid;

C, the 3mol/L phosphate buffer that adds 5 times of volumes in described zymotic fluid mix, and are heated to 100 ℃ of insulations 48 hours, obtain mixed solution;

D, the mixed solution temperature is cooled to normal temperature, the pH value of adjusting mixed solution with ammoniacal liquor reaches between the 6-7 then, and acquisition seaweed bio active substance extract is preserved under-4 ℃ of conditions;

V, disease-resistant worm compound extraction step:

A, balsam pear, ginger, garlic, rape flower, ginkgo skin, tree-of-heaven, fish Teng, green peel of walnut, castor bean leaf, bunge pricklyash leaf, tarragon are cleaned up with clear water, dry then;

B, above-mentioned raw materials is prepared burden by weight, mix then to pulverize and form crushed material, wherein, 10 parts in balsam pear, 3 parts in ginger, 7 parts in garlic, 10 parts of rape flowers, 10 parts of ginkgo skins, 10 parts of tree-of-heavens, 10 parts of fish Tengs, 10 parts of green peel of walnut, 10 parts of castor bean leaves, 10 parts of bunge pricklyash leafs, 10 parts of tarragons;

C, with the water that crushed material adds 5 times of parts by weight, add 1 ‰ phosphoric acid again, boiling is 1 hour under 100 ℃ of conditions, soak to place then 8 hours, obtains disease-resistant worm compound soup;

D, enantiopathy worm compound soup use 4 layers of filtered through gauze, filtering liquid medicine is carried out centrifugation handle, and collect supernatant; The centrifugal separator rotating speed is 6000rpm, centrifugal treating 10 minutes;

E, the pH value of using ammoniacal liquor to adjust supernatant reach 6.5-7, obtain disease-resistant worm compound, and disease-resistant worm compound is preserved under-4 ℃ of conditions.

In the present embodiment, above-mentioned raw materials is got all the ready, mixed according to common process and get final product.

Conditioning agent of the present invention detects through China Guangzhou Analysis ﹠. Test Center, and testing result is as follows:

The present invention experimentizes through Sichuan Academy of Agricultural Sciences's soil investigation, and the result is as follows:

One, test site

Paddy rice: the wise 6 society's gold-tinted all responsibilitys in the Lu Jia of Jianyang City town appoint with Pengzhou City Gexie Mountain town 7 Wang Zhi of society and contracted field in.

Corn: wise village, the Lu Jia of Jianyang City town 6 society's gold-tinted all responsibilitys appoint ground.

Lettuce: 2 groups of Wang Zhi of Pengzhou City Gexie Mountain town 7 societies and contracted field in.

Tealeaves: tea plantation, Qinglong Zhen Yu Tai village, Pengshan County.

Two, for examination soil

Paddy rice: reed young shoot of a reed wise 6 society's gold-tinted all responsibilitys in Jianyang City's are the paddy soil of purple soil parent material development with appointing, the 2 groups of Wang Zhi in 7 villages, Pengzhou City Gexie Mountain town and contracted field be the paddy soil of new alluviation parent material development.

Corn: wise village, the Lu Jia of Jianyang City town 6 society's gold-tinted all responsibilitys are the dryland soil of purple soil parent material development with appointing.

Lettuce: the 2 groups of Wang Zhi in 7 villages, Pengzhou City Gexie Mountain town and contracted field be the paddy soil of new alluviation parent material development.

Tealeaves: tea plantation, Qinglong Zhen Yu Tai village, Pengshan County is a hilly upland, and soil is yellow earth.

Above soil fertility is medium, irrigation and drainage good two season field or nonirrigated farmland, previous crops rape or wheat.

Three, experimental scheme:

1, cK 2, (mu is executed clear water 50kg) 3, the present invention's (mu is executed 100 times of liquid 50kg) 4, potassium dihydrogen phosphate (mu is executed 150 times of liquid 50kg)

Four, test is implemented:

(1), paddy rice

Test region, Jianyang City

District's group arrangement is at random adopted in test, triplicate, and sub-district area 0.027-0.003 mu, the aisle is 0.5 meter between repetition, and the aisle is 0.25 meter between processing, establishes guard row more than 1 meter all around.

1, experimental cultivar: suitable fragrant 3003.

2, the seedling raising and transplanting time, 200X grew seedlings March 25, and May 16 transplanted.

