CN115404255A - Preparation method of amino-oligosaccharin, amino-oligosaccharin composition and preparation method thereof, and application of amino-oligosaccharin composition in biologically inducing crop yield increase - Google Patents
Preparation method of amino-oligosaccharin, amino-oligosaccharin composition and preparation method thereof, and application of amino-oligosaccharin composition in biologically inducing crop yield increase Download PDFInfo
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- CN115404255A CN115404255A CN202211149921.5A CN202211149921A CN115404255A CN 115404255 A CN115404255 A CN 115404255A CN 202211149921 A CN202211149921 A CN 202211149921A CN 115404255 A CN115404255 A CN 115404255A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/06—Coating or dressing seed
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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
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract
The invention belongs to the technical field of agriculture, and particularly relates to a preparation method of amino-oligosaccharin, an amino-oligosaccharin composition, and a preparation method and application thereof. The invention provides a preparation method: mixing water, a chitosan enzyme aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction; mixing the first zymolyte and the second batch of chitosan for a second enzymolysis reaction; mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; inactivating the chitosanase in the third zymolyte, and then carrying out solid-liquid separation to obtain an acetic acid solution of the amino-oligosaccharin. The acetic acid solution of the amino-oligosaccharin obtained by the preparation method provided by the invention is beneficial to the absorption of crops, and the application of the acetic acid solution to the crops through a biological induction technology is beneficial to exciting the crops to generate various adverse-resistant substances, improving the environmental resistance of the crops to the growth of adverse plants such as herbicide phytotoxicity, cold injury and waterlogging and has a positive effect on the yield increase of the crops.
Description
Technical Field
The invention belongs to the technical field of agriculture, and particularly relates to a preparation method of amino-oligosaccharin, an amino-oligosaccharin composition, a preparation method of the amino-oligosaccharin composition and application of the amino-oligosaccharin composition in biologically inducing crop yield increase.
Background
Amino-oligosaccharin (chitosan oligosaccharide) refers to an oligosaccharide in which D-glucosamine is linked by β -1,4 glycosidic bonds. The amino-oligosaccharin can change the soil microflora, promote the growth of beneficial microorganisms and inhibit some plant pathogenic bacteria; can induce the disease resistance of plants, and enable the plants to have immunity and killing effects on various fungi, bacteria and viruses, thereby increasing the yield of crops and fruits and vegetables.
Meanwhile, the amino-oligosaccharin has the characteristics of trace (PPM) level, high efficiency, low cost, no public nuisance and the like in application, is widely popularized and applied in China, and has important significance in sustainable development of agriculture in China.
At present, the commonly used preparation method of amino-oligosaccharin adopts acid to degrade chitosan, chitin and the like, or adopts enzyme to degrade chitosan, chitin and the like. However, both acid degradation of chitosan and chitin or enzymatic degradation of chitosan and chitin have the disadvantages of difficult control of reaction conditions, difficulty in control of the molecular weight of amino-oligosaccharin, high viscosity of reaction products, influence on the efficacy of amino-oligosaccharin during use, and difficulty in purification.
Disclosure of Invention
The invention aims to provide a preparation method of amino-oligosaccharin, an amino-oligosaccharin composition, a preparation method of the amino-oligosaccharin composition and application of the amino-oligosaccharin composition in biologically inducing crop yield increase.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a preparation method of amino-oligosaccharin, which comprises the following steps:
mixing water, a chitosanase aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction to obtain a first zymolyte; the viscosity of the first zymolyte is less than or equal to 30mpa · s;
mixing the first zymolyte and a second batch of chitosan for a second enzymolysis reaction to obtain a second zymolyte; the viscosity of the second zymolyte is less than or equal to 30mpa · s;
mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; the viscosity of the third zymolyte is less than or equal to 30mpa · s;
inactivating the chitosanase in the third zymolyte, and performing solid-liquid separation to obtain an amino-oligosaccharin acetic acid solution, wherein the mass percentage content of the amino-oligosaccharin acetic acid solution is 5-15%, and the molecular weight of the amino-oligosaccharin is 340-3238 Da.
Preferably, the mass percentage content of the chitosanase aqueous solution is 5-15%; the mass ratio of the water to the chitosanase aqueous solution to the acetic acid is 100; the mass ratio of the water to the first batch of chitosan is (500-1000) to (16-32).
Preferably, the mass ratio of the first batch of chitosan to the second batch of chitosan to the third batch of chitosan is 1.
Preferably, the temperature of the first enzymolysis reaction, the temperature of the second enzymolysis reaction and the temperature of the third enzymolysis reaction are independently 35-50 ℃, and the pH value is independently 4-6.
The invention provides an amino-oligosaccharin composition, which comprises an acetic acid solution of amino-oligosaccharin, water and a preservative; the acetic acid solution of the amino-oligosaccharin is the acetic acid solution of the amino-oligosaccharin prepared by the preparation method in the technical scheme; the mass concentration of the amino-oligosaccharin in the amino-oligosaccharin composition is 0.02 +/-0.002 kg/L.
