CN116813816A - A kind of preparation method of compound pullulan - Google Patents

Abstract

The invention discloses a preparation method of compound pullulan, and relates to the technical field of preparation methods of compound pullulan. The preparation method aims to solve the problem of using biological enzymatic hydrolysis for preparation under the existing technology, and its cost is relatively low. High and unable to improve crop stress resistance, the preparation method has the following steps: Step 1: Prepare a 1% acetic acid solution; Step 2: Dissolve pullulan in the acetic acid solution of Step 1, and stir thoroughly uniformly to obtain mixed solution one; step three: add hydrogen peroxide into mixed solution one and stir thoroughly to obtain mixed solution two; step four: set the internal temperature and rotation speed of the reaction kettle, add mixed solution two into the reaction kettle, and add Acid, when the reaction reaches 1 hour, take out 1/10 of the reaction solution. This preparation method has a simple process and has the advantage of low cost compared with biological enzymatic hydrolysis. It has realized large-scale agricultural production and application. The prepared composite Aureobasidium pullulans polymer sugar and improve crop stress resistance.

Description

A kind of preparation method of compound pullulan

Technical field

The invention belongs to the technical field of preparation methods of compound pullulan, and specifically relates to a preparation method of compound pullulan.

Background technique

Pullulan is an extracellular water-soluble mucopolysaccharide fermented from corn, also known as pullulan. In agricultural production, polysaccharide spray can be used as a thickener and preservative. Soil can improve the soil aggregate structure. In the face of various stress conditions, oligosaccharides can stimulate the plant's own immunity, enable the plant to acquire systemic resistance, improve the plant's disease resistance, insect resistance, and stress resistance, and achieve the purpose of preventing pests and diseases. Purpose.

Currently, the invention patent with patent number CN202011443169.6 discloses a method for preparing biotinylated pullulase polysaccharide derivatives, which includes the following steps: (1) Add anhydrous dimethyl sulfoxide once in an Erlenmeyer flask , biotin, N,N'-dicyclohexylcarboimide and 4-dimethylaminopyridine, react at room temperature for 0.5-1h, then add pullulase polysaccharide, continue to react at room temperature for 1-2h, Until the white turbid matter precipitates, a turbid solution is obtained; (2) Filter the turbid solution obtained in step 1, homogenize once to obtain solution 1, let it stand for 3-5 hours, remove the surface impurities and the upper layer solution, and obtain solution 1; (3) ) Filter out the white turbidity in solution 1, collect the filtrate and put it into a dialysis bag with a molecular weight cutoff of 9000Da. Add deionized water to dialyze for 4-8 days. Replace the deionized water every 1 hour. After the dialysis is completed, suction filtrate to obtain white Precipitate, wash the white precipitate with dichloromethane and deionized water in sequence, and dry to obtain a biotinylated pullulan polysaccharide derivative, which can adapt to high-temperature thermal processing processes, but the pullulan is processed by biological enzymatic hydrolysis The preparation cost is high and the preparation process is complicated. Pullulan has no obvious effect on promoting the growth of crops and cannot improve the stress resistance of crops.

Therefore, the above-mentioned problems of high cost and inability to improve crop stress resistance need to be solved urgently to improve the use scenarios of compound pullulan.

Contents of the invention

(1) Technical problems to be solved

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a preparation method of compound pullulan. This preparation method is intended to solve the problem of using biological enzymatic hydrolysis for preparation under the existing technology, which has a high cost and cannot be improved. Technical issues in crop stress resistance.

(2)Technical solutions

In order to solve the above technical problems, the present invention provides such a preparation method of composite pullulan, the steps of which are as follows:

Step 1: Prepare 1% acetic acid solution;

Step 2: Dissolve pullulan in the acetic acid solution of step 1, and stir thoroughly to obtain mixed solution 1;

Step 3: Add hydrogen peroxide into mixed solution one and stir thoroughly to obtain mixed solution two;

Step 4: Set the internal temperature and rotation speed of the reaction kettle, add mixed solution 2 into the reaction kettle, and add acid. When the reaction reaches 1 hour, take out 1/10 of the reaction solution;

Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution;

Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed and time, and mix it thoroughly to obtain mixed solution three;

Step 7: Use pH test paper or a pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value is met. Stop adding potassium hydroxide to obtain mixed solution four;

Step 8: The ultrafiltration equipment concentrates the mixed solution IV, using the pressure difference on both sides of the membrane as the driving force, and the ultrafiltration membrane as the filter medium. Under a certain pressure, when the mixed solution IV flows through the membrane surface, the ultrafiltration The many tiny pores densely covered on the membrane surface only allow water and small molecular substances to pass through and become the permeate, while the substances in the mixed solution 4 that are larger than the pore size on the membrane surface are trapped on the inlet side of the membrane and become concentrated liquid;

Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. Set the spray pressure and the concentrated liquid is atomized to form small droplets. Under the influence of surface tension and hot nitrogen, Under the dual action, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder.

