CN105967761A

CN105967761A - Method for producing amino acid water-soluble fertilizer by utilizing Drawida Japonica Michelsen

Info

Publication number: CN105967761A
Application number: CN201610354443.XA
Authority: CN
Inventors: 陈可夫
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-05-26
Filing date: 2016-05-26
Publication date: 2016-09-28

Abstract

The invention relates to a method for producing an amino acid water-soluble fertilizer by utilizing Drawida Japonica Michelsen. The method comprises the following steps: A. hydrolyzing compound amino acid, namely, adding alkaline protease liquid into crude earthworm protein liquid left after lumbricin is extracted for carrying out enzymolysis, adding acid protease, and further hydrolyzing to prepare hydrolyzed compound amino acid; B. carrying out a chelation reaction, namely, adding ZnSO4.7H2O and Na2SeO3 into the hydrolyzed compound amino acid for carrying out the chelation reaction to obtain compound amino acid chelate; C. separating and purifying, namely, separating and purifying the compound amino acid chelate by using an organic solvent methanol or ethanol, and then drying to obtain the amino acid water-soluble fertilizer. The method has the advantages that the water-soluble fertilizer prepared by carrying out chelation on the compound amino acid and trace elements can be rapidly absorbed by plants, and has the characteristics of increasing yield, improving sporocarp quality, being stress-resistant, improving soil, increasing medicine utilization rate and the like.

Description

Utilize the method that Japan's Dula earthworm produces aminoacid Water soluble fertilizer

Technical field

The present invention relates to water-soluble fertilizer field.

Background technology

After Japan's Dula earthworm extracting lumbricin, the most substantial amounts of Lumbricus crude protein is not used, waste money Source.

Summary of the invention

After the purpose of the present invention is aiming at currently with Japan's Dula earthworm extracting lumbricin, the thick egg of the most substantial amounts of Lumbricus Failing in vain to utilize, cause the wasting of resources, the present invention is then the method utilizing Japan's Dula earthworm crude protein to produce aminoacid Water soluble fertilizer.

The present invention comprises the steps:

A. aminoacids complex is hydrolyzed: from Japan's Dula earthworm, extract lumbricin, the Lumbricus crude protein being left after extracting lumbricin Liquid, is warming up to 48-52 DEG C, adjusts pH8.12-8.14, adds alkaline protease liquid enzymolysis 4.5-6 by enzyme power 9500-11000U/g little Shi Hou, is heated up to the activity of 90-100 DEG C of enzyme denaturing, is cooled to 42-48 DEG C, adjusts pH3.3-3.5, adds by enzyme power 9000-10000U/g After entering acid protease continuation hydrolysis 4-5.5 hour, it is heated up to 90-100 DEG C of enzyme denaturing activity, is subsequently cooled to room temperature, regulation PH7.1-7.3, prepares hydrolysis aminoacids complex;

B. chelatropic reaction: add ZnSO in hydrolysis aminoacids complex₄.7H₂O and Na₂SeO₃, its weight is: hydrolysis Compounded amino acidic group acid: ZnSO₄.7H₂O=2-3:1, hydrolysis compounded amino acidic group acid: Na₂SeO₃=2-3:1;At 75-78 DEG C Under conditions of temperature and pH value 6.2-6.4, chelatropic reaction 1-1.2 hour, prepare chelates of composite amino acid；

The most isolated and purified: by chelates of composite amino acid organic solvent methanol or separation of ethanol purification, its weight is: Chelates of composite amino acid: organic solvent=5-6:1, is then dried and prepares aminoacid Water soluble fertilizer.

The method have the advantages that:

1. this project utilization " method extracting lumbricin from Japan's Dula earthworm " patent of invention technology (patent No.: 97111350.5), from the Japanese Dula earthworm (also known as leech-earthworm) being grown in area, Jingmen, " lumbricin (Fe-Zn is extracted Glycoprotein) ".Also having substantial amounts of Lumbricus crude protein after extracting Fe-Zn glycoprotein, these substantial amounts of Lumbricus crude protein are through double-enzyme hydrolysis Rear acquisition aminoacids complex, of many uses.