3, planting density: capable transplantings of width adopted in test, and wide capable 36cm, narrow row 18cm, nest be apart from 15cm, and then every sub-district plants the nest/district of 10 row/districts * 36 nests/OK=360, amounts to and mu plants 1.33 ten thousand nests.

4, Fertilization Level: 0.58 kilogram in urea is executed in every sub-district, 1.62 kilograms of superphosphate, 0.27 kilogram in potassium chloride.

5, field management: spray fertilizer once by test requirements document respectively at seedling stage April 16, spray fertilizer once tillering stage April 29, and shooting stage June 25 spray fertilizer once.And add phoxim control snout moth's larva of rice secondary with dimehypo respectively May 18 and July 18.

The test region, Pengzhou City

Small plot experiment is provided with 3 repetitions, and district's group is arranged at random.5.0 meters of zone leaders, wide 4.0 meters, 20 square metres of areas, between repetition, the aisle, minizone is 50cm, seal stays guard row in week.

1, kind: D excellent 63

2, the seedling raising and transplanting time: 200X sowed April 5, and May 12 transplanted.

3, planting density: transplant specification and be (10+6) very little * 4 cun, every nest is planted individual plant, every sub-district plants 600 strains, mu plants 2.0 ten thousand nests.

4, Fertilization Level: mu is made base fertilizer for 40 kilograms with 25% compound fertilizer dedicated to paddy, transplants back 7 days mu and topdresses once for 20 kilograms with the carbon ammonium.

5, field management: rice striking root phase, shooting stage, blooming stage mu are converted clear water with 200ml conditioning agent of the present invention and are sprayed for 40 kilograms, spray altogether three times.

After transplanting is turned green, the fine shallow irrigation that adopts, low temperature overcast and rainy day weather is with the Tian Weizhu that dries in the air, and the heading filling stage is based on intermittent irrigation.Seedling stage, booting stage, each prevented and treated rice blast once with Fujione.

(2), corn

Small plot experiment is provided with 3 repetitions, and district's group is arranged at random.6.7 meters * 3.0 meters=20.1 meters of every zone leaders ²=0.03 mu.Stay 50 centimetres in aisle between repetition, every sub-district kind 4 row, every row 25 strains, 100 strains of every district.Establish guard row all around.

1, corn variety: become single 26

2, seedling raising and transplanting: 200X grew seedlings March 22.April 2 transplanted.

3, planting density: adopt the cultivation of single file individual plant, line-spacing 2.25 chis, nest are apart from 0.80 chi, and mu is planted 3333 strains.

4, Fertilization Level: 50 kilograms in urea of mu, 50 kilograms in phosphate fertilizer, farmyard manure 30 is chosen.Phosphate fertilizer is made base fertilizer entirely, and the urea bottom application accounts for 30% (0.45 kilogram/district), carries seed manure and accounts for 20% (0.3 kilogram/district), attacks luxuriant fertilizer and accounts for 50% (0.75 kilogram/district).

5, field management: conditioning agent of the present invention is by test requirements document, April 16 seedling spraying for the first time, shooting stage May 2 sprays for the second time, typhon mouth phase May 15 sprays for the third time.In May 20 with phoxim baythion control of maize snout moth's larva once.

(3), lettuce

Small plot experiment is provided with 4 and handles 3 repetitions, and district's group is arranged at random.Be provided with 4 and handle 3 repetitions, district's group is arranged at random, and 6.67 meters of zone leaders are wide 3.0 meters, 20.0 square metres of areas.Every sub-district plants 180 strains, and specification is 1.0 chis * 1.0 chis, amounts to mu and plants 6000 strains.Between processing, handle between, repeat between the aisle be 50 centimetres, stay guard row all around, select the transplantation of seedlings of ad eundem lettuce.Gathering in the crops note separately by the sub-district produces.

1, kind: the blue or green silk of hanging

2, management: carry out according to the land for growing field crops high-yield culture technique.Conditioning agent of the present invention is converted clear water and is sprayed for 40 kilograms, transplants back 10 days, opening quotation phase, expanding stage and respectively sprays once.

(4), tealeaves

Test is provided with three processing, three repetitions, and the sub-district area is 8 * 1.3m.200X sprayed for the first time April 26, investigation on May 29.For the second time spray May 29 the same year, investigation on June 25.Because of this year arid is serious, can not pluck again later on, test there is certain influence.