Preferably, the mass ratio of the amino-oligosaccharin to the acetic acid to the preservative is 20 (6-7) to (0.5-1.5).
The invention provides a preparation method of the amino-oligosaccharin composition, which comprises the following steps: mixing the acetic acid solution of the amino-oligosaccharin prepared by the preparation method in the technical scheme, water and a preservative.
The invention provides an application of the amino-oligosaccharin composition in the technical scheme or the amino-oligosaccharin composition prepared by the preparation method in the technical scheme in promoting the yield increase of crops.
The invention provides a method for improving crop yield, which comprises the following steps:
diluting the amino-oligosaccharin composition with water to obtain a diluted medicinal liquid; the amino-oligosaccharin composition is the amino-oligosaccharin composition described in the technical scheme or the amino-oligosaccharin composition prepared by the preparation method described in the technical scheme;
dressing the diluted liquid medicine to obtain crop seeds subjected to seed dressing treatment;
after the crop seeds subjected to seed dressing treatment are sowed to obtain crop plants, the diluted liquid medicine is respectively sprayed on the crop plants in the three-leaf period, the flowering period and the grain swelling period of the crops.
Preferably, the dilution multiple is 10-200 times; the mass ratio of the diluted liquid medicine to the crop seeds in the seed dressing process is 1 (50-68); the ratio of the volume of the diluted liquid medicine to the planting area of the crops during spraying is 330mL.
The invention provides a preparation method of amino-oligosaccharin, which comprises the following steps: mixing water, a chitosanase aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction to obtain a first zymolyte; the viscosity of the first zymolyte is less than or equal to 30mpa · s; mixing the first zymolyte and a second batch of chitosan for a second enzymolysis reaction to obtain a second zymolyte; the viscosity of the second zymolyte is less than or equal to 30mpa · s; mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; the viscosity of the third zymolyte is less than or equal to 30mpa & s; inactivating the chitosanase in the third zymolyte, and performing solid-liquid separation to obtain an amino-oligosaccharin acetic acid solution, wherein the molecular weight of the amino-oligosaccharin is 340-3238 Da, and the mass percentage content of the amino-oligosaccharin acetic acid solution is 5-15%. According to the preparation method provided by the invention, the chitosan is divided into three batches to be subjected to enzymolysis reaction by adopting the chitosan enzyme, and the control on the enzymolysis reaction of the chitosan is effectively realized by controlling the viscosity of an enzymolysis product obtained by the three batches of enzymolysis reaction, so that the molecular weight of the amino-oligosaccharin in the prepared acetic acid solution of the amino-oligosaccharin is 340-3238 Da, the mass percentage content is 5-15%, the subsequent solid-liquid separation is simple and easy, and the yield of the amino-oligosaccharin is high (the average yield is more than or equal to 98.0%); the amino-oligosaccharin with the molecular weight of 340-3238 Da is beneficial to the absorption of crops, and is also beneficial to exciting the crops to generate various stress-resistant substances including superoxide dismutase (SOD), peroxidase (POD) and soluble sugar, so that the environmental resistance of the crops to the growth of unfavorable plants such as herbicide phytotoxicity, cold injury, waterlogging and the like is improved, and the active effect on the yield increase of the crops is achieved.
The invention provides an amino-oligosaccharin composition, which comprises an acetic acid solution of amino-oligosaccharin, water and a preservative; the acetic acid solution of the amino-oligosaccharin is the acetic acid solution of the amino-oligosaccharin prepared by the preparation method in the technical scheme; the mass concentration of the amino-oligosaccharin in the amino-oligosaccharin composition is 0.02 +/-0.002 kg/L. The amino-oligosaccharin in the amino-oligosaccharin composition provided by the invention is beneficial to the absorption of crops, is beneficial to exciting the crops to generate various stress-resistant substances, improves the environmental resistance of the crops to the growth of unfavorable plants such as herbicide phytotoxicity, cold injury and waterlogging, and has a positive effect on the yield increase of the crops. The results of the examples show that the soybean treated by the amino-oligosaccharin composition has the SOD content of 136.35U/g in the soybean which is not treated by the amino-oligosaccharin composition, the SOD content of the soybean treated by the amino-oligosaccharin composition can reach 148.37U/g to the maximum extent, and the SOD content increase rate is 8.82 percent; the content of POD in the soybeans which are not treated by the amino-oligosaccharin composition is 59.93U/g, the content of POD in the soybeans which are treated by the amino-oligosaccharin composition is up to 69.37U/g, and the increase rate of the content of POD is 15.75%; the content of soluble sugar in the soybeans which are not treated by the amino-oligosaccharin composition is 5.97 percent, the content of soluble sugar in the soybeans which are treated by the amino-oligosaccharin composition can reach 6.45 percent at most, and the increase rate of the content of soluble sugar is 8.04 percent; therefore, the amino-oligosaccharin composition provided by the invention can promote the yield increase of soybeans by 3.9-15%.