2. aminoacids complex mainly quickly can be absorbed by plant by this project with the trace element chelated Water soluble fertilizer that is fabricated to, There is volume increase, improve the feature such as Fruitbody, degeneration-resistant, improvement soil, raising utilization ratio of drug, big to solving in agricultural production Amount uses chemical fertilizer and pesticide causes the problems such as soil quality decline, environmental pollution and pesticide residues to play positive role.

3. Lumbricus deep processing and application, can drive 1,100 peasant households to become vermiculture specialist, and social benefit is obvious.

Detailed description of the invention

The present invention comprises the steps:

A. aminoacids complex is hydrolyzed: from Japan's Dula earthworm, carry extracting lumbricin, the thick egg of Lumbricus being left after extracting lumbricin White liquor, is warming up to 50 DEG C, adjusts pH8.13, after adding alkaline protease liquid enzymolysis 6 hours by enzyme power 10000U/g, and, heat 95 DEG C The activity of enzyme denaturing, is cooled to 46 DEG C, adjusts pH3.4, after adding acid protease liquid enzymolysis 5 hours by enzyme power 9500U/g, is heated up to 95 DEG C of enzyme denaturing activity, are subsequently cooled to room temperature, regulate pH7.2, prepare hydrolysis aminoacids complex;

B. chelatropic reaction: add ZnSO in hydrolysis aminoacids complex₄.7H₂O and Na₂SeO₃, its weight is: hydrolysis Compounded amino acidic group acid: ZnSO₄.7H₂O=2-3:1, hydrolysis compounded amino acidic group acid: Na₂SeO₃=2-3:1;76 DEG C of temperature Under conditions of degree and pH value 6.3, chelatropic reaction 1 hour, prepare chelates of composite amino acid；

Specific embodiment

Concrete technology flow process: Lumbricus → rinsing → homogenate → centrifugal → supernatant → extraction lumbricin → extracting solution → decompression Concentration → Lumbricus crude protein → alkaline protease → acid protease → aminoacids complex → add slaine → tune pH → heating a flat iron plate for making cakes Close → add organic solution precipitation → separation → dry → product.

1. Lumbricus rinsing: rinsing 1 hour with surfing type, will rinse well outside fresh Lumbricus epidermis, the food in gastrointestinal tract is through water logging After be drained transparent shape；

2. homogenate: joined by the Lumbricus rinsed in pH8.14 Tris-HCl buffer, refiner is homogenized；

3. extracting lumbricin (Fe-Zn glycoprotein): its method for extracting is shown in patent of invention (Patent No. 97111350.5).

4. hydrolysis aminoacids complex: the Lumbricus crude protein liquid being left after extracting, concentrates in colloid solution, by temperature liter To 50 DEG C, adjust pH8.3, after adding alkaline protease liquid enzymolysis 6 hours by enzyme power 10000U/g,.It is warmed to 95 DEG C of alkalescence enzymes that go out Activity, be cooled to 46 DEG C, adjust pH3.4, add acid protease liquid enzymolysis 5 hours by enzyme power 1000U/g, be warmed to 95 DEG C and go out After the activity of acidicenzym so that it is cooling.

Acid protease (30000u/g), alkaline protease (30000u/g) is purchased from that such to tie up letter (Chinese) biotechnology limited Company.