Four, result of the test

(1), rice test result

Two rice test results find out: use conditioning agent of the present invention and have good effect.Jianyang City is than 39.5 kilograms of spray clear water volume increase, and volume increase 11.51% is increased production 21.0 kilograms than spraying potassium dihydrogen phosphate, and rate of growth is 5.81%.

The paddy rice result of Pengzhou City compares according to 47.8 kilograms of volume increase, volume increase 10.62%; Than 36.7 kilograms of spray clear water volume increase, rate of growth is 7.96%, and than spraying 14.5 kilograms of potassium dihydrogen phosphate volume increase, rate of growth is 3.00%.

The survey showed that: spray the percentage of earbearing tiller that helps improving paddy rice behind the conditioning agent of the present invention, increase grain number per spike and thousand kernel weight.

(2), corn result of the test

The corn result of the test of Jianyang City is found out: using conditioning agent of the present invention also has good effect of increasing production, and than 41.0 kilograms of spray clear water volume increase, rate of growth is 10.81%, and than 24.6 kilograms of spray potassium dihydrogen phosphate volume increase, rate of growth is 6.22%.

The survey showed that in test: spray the grain number per spike and the 100-grain weight that help increasing corn behind the conditioning agent of the present invention.

(3), lettuce result of the test

The lettuce result of the test of Pengzhou City is found out: using conditioning agent of the present invention also has good effect of increasing production, and comparison is according to 306 kilograms of mu volume increase, volume increase 14.62%; Than 268 kilograms of spray clear water mu volume increase, volume increase 12.58%, than 88 kilograms of spray potassium dihydrogen phosphate mu volume increase, rate of growth is 3.81%.

The survey showed that: spray the growth that helps lettuce behind the conditioning agent of the present invention, individual plant weight and commodity individual plant weight all increase to some extent.

(3), tealeaves result of the test

Pengshan County tealeaves result of the test is found out: use conditioning agent of the present invention and help tealeaves increase bud tip quantity and improve hundred bud weight, thereby improve output, increase production 6.38% than spray clear water mu, than spray nutrient volume increase 2.78%.

Conclusion

1, rice application conditioning agent of the present invention has good effect.The Pengzhou City comparison is increased production 38.1 kilograms/mu of 10.62%: two some average specific spray clear water volume increase according to 47.8 kilograms/mu of volume increase, volume increase 9.74%, and than 17.75 kilograms/mu of application of phosphoric acid potassium dihydrogen volume increase, the training productive rate is 4.41%.

2, corn is used conditioning agent of the present invention, and than 41.0 kilograms/mu of spray clear water volume increase, rate of growth is 10.81%, and than 24.6 kilograms/mu of application of phosphoric acid potassium dihydrogen volume increase, rate of growth is 6.22%.

3, lettuce is used conditioning agent of the present invention, and comparison increases production 14.62% according to 306 kilograms/mu of volume increase; Than 268 kilograms/mu of spray clear water volume increase, volume increase 12.58%, than 88 kilograms/mu of application of phosphoric acid potassium dihydrogen volume increase, rate of growth is 3.81%.

4, conditioning agent of the present invention helps improving the percentage of earbearing tiller of paddy rice, and it is heavy with grain to increase grain number per spike.

5, conditioning agent of the present invention helps the growth of lettuce and improves individual plant weight and commodity individual plant weight.

6, conditioning agent of the present invention helps the increase of tea bud tip quantity and improves hundred bud weight, thereby improves output.

The present invention experimentizes through Dayi County rural development office, and the result is as follows:

One, basic condition

(1) place and scale

1, test: with the ground comparative trial be distributed in out Jiang Zhenxiang osmanthus village 12 societies (2 groups), go out Jiang Zhen vegetable garden village 12 societies (2 groups), Mu Jiaying village, He Ming township 13 societies (2 groups), totally 6 groups, 3.2 mu of test area are wherein handled 1.83 mu.

2, demonstration: Maize Production use exemplary distribution former in the Shanxi, please, the crane ring, go out 4 small towns, river, 52 mu of areas.