The invention provides a method for improving crop yield, which comprises the following steps: diluting the amino-oligosaccharin composition with water to obtain a diluted medicinal liquid; the amino-oligosaccharin composition is the amino-oligosaccharin composition described in the above technical scheme or the amino-oligosaccharin composition prepared by the preparation method described in the above technical scheme; dressing the diluted liquid medicine to obtain crop seeds subjected to seed dressing treatment; after the crop seeds subjected to seed dressing treatment are sowed to obtain crop plants, the diluted liquid medicine is respectively sprayed on the crop plants in three leaf-compound periods, flowering periods and kernel-swelling periods of the crops. The method provided by the invention adopts the amino-oligosaccharide composition or the amino-oligosaccharide composition prepared by the preparation method in the technical scheme to treat crop seeds and three compound leaf periods, flowering periods and drum grain periods during growth respectively, so that the method can effectively stimulate crops to generate various adverse environment resistant substances, and has a positive effect on increasing the crop yield by improving the environmental resistance of the crops to adverse plant growth such as phytotoxicity, cold injury, waterlogging and the like. The results of the embodiment show that the method provided by the invention can promote the yield increase of the Jianshan soybeans by 3.9-15%.
Detailed Description
The invention provides a preparation method of amino-oligosaccharin, which comprises the following steps:
mixing water, a chitosanase aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction to obtain a first zymolyte; the viscosity of the first zymolyte is less than or equal to 30mpa · s;
mixing the first zymolyte and a second batch of chitosan for a second enzymolysis reaction to obtain a second zymolyte; the viscosity of the second zymolyte is less than or equal to 30mpa · s;
mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; the viscosity of the third zymolyte is less than or equal to 30mpa · s;
inactivating the chitosanase in the third zymolyte, and performing solid-liquid separation to obtain an amino-oligosaccharin acetic acid solution, wherein the mass percentage content of the amino-oligosaccharin acetic acid solution is 5-15%, and the molecular weight of the amino-oligosaccharin is 340-3238 Da.
In the present invention, all the preparation starting materials/components are commercially available products well known to those skilled in the art unless otherwise specified.
Mixing water, a chitosanase aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction to obtain a first zymolyte; the viscosity of the first zymolyte is less than or equal to 30mpa · s.
In the present invention, the chitosan is preferably food grade.
In the present invention, the chitosanase aqueous solution preferably has a mass percentage of 5 to 15%, more preferably 5.5 to 13.5%.
In the present invention, the acetic acid is particularly preferably glacial acetic acid.
In the present invention, the acetic acid is preferably food grade acetic acid.
In the present invention, the acetic acid preferably provides an acidic environment for the enzymatic reaction.
In the present invention, the enzymatic reaction is preferably carried out in an enzymatic kettle.
In the specific embodiment of the invention, the volume of the enzymolysis kettlePreferably 1.5m 3 。
In the present invention, the mass ratio of the water, the aqueous chitosanase solution and the acetic acid is preferably 100.
In the present invention, the mass ratio of the water to the first batch of chitosan is preferably (500-1000) to (16-32), more preferably 1000.
In the present invention, the mixing and mixing order of the raw materials in the first enzymatic hydrolysis reaction is preferably: and adding the water into the enzymolysis reaction kettle, heating the water to the temperature of the enzymolysis reaction, and sequentially adding the first batch of chitosan, acetic acid and chitosan enzyme aqueous solution.
In the present invention, the temperature of the first enzymatic hydrolysis reaction is preferably 35 to 50 ℃, more preferably 38 to 45 ℃.
In the present invention, the pH of the first enzymatic hydrolysis reaction is preferably 4 to 6, and more preferably 4.5 to 5.5.
In the invention, the first enzymolysis reaction is preferably carried out under the condition of stirring, and the invention has no special requirement on the specific implementation process of the stirring.
After the first zymolyte is obtained, mixing the first zymolyte and a second batch of chitosan for a second enzymolysis reaction to obtain a second zymolyte; the viscosity of the second zymolyte is less than or equal to 30mpa · s.
In the present invention, the mass ratio of the first batch of chitosan, the second batch of chitosan and the third batch of chitosan is preferably 1.
In the present invention, the temperature of the second enzymatic hydrolysis reaction is preferably 35 to 50 ℃, more preferably 38 to 45 ℃.
In the present invention, the pH of the second enzymatic reaction is preferably 4 to 6, and more preferably 4.5 to 5.5.
In the invention, the second enzymolysis reaction is preferably carried out under the condition of stirring, and the invention has no special requirement on the specific implementation process of the stirring.
After the second zymolyte is obtained, mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; the viscosity of the third zymolyte is less than or equal to 30mpa · s.
In the present invention, the temperature of the third enzymatic hydrolysis reaction is preferably 35 to 50 ℃, and more preferably 38 to 45 ℃.
In the present invention, the pH of the third enzymatic reaction is preferably 4 to 6, and more preferably 4.5 to 5.5.