4. concentrating under reduced pressure: being cooled to 70 DEG C of insulations in temperature, be evaporated to 60%, aminoacids complex is 28.3g/ 100ml, adjusts pH7.2, prepares hydrolysis aminoacids complex;

5. chelatropic reaction: add ZnSO in hydrolysis aminoacids complex₄.7H₂O and Na₂SeO₃, its weight is: hydrolysis is multiple Close amino acidic group acid: ZnSO₄.7H₂O=2-3:1, hydrolysis compounded amino acidic group acid: Na₂SeO₃=2-3:1;At 76 DEG C of temperature and pH Under conditions of value 6.3, chelatropic reaction 1 hour, prepare chelates of composite amino acid；

Experimental technique and result

1. experiment material

A. the Japanese Dula earthworm of autotrophy, is commonly called as " leech-earthworm ".

B. exogenous protease: acid protease (30000U/g), neutral protease (30000U/g), alkaline protease (30000U/g) it is purchased from and such ties up letter (Chinese) Bioisystech Co., Ltd.

2 test methods

A. the preparation of Lumbricus serosity: be placed in by fresh Lumbricus in surfing type potcher after rinsed clean, the homogenate of high-speed homogenization machine is stand-by.

B. the exogenous protease hydrolysis to Lumbricus crude protein: the Lumbricus crude protein of quality such as take, by enzyme activity 10000U/g, Be separately added into acid protease, neutral protease and hydrolysis by novo 5 hours, do not add exogenous protease for comparison, the end Substrate concentration 1:2, pH7.0, temperature 50 C.Result of the test is shown in Table 1.

The effect that Lumbricus crude protein is hydrolyzed by the different protease of table 1

Result shows that adding three kinds of protease can improve hydrolysis rate.The amino-acid nitrogen content of hydrolyzed solution represents in hydrolyzed solution The quantity of actual amino state, content is the highest, and the value of hydrolyzed solution is the biggest.Degree of hydrolysis then represents that the albumen hydrolyzed accounts for The percentage ratio of raw material total protein quantity, in degree of hydrolysis the highest explanation raw material, Proteolytic enzyme is the most thorough, and the application of this hydrolysis process is dived Power is the biggest.Being not added with in the case of exogenous enzyme relies only on self enzyme hydrolysis, amino-acid nitrogen content only has 0.084%, protein degree 3.19%, enzymolysis efficiency is the lowest, does not substantially have using value.

The effect that Lumbricus crude protein is hydrolyzed by different temperatures

With the rising of temperature, degree of hydrolysis first rises, and is slowly declining.Enzyme thermostability is preferable, when 50 DEG C, and hydrolysis by novo It is basonuclin enzyme optimum temperature that degree is up to 18.85,50 DEG C；When 45 DEG C, acid protease hydrolysis degree is up to 58.04,45 DEG C it is pepsin optimum temperature.Result of the test is shown in Table 2.

The effect that Lumbricus crude protein is hydrolyzed by table 2 different temperatures

The effect that Lumbricus crude protein is hydrolyzed by enzymolysis time

Constantly raise along with enzymolysis time extends degree of hydrolysis, but different time reaction tendency is different.At 5 hours inner acidic protease Hydrolysis rate is the highest；In 6 hours, hydrolysis by novo speed is the highest.Result of the test is shown in Table 3.

The effect that Lumbricus crude protein is hydrolyzed by the different enzymolysis time of table 3

The rate of charge impact on chelation percent

Chelates of Amino Acids And Trace Elements can be formed chelate by a kind of or class of amino acid with metal ion, it is possible to hydrolyzed solution In some little peptide molecule formed chelate.Metal ion usually adds ZnSO₄.7H₂O and Na₂SeO₃, mainly cultivation of crops is produced Raw Zinc-enriched selenium-enriched effect.Zinc and selenium are all essential trace elements in animals and plants, participate in a series of metabolic process of body, strengthen immunity Power, can increase the quality of sporophore, has the strongest degeneration-resistant effect.

Molar ratio, amino acid ligand and the mol ratio i.e. rate of charge of metal ion, be the important of formation chelatropic reaction Factor.Generally, coordination ratio the least then chelating degree is the highest, less stable；Coordination ratio too big then chelating degree is stable Property is too high, it is difficult to absorbed by organism.