(2) experimental cultivar and breeding time

1, test: the corn variety for examination is respectively " Great Wall 799 " 3 groups 1.6 mu, April 2～20 sowing, August 2～5 maturation, average 116 days of the time of infertility; " become single 21 " 1 group 0.3 mu, sowing on March 27, August 1 maturation, the 128 days time of infertility; " refined beautiful No. 10 " 1 group 1 mu, March 27 sowing, August 1 maturation, the 134 days time of infertility; " difficult to understand beautiful No. 13 " 1 group 0.3 mu, sowing on March 27, August 15 maturation, the 132 days time of infertility.

2, demonstration: kind has " refined beautiful No. 10 ", " river single 15 ", " No. 2 at prime ", " Great Wall 799 ", " Dongdan 60 " etc., the 25-4 month in March 30 sowing, 28-August 15 July maturation, average 130 days of the time of infertility.

(3) fertilising situation

Execute 12.5 kilograms of purity nitrogens with ground comparative trial mu is flat, 8 kilograms of phosphorus pentoxides, 6.5 kilograms of potassium oxides, demonstration ground fertilizing amount can be represented the Fertilization Level of whole county Maize Production fully with experimental field basic identical.

Two, materials and methods

(1) with the ground comparative trial

1, time of application and number of times: according to maize growth process and " paddy rice, wheat, corn are used " failing to give attention to " plant growth regulator testing program " design, respectively in corn seedling stage and pollination phase application in two dressings.The concrete time is: on May 14,15 seedling stage, the pollination phase is 10-23 day in June.

2, amount of application and concentration: seedling stage and pollination phase all use by 1: 100 times of liquid, and mu is converted 50 kilograms in water.3、

Be used to shake the formula atomizer spray.

(2) demonstration

Time of application, number of times, consumption, concentration and method etc. are with identical with the ground comparative trial.

Three, test results and analysis

(1) experimental yield result

Find out that from the output statistics contrast processing average yield per mu of using plant growth regulator of the present invention reaches 410.96 kilograms, comparison is according to 356.14 kilograms of increases by 54.82 of (CK), volume increase 15.39%.6 groups is group group volume increase with the ground comparative trials, the amplitude maximum reach 133.5 kilograms, volume increase 38.36%; Increase production minimum reach 30 kilograms, volume increase 11.54%.By the hypothesis test of paired data, t=4.889＞t0.01-4.032.Illustrate to handle to have real difference that processing has extremely significant effect of increasing production than CK with CK.

The demonstration effect of increasing production with fit like a glove with the ground comparative test result, do not repeat comparison (down with) at this.

(2) volume increase factor analysis

Find out that from three factor analyses that spike number, a number and the heavy corn yield of grain constitute handle the average mu of spike number that produces effect and reach 2946.8, maintain an equal level with CK: single grain number per spike reaches 422.8, increases by 327.43 grams than CK, increases by 39.07 than CK, increase by 10.18%; Thousand kernel weight reaches 327.43 grams, increases by 16.02 grams than CK, increases by 5.14%.From a grain number and an effect of heavily increasing production,, grain number per spike accounts for 65.89% of total units increased in production because of increasing production 36.78 kilograms (theoretical calculating, down together); Increase production 19.04 kilograms because of thousand kernel weight, account for 34.1% of total units increased in production.Analysis result explanation: it is the increase that comes from grain number per spike that nearly 2/3rds production-increasing function is arranged, 1st/3rd, because the raising of thousand kernel weight, and spike number does not have notable difference.

(3) increasing yield initial analysis

Because plant growth regulating of the present invention is the conditioning agent of a kind of medicine, fertile dual-purpose type, just corn growth has been produced active influence since seedling stage.At first be to use conditioning agent seedling stage, the nutriment in the middle of the conditioning agent that made Maize Absorption, well developed root system, the leaf look dark green, the suction fertilizer of crop, water absorbing capacity and photosynthesis ability, and disease-resistant (northern and southern leaf blight, banded sclerotial blight) ability strengthens greatly, guaranteed the normal differentiation of male and female flowering, and striving for high yield for big fringe of later stage lays the foundation.Secondly, be to use taking out the hero phase of weaving silk, help the pollination of normally blooming, promote pollen germination and pollen tube growth, prolong the pollen life-span, improve the fertilization ripening rate, realize increasing grain and increase production.According to the species test data analysis, why great disparity is very big for grain number per spike, and reason is not that tassel row number increases (handle with CK and be 14.1 row), but row grain digital display work increases (be treated to 29.98, increase by 2.9 than CK, increase by 10.71%).In addition, the pollination phase uses the sea and helps transfer and the result of photosynthetic product to seed, promotes full seed, and grain heavily increases.