In the invention, the third enzymolysis reaction is preferably carried out under the condition of stirring, and the invention has no special requirements on the specific implementation process of the stirring.
After the third zymolyte is obtained, the chitosanase in the third zymolyte is inactivated and then subjected to solid-liquid separation to obtain an acetic acid solution of the amino-oligosaccharin, wherein the mass percentage content of the acetic acid solution of the amino-oligosaccharin is 5-15%, and the molecular weight of the amino-oligosaccharin is 340-3238 Da.
In the present invention, the specific implementation process of the chitosanase inactivation is preferably as follows: and heating the first zymolyte to the inactivation temperature, and then cooling to the room temperature. In the present invention, the inactivation temperature is specifically preferably 90 to 95 ℃, and more preferably 90 ℃. In the present invention, the biological rate of raising the temperature from the temperature of the third enzymatic reaction to the inactivation temperature is preferably 0.8 to 0.9 ℃/min.
In the present invention, the solid-liquid separation specifically preferably includes performing screen filtration and nanofiltration membrane filtration as follows. In the invention, the diameter of the screen hole of the filter screen used in the screen filtration is preferably 3-5 μm, and the invention has no special requirements on the specific implementation process of the screen filtration. The amino-oligosaccharin with the molecular weight of 340-3238 Da is obtained through excellent filtration by a nanofiltration membrane, and macromolecular substances obtained through the filtration by the nanofiltration membrane are preferably subjected to enzymolysis reaction continuously according to the technical scheme. The present invention preferably removes the inactivated chitosanase by the solid-liquid separation. In the invention, the liquid product is obtained by filtering with the screen, and preferably, the liquid product is filtered by a nanofiltration membrane to obtain the acetic acid solution of the amino-oligosaccharin. The invention preferably intercepts amino-oligosaccharin with the molecular weight of 340-3238 Da through nanofiltration.
In the invention, the macromolecular substance obtained after nanofiltration is preferably used as a raw material to continue enzymolysis reaction to obtain the amino-oligosaccharin. In the invention, the first kettle yield of the amino-oligosaccharin is 88.5 percent, and the second kettle yield of the enzymolysis reaction of the high molecular weight substances intercepted by nanofiltration is 97.5 percent; the yield of the 3 rd kettle and the subsequent kettles is 98.4 percent, and the average yield of the amino-oligosaccharin obtained by the preparation method provided by the invention is more than or equal to 98.0 percent.
In the invention, the content of the amino-oligosaccharin in acetic acid solution is 5-15% by mass, preferably 7.5-15% by mass.
In the present invention, the molecular weight of the amino-oligosaccharin is 340-3238 Da, preferably 340-2574 Da.
In the present invention, the chemical name of the amino-oligosaccharin is β - (1, 4) -2-amino-2-deoxy-D-glucan.
In the invention, the molecular structural formula of the amino-oligosaccharin obtained by the invention is shown as formula 1:
in the present invention, n in formula 1 is an integer of 0 to 18, preferably an integer of 0 to 14.
The invention adopts the chitosanase to carry out biological enzymolysis degradation on the chitosan, has mild reaction conditions, low energy consumption, less side reaction, no environmental pollution and easy industrialization.
The molecular weight of the amino-oligosaccharin in the acetic acid solution of the amino-oligosaccharin prepared by the preparation method provided by the invention is 340-3238 Da, more preferably 340-2574 Da, and the molecular weight of the amino-oligosaccharin is less than 2600Da, so that the amino-oligosaccharin can effectively stimulate plants to generate various stress-resistant substances, improves the environmental resistance of the plants to the growth of unfavorable plants such as cold damage, waterlogging damage and the like, and is suitable for being absorbed and utilized by the plants.
The invention provides an amino-oligosaccharin composition, which comprises an acetic acid solution of amino-oligosaccharin, water and a preservative; the acetic acid solution of the amino-oligosaccharin is the acetic acid solution of the amino-oligosaccharin prepared by the preparation method of the technical scheme; the mass concentration of the amino-oligosaccharin in the amino-oligosaccharin composition is 0.02 +/-0.002 kg/L.
In the present invention, the preservative is particularly preferably potassium sorbate.
In the present invention, the potassium sorbate is preferably food grade.
In the invention, the mass ratio of the amino-oligosaccharide, the acetic acid and the preservative is preferably 20 (6-7) to (0.5-1.5), and more preferably 20 (6.5-7) to (0.5-1.5).
The invention provides a preparation method of the amino-oligosaccharin composition, which comprises the following steps: mixing the acetic acid solution of the amino-oligosaccharin prepared by the preparation method in the technical scheme, water and a preservative.
In the invention, the amino-oligosaccharin prepared by the preparation method of the technical scheme is directly mixed with the water and the preservative without further treatment. In the present invention, the temperature of the mixing is preferably room temperature.
The invention provides an application of the amino-oligosaccharin composition in the technical scheme or the amino-oligosaccharin composition prepared by the preparation method in the technical scheme in promoting the yield increase of crops.