The different rate of charge impact on chelation percent of table 4

As shown in Table 4, above-mentioned several aminoacid rate of charge 2:1 with 3:1 chelating degree is more or less the same, the throwing of this experimental selection 2:1 Material ratio is the optimum charging ratio preparing Chelates of Amino Acids And Trace Elements, both can guarantee that and has obtained stable chelate, again can be abundant Utilize aminoacid.

Chelatropic reaction is affected relatively big, under the acid condition that pH value is relatively low, due to the shadow of Radiation grafting by acid-base value condition Ringing, aminoacid is difficult to dissociate, it is impossible to form stable Chelates of Amino Acids And Trace Elements with metallic ion coordination；And at pH value relatively Under the conditions of high alkalescence, metal ion then can hydrolyze, and chelate stability is poor.Test result indicate that pH value when 6.3, chela It is very fast that conjunction reaction is carried out, and chelation percent is maximum.

Reaction temperature and time: aminoacid is the endothermic reaction with the chelatropic reaction of metal ion, and reaction temperature is the highest to instead Should be the most favourable, if but reaction temperature is the highest, and the response time is too short will cause reacting thorough, and temperature is the highest is easily destroyed amino Acid and chelate thereof；If reaction temperature is the lowest, then chelatropic reaction is too slow, and yield is relatively low.Experiment shows, reaction temperature controls 76 About DEG C, the response time controls at about 1 hour, and it is very fast more complete that chelatropic reaction is carried out.

Chelate isolated and purified: due to molten in the organic solvent such as ethanol, methanol of Chelates of Amino Acids And Trace Elements Xie Du is minimum, and this chelate i.e. dissolves in the organic solvents such as ethanol, thus Chelates of Amino Acids And Trace Elements is just separated Purification, organic solvent can be recycled by recycling, will not increase the biggest cost.

The detection of chelate: Chelates of Amino Acids And Trace Elements is mainly and the amino on amino acid molecular and carboxyl chelating Exist, infrared spectrometer occurs in that the stretching vibration absworption peaks such as Se-N, Zn-N, Se-O, Zn-O respectively, specific according to these Absworption peak determines the biochemical stability of Chelates of Amino Acids And Trace Elements.Aminoacid is not less than 18g%, trace element Se, Zn content It is not less than 2mg%.

Using method: this aminoacid Water soluble fertilizer 50mg adds water 50 kilograms of drip irrigations 667m²；Also can use as foliage application.

Claims

1. utilize the method that Japan's Dula earthworm produces aminoacid Water soluble fertilizer, it is characterised in that comprise the steps:

A. aminoacids complex is hydrolyzed: from Japan's Dula earthworm, extract lumbricin, the thick egg of Lumbricus being left after extracting lumbricin White liquor, is warming up to 48-52 DEG C, adjusts pH8.12-8.14, adds alkaline protease liquid enzymolysis 5-7 by enzyme activity 9500-11000U/g After hour, it is heated up to the activity of 90-100 DEG C of enzyme denaturing, is cooled to 42-48 DEG C, adjust pH3.3-3.5, by enzyme activity 9000- After 10000U/g adds acid protease continuation hydrolysis 4-6 hour, it is heated up to 90-100 DEG C of enzyme denaturing activity, is often subsequently cooled to Temperature, regulates pH7.1-7.3, prepares hydrolysis aminoacids complex;

B. chelatropic reaction: add ZnSO in hydrolysis aminoacids complex₄.7H₂O and Na₂SeO₃, its weight is: hydrolysis is multiple Close amino acidic group acid: ZnSO₄.7H₂O=2-3:1, hydrolysis compounded amino acidic group acid: Na₂SeO₃=2-3:1;75-78 DEG C of temperature Under conditions of degree and pH value 6.2-6.4, chelatropic reaction 1-1.2 hour, prepare chelates of composite amino acid；