Four, benefit analysis

(1) economic benefit

Calculate by actual production, handling than CK mu is flat increases by 54.82 kilograms, and by 1.4 yuan of calculating of market price per kilogram, mu is flat to increase income 76.75 yuan.Remove 15 yuan of conditioning agent costs (2 times, totally 1000 milliliters), the peasant uses and throws 1 15 yuan of workers, and the flat net income of mu can reach 46.75 yuan, goes into operation than reaching 1: 2.56.The economic benefit highly significant.

(2) social benefit

Grain security is the cardinal task of our country, and corn is the third-largest cereal crops in the county, whole county cereal crops, occupies critical role in the production.Test, the demonstration proof is used " failing to give attention to " plant growth substance and have significant effect of increasing production on Maize Production.By more than 50 mu of calculating of this year demonstration, can increase kilogram surplus the corn 2700, if on more than the 50000 mu of corn in the whole county, generally apply, can increase more than 250 ten thousand kilograms of total grain outputs newly.This is to guarantee that grain aggregate does not subtract, and realizes the effective way of grain security.

(3) ecological benefits

Plant growth regulator of the present invention is to adopt biochemical synthetic method and add natural drug (containing alkaloid compositions such as sterilization, desinsection) to develop.Analyze through relevant departments' quality inspection, nuisanceless material and toxin composition, noresidue, pollution-free, be ecological, environmental protective class fertilizer, the medicine dual-purpose type conditioning agent that meets national environmental standard fully and produce the non-polluted farm product requirement, its wide popularization and application, very favourable to environmental and ecological environment, have significant ecological benefits.

Five, discuss and build

(1) test, demonstration fully prove: plant growth regulator of the present invention is used on corn, has significant effect of increasing production, 30～133.5 kilograms of the flat volume increase of mu, amplification 11.54～38.36%.The main cause of volume increase is: effectively promoted nourishing and growing of corn seedling stage, guaranteed that the normal differentiation of bud and the hero of taking out of middle and later periods weave silk, helped improving the fertilization ripening rate, realized increasing the grain weightening finish and increase production.

(2) since this year the county influenced by natural calamities such as high temperature rarely seen in the history, dry season, regional area (mainly be hills and along the mountain one band) corn is disaster-stricken serious, output is lower, and situation about being reflected can not be represented the long-term level of production, also needs the coming year carry out test, demonstration in nearly step.

(3) except that the experiment and demonstration of on paddy rice, wheat, corn three generalized grain crops, having carried out plant growth regulator of the present invention and, suggestion also should be carried out test, demonstration on economic crops such as rape, vegetables, fruit tree, edible mushroom, traditional Chinese medicine, flowers, extensively explore application effect and application process on Different Crop, for large scale application provides scientific basis.

Claims (4)

1, a plant growth regulators is characterized in that: comprise the following weight proportion raw material:

Copper sulphate 1-20

Zinc sulphate 1-50

Manganese sulphate 1-30

Iron sulfate 1-40

Molybdic acid is received 1-25

Amino acid 0.1-1

Nucleic acid-protein 1-20

Vitamin E 0.001-0.002

Agricultural rare-earth 2-6

Silicic acid 1-30

Sugar 5-20

Water 1000-2000;

Contain the potato PPO compound of 0.5 weight portion, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 weight portion, the earthworm superoxide dismutase of 0.5 weight portion, the seaweed bio active substance of 0.5 weight portion, the disease-resistant worm compound of 0.5 weight portion in the described conditioning agent of per 100 weight portions.

2, plant growth regulator according to claim 1 is characterized in that: comprise the following weight proportion raw material:

Copper sulphate 10

Zinc sulphate 25

Manganese sulphate 15

Iron sulfate 20

Molybdic acid receives 12

Amino acid 0.5

Nucleic acid-protein 10

Vitamin E 0.0015

Agricultural rare-earth 4

Silicic acid 20

Sugar 12

Water 1500.

3, plant growth regulator according to claim 1 is characterized in that: comprise the following weight proportion raw material:

Copper sulphate 20

Zinc sulphate 50

Manganese sulphate 30

Iron sulfate 40

Molybdic acid receives 25

Amino acid/11

Nucleic acid-protein 20

Vitamin E 0.002

Agricultural rare-earth 6

Silicic acid 30

Sugar 20

Water 2000.