The invention provides a method for improving crop yield, which comprises the following steps:
diluting the amino-oligosaccharin composition with water to obtain a diluted medicinal liquid; the amino-oligosaccharin composition is the amino-oligosaccharin composition described in the above technical scheme or the amino-oligosaccharin composition prepared by the preparation method described in the above technical scheme;
dressing the diluted liquid medicine to obtain crop seeds subjected to seed dressing treatment;
after the crop seeds subjected to seed dressing treatment are sowed to obtain crop plants, the diluted liquid medicine is respectively sprayed on the crop plants in the three-leaf period, the flowering period and the grain swelling period of the crops.
The invention dilutes the amino-oligosaccharin composition with water to obtain diluted liquid medicine; the amino-oligosaccharin composition is the amino-oligosaccharin composition in the technical scheme or the amino-oligosaccharin composition prepared by the preparation method in the technical scheme.
In the present invention, the dilution ratio is preferably 10 to 200 times, and more preferably 10 or 200 times.
After the diluted liquid medicine is obtained, the diluted liquid medicine is used for dressing seeds to obtain the crop seeds treated by dressing the seeds.
In the present invention, the crop seed is particularly preferably a soybean seed.
In the present invention, in the case of the present invention, the soybeans are particularly preferably soybeans planted in Hill farm Hill science and technology gardens of nine-three administration offices in Heilongjiang province.
In the present invention, the variety of soybean is particularly preferably Longnam 306.
In the present invention, the mass ratio of the diluted chemical solution to the crop seeds in dressing is preferably 1 (50-68), more preferably 1 (52.5-65).
In the invention, after the diluted medicament is used for dressing seeds, the invention preferably further comprises the step of dressing the aired crop seeds by using a seed coating agent. In the invention, the seed dressing agent is preferably used for dressing seeds: and (3) dressing the seeds by using Liangdun and ammonium molybdate in sequence. In the invention, when the light shield is used for dressing seeds, the mass ratio of the volume of the light shield to the crop seeds is preferably 100mL to 50kg, and the invention has no special requirements on specific purchase sources of the light shield. In the present invention, when the ammonium molybdate is used for dressing seeds, the mass ratio of the ammonium molybdate to the crop seeds is preferably 0.01.
In the invention, after the seed coating agent is used for seed dressing, the invention preferably dries the wet crop seeds after the seed coating agent is used for seed dressing to obtain the crop seeds subjected to seed dressing treatment, and then seeds are sowed.
After the crop seeds treated by seed dressing are obtained, the invention seeds the crop seeds treated by seed dressing to obtain crop plants, and sprays the diluted liquid medicine on the crop plants respectively in three leaf-compound periods, flowering periods and kernel-swelling periods of the crops.
In the present invention, the seeding is particularly preferably 39 ten thousand seeds per hectare.
In the invention, the spraying is preferably carried out by adopting unmanned aerial vehicle.
In the invention, the unmanned aerial vehicle has the pesticide application capacity of 20L.
In the invention, the flying height of the unmanned aerial vehicle for pesticide application is 1.8m.
In the invention, the ratio of the volume of the diluted liquid medicine to the planting area of crops in the spraying process is 330mL.
The method for improving the crop yield provided by the invention comprises the steps of firstly diluting the amino-oligosaccharin composition with water, then dressing crop seeds with seeds, and performing primary induction; then inducing for the second time in three leaf-compound periods of crop plant production, wherein the three leaf-compound periods are the initial stages of independent photosynthesis of crops and are also the key stages of growth of leaves and roots; then, the third induction is carried out in the flowering period of crops, the flowering and pod bearing effects are directly related to the yield, and the amino-oligosaccharin composition provided by the invention can promote the flowering and pod bearing number, reduce the number of empty pods and increase the number of seeds of a single plant at the stage; finally, the fourth induction is carried out in the grain swelling period, the nutrient demand in the grain swelling period is extremely high, and simultaneously, the plant is at the stage of aging, the fourth induction is carried out in the grain swelling period by adopting the amino oligosaccharide composition, so that the functional period of the leaves can be at least prolonged, the regulation and control function can accelerate the absorption and conversion of nutrients, the timely supply of nutrients is ensured, the full grain is promoted, and the grain weight is increased.
The method for preparing the chitosan oligosaccharide by degrading chitosan with the chitosan enzyme has the advantages of reaction temperature of 35-50 ℃, pH value of 4.0-6.0, mild conditions, no harsh requirements on equipment, high yield rate (more than or equal to 98.0 percent) of the chitosan oligosaccharide, no environmental pollution and easy industrialization; the degradation process and the relative molecular mass distribution of degradation products are easy to control, the molecular weight is 340-3238 Da, preferably 340-2574 Da, and the amino-oligosaccharin can excite plants to generate various substances resisting the adverse environment within the molecular weight range, improve the environmental resistance of the plants to the growth of unfavorable plants such as cold damage, waterlogging damage and the like, and is suitable for being absorbed and utilized by the plants.