4, the production method of a plant growth regulators, it is characterized in that, comprise the following weight proportion raw material, copper sulphate 1-20, zinc sulphate 1-50, manganese sulphate 1-30, iron sulfate 1-40, molybdic acid are received 1-25, amino acid 0.1-1, nucleic acid-protein 1-20, vitamin E 0.001-0.002, agricultural rare-earth 2-6, silicic acid 1-30, sugared 5-20, water 1000-2000; Contain the potato PPO compound of 0.5 weight portion, the rhodotorula glutinis L-phenylalnine ammonialyase compound of 0.5 weight portion, the earthworm superoxide dismutase of 0.5 weight portion, the seaweed bio active substance of 0.5 weight portion, the disease-resistant worm compound of 0.5 weight portion in the described conditioning agent of per 100 weight portions;

The extraction step of I, rhodotorula glutinis L-phenylalnine ammonialyase compound:

A, preparation medium comprise following weight percentages in the medium: sucrose 1%, corn steep liquor 3%, earthworm slurry 2%, sodium chloride (NaCL) 0.5%, L-tyrosine 0.1%, dipotassium hydrogen phosphate (K ₂HPO ₄) 0.1%, defoamer 0.01%, all the other are clean waters, regulate the pH value 5.0～5.5 of medium;

B, medium is carried out sterilization processing, under 30 ℃ of temperature, inoculate bacterial classification then, under 30 ℃ of temperature, cultivated 12-15 hour, under 40 ℃ of temperature, cultivated 5-7 hour, generate zymotic fluid;

C, zymotic fluid is carried out centrifugal treating, collect wet cell;

D, working concentration are that 0.9% physiological saline carries out carrying out washing treatment to wet cell, add 1 gram wet cell in every ml physiological saline, then physiological saline are carried out centrifugal treating, collect wet cell;

E, wet cell and buffer solution are mixed into cell suspending liquid, add 1 gram wet cell in per 4 milliliters of buffer solutions; In cell suspending liquid, add concentration then again and be 10% triton x-100 osmotic treated; The concentration of described buffer solution is 50mmol/L, pH value 8.8;

F, use processor for ultrasonic wave are handled described cell suspending liquid ultrasonic disruption, produce broken liquid;

G, to described broken liquid multilayer filtered through gauze, produce filtered fluid, filtered fluid is carried out centrifugal treating, get supernatant;

H, select molecular cut off 20,000 PS milipore filters to carry out ultrafiltration and concentration to supernatant to handle, obtain once concentration liquid;

The degree of saturation scale of I, the actual volume according to once concentration liquid, temperature and sulfate of ammoniac calculates and reaches 38% degree of saturation and should add sulfate of ammoniac consumption in the concentrate, prepares out solid sulphuric acid iron;

J, once concentration liquid is directly poured in the beaker, added solid sulphuric acid iron, then once concentration liquid is carried out refrigerated centrifuge and handle, obtain the secondary supernatant;

K, to secondary supernatant ultrafiltration and concentration, obtain secondary concentration liquid;

The degree of saturation scale of L, the actual volume according to secondary concentration liquid, temperature and sulfate of ammoniac calculates from the required sulfate of ammoniac consumption of 38%-75% degree of saturation, prepares out solid sulphuric acid iron;

M, secondary concentration liquid is directly poured in the beaker, added solid sulphuric acid iron, then secondary concentration liquid is carried out refrigerated centrifuge and handle, abandoning supernatant keeps sediment, obtains rhodotorula glutinis L-phenylalnine ammonialyase compound;

N, with the freeze-drying of rhodotorula glutinis L-phenylalnine ammonialyase compound, preserve down at-20 ℃ then;

II, potato PPO (PPO) compound extraction step:

A, with potato clean, section, carry out homogenized then; In the homogenized process, add the 0.03M phosphate buffer of 1 liter of parts by volume in the potato pulp of per 1 kilogram-mass part, mix, obtain potato pulp; The temperature of described 0.03M phosphate buffer is 1 ℃, pH value 5.5;

After B, the homogenized, use the multilayer gauze that potato pulp is filtered, then filtered fluid is carried out centrifugation and handle, obtain the polyphenol oxidase zyme extract;