The invention provides application of amino-oligosaccharin composition in crop yield increase, which preferably gives crop induced resistance in the initial stage of crop growth by a biological induction method, and achieves crop yield increase of 3.9-10.63% by sequentially passing through the biological induction method in three-leaf compound period, flowering period and drum grain period of 'growth node'.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Adding 1000L of water into a 1500L enzymolysis kettle, heating to 40 ℃ under stirring, sequentially adding 30kg of a chitosanase aqueous solution with the mass percentage of 10%, 30kg of food grade glacial acetic acid, adding 32kg of food grade chitosan, maintaining the kettle temperature at 38-40 ℃, lasting for about 24-30 hours, and obtaining a first enzymolysis product when the viscosity of the materials in the kettle is less than or equal to 30mpa & s through enzymolysis; keeping the temperature at 38-40 ℃, adding 32kg of food-grade chitosan again while stirring, and carrying out enzymolysis until the viscosity of the materials in the kettle is less than or equal to 30mpa · s to obtain a second enzymolysis product; keeping the temperature at 38-40 ℃, stirring, finally adding 32kg of food-grade chitosan, and carrying out enzymolysis until the viscosity of the materials in the kettle is less than or equal to 30mpa · s to obtain a third enzymolysis product;
heating the third enzymolysis product to 90 ℃ within 1 hour, cooling to room temperature, inactivating the chitosanase, filtering by using a filter sieve with the diameter of 3-5 mu m to obtain a homogeneous chitosan oligosaccharide aqueous solution, filtering by using a nanofiltration membrane, and collecting an acetic acid solution of amino-oligosaccharide with the molecular weight of 340-3238 Da and the mass percentage content of 8.5%, wherein the yield of a first kettle is 88.5%, and the yield of a second kettle for carrying out enzymolysis reaction by recycling high molecular weight substances intercepted by nanofiltration is 97.5%; the yields in the 3 rd kettle and the subsequent kettles are 98.4%, and the average yield of the amino-oligosaccharins obtained by the preparation method provided by the embodiment is more than or equal to 98.0%.
Example 2
Conveying the acetic acid solution of the amino-oligosaccharin prepared in the example 1 to a preparation kettle, adding 3678L of water and 5.6kg of potassium sorbate to the preparation kettle at room temperature, and uniformly stirring to obtain 4800L of stable homogeneous liquid of the amino-oligosaccharin composition, wherein the weight of the homogeneous liquid is 1008.7 +/-0.2 g/L (20 ℃); wherein, each 1000L of the amino-oligosaccharin composition comprises 20kg of amino-oligosaccharin, 6.25kg of glacial acetic acid and 1.17kg of potassium sorbate;
example 3
The test conditions are as follows:
1. test site: hill farm Hill science and technology park of Jishan company of nine thirds of northern wasteland masses;
2. overview of the test site: the test site is located in the Hill farm science and technology park with 1.1 m large ridges. The elevation is 288.1 meters, the longitude is 125.17 degrees, the latitude is 48.51 degrees, the soil type is eluviated black calcium soil, the organic matter content is 40.4g/kg, the PH value is 5.9, the alkaline hydrolysis nitrogen is 198mg/kg, the available phosphorus is 37mg/kg, the available potassium is 274mg/kg, and the tilling depth is 35cm. In the fourth heat accumulation zone;
3. the test varieties are: cultivating Longnao 306;
4. planting management: sowing about 39 ten thousand seeds per mu of land; the unmanned aerial vehicle is adopted for pesticide application in a test field, the pesticide box is 20 kilograms, and the spraying area is 15 mu; sowing after self-dressing on the farm;
thirdly, the experimental process:
diluting the amino-oligosaccharide composition prepared in example 2 with water, diluting treatment 1 with water 200 times, and diluting treatment 2 with water 10 times to obtain diluted medicinal solution;
1. seed treatment and primary induction;
mixing the diluted liquid medicine obtained in the step 1 with the Longreclaimed 306 seeds according to a mass ratio of 1.
Mixing the diluted liquid medicine obtained in the step 2 with the Longreclaimed 306 seeds in a mass ratio of 1:50 (namely, mixing the diluted liquid medicine with the Longreclaimed 306 seeds by 1 jin), air-drying, mixing the seeds by using 100mL of Liangdun and 50kg of Longreclaimed 306 seeds by using 10g of ammonium molybdate, mixing the seeds by using 50kg of Longreclaimed 306 seeds by using ammonium molybdate, and air-drying.
And (3) airing and sowing the Longreclaimed 306 seeds mixed with the diluted liquid medicine obtained in the treatment 1 and the treatment 2. 70 kilograms of seeds are sowed in each hectare, the seeding rate per mu is 4.6 kilograms, and 39 thousands of seeds are sowed in each hectare.
2. Method and period for spraying foliage
And (3) merging the experimental fields of the Longjin 306 seeds after the seeds are mixed with the diluted liquid medicine obtained by the treatment 1 and the treatment 2 into 1 plot, wherein the sowing area is 4.95 mu.
The unmanned aerial vehicle pesticide spraying (the capacity is 20 liters) is adopted in the test, and the flying height is 1.8 meters;
the dosage per hectare is 330mL, and the dosage per mu is 22mL.
The spraying period is as follows:
three leaf-recovering stages: spraying corresponding Longnam 306 test plots with the diluted liquid medicine obtained by the treatment 1 and the treatment 2 for the first time in 21 days after 6 months;
and (3) flowering period: 7, 7 months and 7 days, respectively spraying corresponding Longkelen 306 experimental fields with the diluted liquid medicines obtained by the treatment 1 and the treatment 2 for the second time;
and (3) a granulation period: spraying corresponding Longnam 306 test plots with the diluted liquid medicine obtained by the treatment 1 and the treatment 2 for the third time in 23 days after 8 months;
analysis of results
1. Analysis of results in different growth periods
The dragon reclamation 306 was treated with the amino-oligosaccharide composition prepared in example 2 in place of water as a blank control group, and the results of the analysis on emergence and number of days of growth are shown in table 1.
TABLE 1 examination table for growth period
From table 1 it can be derived: the amino-oligosaccharin composition obtained by the preparation method provided by the invention can obviously help soybeans enter the next growth stage in advance and prolong the growth days when the dragon cultivating 306 is treated. From the seedling emergence condition, because the low temperature is continuously carried out in the last ten days of May, the low temperature has certain influence on the seedling emergence of the soybeans when the seedling emergence stage of the soybeans is met, and the seedling emergence of the soybeans is slow, irregular and even pink; the adverse resistance (cold resistance) of the amino-oligosaccharin composition provided by the invention can bring the seedling emergence ahead of time by 2-3 days; can reduce the number of the powder and improve the rate of emergence by about 11 percent (the effect of seedling protection can be considered to a certain degree).
In 7 and 8 months, the local rainfall is too large, rainstorm weather occurs, and the growth of the soybeans is retarded under the influence of waterlogging. The adverse resistance (waterlogging resistance) of the amino-oligosaccharide composition provided by the invention can reduce the influence of waterlogging and enable soybeans to recover to a normal physiological state more quickly.
In the pod-bearing and grain-swelling stage of the soybean, reproductive growth is dominant, and nutrient substances are transferred to grains, so that the grains are expanded; during the period from the initial grain stage to the mature stage, the early stress resistance effect of the amino-oligosaccharide composition provided by the invention prolongs the functional period of the leaves (prolongs 2 days), so that more nutrients are accumulated in the grains, and the improvement of the yield is directly facilitated.
The results of the analysis of the herbicide resistance in dragon reclamation 306, which was treated with the amino-oligosaccharide composition prepared in example 2 in place of water as a blank control group, are shown in table 2.
TABLE 2 ratio of foliar herbicide to soybean true leaf hazard
From the results of table 2, it can be concluded that: as can be seen from the data in Table 2, the amino-oligosaccharin composition provided by the invention has a remarkable effect on resisting the herbicide damage of postemergence stems and leaves in the period of 2-3 leaves of soybeans. The adverse resistance (herbicide hazard resistance) of the amino-oligosaccharin composition provided by the invention can reduce the incidence rate of soybean hazard by about 40-61%, and the affected area is obviously smaller. At the initial stage of soybean photosynthesis, true leaves damage has great influence on photosynthetic efficiency, so that growth and development are stagnated and delayed; the anti-stress effect of the amino-oligosaccharin composition provided by the invention can reduce the influence time of phytotoxicity on leaf damage and increase the effective time of photosynthesis, so that the accumulated amount of dry matters is increased and the growth and development of plants at the next stage are promoted.
Note: when the control leaves were damaged, assuming 10 days of recovery were required, only 2 days of treatments 1 and 2 were required for recovery, and the remaining 8 days were photosynthetic, and the leaves performed more work.
The dragon reclamation 306 was treated with the amino-oligosaccharin composition prepared in example 2 replaced with water as a blank control group, and the analysis of the external morphology results during the growth process is shown in table 3.
TABLE 3 external form questionnaire for different periods
From the results of table 3, it can be concluded that: from the data in table 3, it can be seen that the stress resistance effect of the seed dressing treatment in the seedling emergence stage and the herbicide phytotoxicity stage in the early stage lays a good foundation for the soybean growth, and the amino-oligosaccharin composition provided by the invention is continuously induced to the soybean by the foliar spraying for three times in the following stage, so that the stress resistance of the soybean is continuously enhanced; meanwhile, the growth of plant leaves and roots is promoted well in the growth stage, so that the photosynthesis of the overground part and the nutrient absorption capacity of the underground part are improved simultaneously, the soybean keeps good growth vigor, the leaves delay senescence, and the increased photosynthesis time provides nutrition guarantee for later pod formation and seed swelling.