C, in the polyphenol oxidase zyme extract, add solid ammonium sulfate and make it reach 40% degree of saturation, carry out centrifugation then and handle, obtain supernatant;

D, in supernatant, add solid ammonium sulfate and make it reach 70% degree of saturation, carry out centrifugation then and handle, collect thick enzyme sediment, obtain the potato PPO compound;

The extraction step of III, earthworm superoxide dismutase:

A, the earthworm that will live place light salt brine to clean, and the content of salt is no more than 0.3% in the light salt brine; The earthworm that will live is rubbed processing, and in the rubbing process, the lactose that adds about 0.5 vitamin C that restrains and 0.5 gram in per 1000 gram earthworms obtains earthworm juice as protectant;

B, earthworm juice is obtained earthworm freeze after freezing processing under-4 ℃～-1 ℃ the temperature;

C, earthworm frozen put into the phosphate buffer homogenized, the earthworm of per 1 kilogram-mass part is frozen the phosphate buffer of 3 liters of parts by volume of configuration, and the parameter of phosphate buffer is 0.05mol/L, pH value 7.8, and homogenized finishes, carry out centrifugation and handle, obtain one time supernatant;

D, supernatant is used the multilayer filtered through gauze, then filtered fluid is carried out centrifugation and handle, obtain the secondary supernatant;

E, will be in the above-mentioned clear liquid of secondary add the acetone mixed processing of 1.25 times of parts by volume, leave standstill processing after, the collecting precipitation thing;

F, described sediment joined in the phosphate buffer dissolve, obtain lysate; The phosphate buffer of 3 liters of parts by volume of sediment configuration of per 1 kilogram-mass part, the parameter of phosphate buffer is 0.05mol/L, pH value 7.8;

G, to the water-bath heating 20 minutes under 65 ℃ of conditions of described lysate, left standstill 10 minutes, water-bath is heated to 65 ℃ again, be cooled to 4 ℃ then rapidly, lysate is carried out centrifugation handle, obtain three times supernatant, contain the earthworm superoxide dismutase in the supernatant;

H, the earthworm superoxide dismutase is preserved under-4 ℃ of conditions;

IV, seaweed bio active substance extraction step:

A, get 1 kilogram of clean marine alga, add 1 kilogram of wash clean earthworm, add one piece of raw egg and carry out homogenized, obtain mixed serum;

B, by per 1 kilogram of mixed serum add sucrose 40 grams, dusty yeast 20 grams, ammonium sulfate 1.0 grams, 1000 gram water stir the back, be warmed up to 40 ℃, fermentation process obtains zymotic fluid;

C, the 3mol/L phosphate buffer that adds 5 times of volumes in described zymotic fluid mix, and are heated to 100 ℃ of insulations 20-48 hour, obtain mixed solution;

D, the mixed solution temperature is cooled to normal temperature, the pH value of adjusting mixed solution with ammoniacal liquor reaches between the 6-7 then, and acquisition seaweed bio active substance extract is preserved under-4 ℃ of conditions;

V, disease-resistant worm compound extraction step:

A, balsam pear, ginger, garlic, rape flower, ginkgo skin, tree-of-heaven, fish Teng, green peel of walnut, castor bean leaf, bunge pricklyash leaf, tarragon are cleaned up with clear water, dry then;

B, above-mentioned raw materials is prepared burden by weight, mix then to pulverize and form crushed material, wherein, 10 parts in balsam pear, 3 parts in ginger, 7 parts in garlic, 10 parts of rape flowers, 10 parts of ginkgo skins, 10 parts of tree-of-heavens, 10 parts of fish Tengs, 10 parts of green peel of walnut, 10 parts of castor bean leaves, 10 parts of bunge pricklyash leafs, 10 parts of tarragons;

C, with the water that crushed material adds 5 times of parts by weight, add 1 ‰ phosphoric acid again, under 100 ℃ of conditions boiling 0.5-1 hour, soak then and placed 5-8 hour, obtain disease-resistant worm compound soup;

D, enantiopathy worm compound soup use the multilayer filtered through gauze, filtering liquid medicine is carried out centrifugation handle, and collect supernatant;

E, the pH value of using ammoniacal liquor to adjust supernatant reach 6.5-7, obtain disease-resistant worm compound, and disease-resistant worm compound is preserved under-4 ℃ of conditions.