Production test analysis of soybean using the composition prepared in example 2 in place of water as a control blank, longnam 306 was subjected to
TABLE 4 indoor examination result questionnaire table
As can be seen from the results of the seed test data in Table 4, the data directly related to yield: the three data of the hundred grain weight, the pod number and the grain number are obviously higher than those of a control group, and the results prove that the biological induction technology achieves the effects of promoting growth and increasing accumulation of nutrients by improving stress resistance in a continuous and stable induction process in the previous period, and the SOD content in the soybeans which are not treated by the amino-oligosaccharin composition is 136.35U/g, the SOD content in the soybeans which are treated by the amino-oligosaccharin composition in the treatment 1 and the treatment 2 is 148.37U/g and the SOD content increase rate is 8.82 percent when the soybeans are detected in the branching period of the soybeans; the content of POD in the soybeans which are not treated by the amino-oligosaccharin composition is 59.93U/g, the content of POD in the soybeans which are treated by the amino-oligosaccharin composition is 69.37U/g, and the increase rate of the content of POD is 15.75 percent; the content of soluble sugar in the soybeans which are not treated by the amino-oligosaccharin composition is 5.97 percent, the content of soluble sugar in the soybeans which are treated by the amino-oligosaccharin composition is 6.45 percent, and the increase rate of the content of soluble sugar is 8.04 percent; the nutrient requirement of later reproductive growth is ensured, and the effect of increasing the yield is finally achieved.
Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and all of the embodiments belong to the protection scope of the present invention.
Claims (10)
1. A preparation method of amino-oligosaccharin is characterized by comprising the following steps:
mixing water, a chitosanase aqueous solution, acetic acid and a first batch of chitosan to perform a first enzymolysis reaction to obtain a first zymolyte; the viscosity of the first zymolyte is less than or equal to 30mpa · s;
mixing the first zymolyte and a second batch of chitosan for a second enzymolysis reaction to obtain a second zymolyte; the viscosity of the second zymolyte is less than or equal to 30mpa · s;
mixing the second zymolyte and a third batch of chitosan for a third enzymolysis reaction to obtain a third zymolyte; the viscosity of the third zymolyte is less than or equal to 30mpa · s;
inactivating the chitosanase in the third zymolyte, and performing solid-liquid separation to obtain an amino-oligosaccharin acetic acid solution, wherein the mass percentage content of the amino-oligosaccharin acetic acid solution is 5-15%, and the molecular weight of the amino-oligosaccharin is 340-3238 Da.
2. The preparation method according to claim 1, characterized in that the mass percentage content of the chitosanase aqueous solution is 5-15%; the mass ratio of the water to the chitosanase aqueous solution to the acetic acid is 100; the mass ratio of the water to the first batch of chitosan is (500-1000) to (16-32).
3. The preparation method according to claim 2, wherein the mass ratio of the first batch of chitosan to the second batch of chitosan to the third batch of chitosan is 1.
4. The method according to claim 1 or 2, wherein the first, second and third enzymatic reactions independently have a temperature of 35 to 50 ℃ and a pH of 4 to 6.
5. An amino-oligosaccharin composition comprising an acetic acid solution of amino-oligosaccharin, water and a preservative; the acetic acid solution of the amino-oligosaccharin is the acetic acid solution of the amino-oligosaccharin prepared by the preparation method of any one of claims 1 to 4; the mass concentration of the amino-oligosaccharin in the amino-oligosaccharin composition is 0.02 +/-0.002 kg/L.
6. The amino-oligosaccharin composition according to claim 5, wherein the mass ratio of amino-oligosaccharin, acetic acid and preservative is 20 (6-7) to (0.5-1.5).
7. The method of producing the amino-oligosaccharide composition of claim 5 or 6, comprising the steps of: mixing the solution of amino-oligosaccharin in acetic acid prepared by the process according to any one of claims 1 to 4, water and a preservative.
8. Use of the amino-oligosaccharin composition of claim 5 or 6 or the amino-oligosaccharin composition prepared by the method of claim 7 for increasing the yield of crops.
9. A method of increasing crop yield, comprising the steps of:
diluting the amino-oligosaccharin composition with water to obtain a diluted medicinal liquid; the amino-oligosaccharin composition is the amino-oligosaccharin composition according to claim 5 or 6 or the amino-oligosaccharin composition prepared by the preparation method according to claim 7;
dressing the diluted liquid medicine to obtain crop seeds subjected to seed dressing treatment;
after the crop seeds subjected to seed dressing treatment are sowed to obtain crop plants, the diluted liquid medicine is respectively sprayed on the crop plants in three leaf-compound periods, flowering periods and kernel-swelling periods of the crops.
10. The method of claim 9, wherein the dilution factor is 10 to 200 times; the mass ratio of the diluted liquid medicine to the crop seeds in the seed dressing process is 1 (50-68); the ratio of the volume of the diluted liquid medicine to the planting area of the crops during spraying is 330mL.
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