Further, the specific steps for preparing a 1% acetic acid solution are: prepare acetic acid and water in a ratio of 1:100, and then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution.

Further, the weight ratio of the pullulan to the acetic acid solution is 1:0.5-20.

Further, the weight ratio of the mixed solution 1 and hydrogen peroxide is 1:10.

Further, the acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid, and the weight ratio of pullulan and acid is 1-4:10-100.

Further, the set internal temperature of the reaction kettle is 50-99°C, and the rotation speed is 400 r/min.

Further, the set stirring speed is 30-200r/min and the time is 5-10 minutes.

Further, the pH value requirement is that the pH value is equal to 7.0.

Further, the operating pressure of the ultrafiltration equipment is 0.11-0.6Mpa, and the membrane flux of the ultrafiltration membrane is 1-500L/㎡h.

Further, the spray pressure is 0.65-1.2MPa.

(3) Beneficial effects

Compared with the existing technology, the beneficial effects of the present invention are: the preparation method of the present invention has a simple process, has the advantage of low cost compared with biological enzymatic hydrolysis, and the production process is safe, no harmful gas is generated, and does not cause any harm to the environment. Impact, the process is simple, and large-scale agricultural production applications have been realized. The prepared compound pullulan can gradually degrade into oligosaccharides under the action of soil microorganisms, thereby prolonging and promoting crop growth and improving crop stress resistance. .

Detailed ways

This specific embodiment is a preparation method of composite pullulan, and the steps are as follows:

Step 1: Prepare 1% acetic acid solution;

Step 2: Dissolve pullulan in the acetic acid solution of step 1, and stir thoroughly to obtain mixed solution 1;

Step 3: Add hydrogen peroxide into mixed solution one and stir thoroughly to obtain mixed solution two;

Step 4: Set the internal temperature and rotation speed of the reaction kettle, add mixed solution 2 into the reaction kettle, and add acid. When the reaction reaches 1 hour, take out 1/10 of the reaction solution;

Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution;

Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed and time, and mix it thoroughly to obtain mixed solution three;

Step 7: Use pH test paper or a pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value is met. Stop adding potassium hydroxide to obtain mixed solution four;

Step 8: The ultrafiltration equipment concentrates the mixed solution IV, using the pressure difference on both sides of the membrane as the driving force, and the ultrafiltration membrane as the filter medium. Under a certain pressure, when the mixed solution IV flows through the membrane surface, the ultrafiltration The many tiny pores densely covered on the membrane surface only allow water and small molecular substances to pass through and become the permeate, while the substances in the mixed solution 4 that are larger than the pore size on the membrane surface are trapped on the inlet side of the membrane and become concentrated liquid;

Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. Set the spray pressure and the concentrated liquid is atomized to form small droplets. Under the influence of surface tension and hot nitrogen, Under the dual action, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder.

Further, the specific steps for preparing a 1% acetic acid solution are: prepare acetic acid and water in a ratio of 1:100, and then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution.

Further, the weight ratio of the pullulan to the acetic acid solution is 1:0.5-20.

Further, the weight ratio of the mixed solution 1 and hydrogen peroxide is 1:10.

Further, the acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid, and the weight ratio of pullulan and acid is 1-4:10-100.

Further, the set internal temperature of the reaction kettle is 50-99°C, and the rotation speed is 400 r/min.

Further, the set stirring speed is 30-200r/min and the time is 5-10 minutes.

Further, the pH value requirement is that the pH value is equal to 7.0.

Further, the operating pressure of the ultrafiltration equipment is 0.11-0.6Mpa, and the membrane flux of the ultrafiltration membrane is 1-500L/㎡h.

Further, the spray pressure is 0.65-1.2MPa.

Example 1

When using the preparation method of this technical solution, the steps are as follows:

Step 1: Prepare a 1% acetic acid solution: Prepare acetic acid and water in a ratio of 1:100, then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution;

Step 2: Dissolve pullulan in the acetic acid solution of step 1. The weight ratio of pullulan to acetic acid solution is 1:0.5, and stir thoroughly to obtain mixed solution 1;

Step 3: Add hydrogen peroxide into mixed solution one. The weight ratio of mixed solution one and hydrogen peroxide is 1:10, and stir thoroughly to obtain mixed solution two;

Step 4: Set the internal temperature of the reaction kettle to 50°C and the rotation speed to 400r/min. Add mixed solution 2 into the reaction kettle and add acid. The acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid. , the weight ratio of pullulan and acid is 1:10, when the reaction reaches 1 hour, take out 1/10 of the reaction solution;

Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution;

Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed to 30r/min and the time to 10 minutes, and mix it thoroughly to obtain mixed solution three;

Step 7: Use pH test paper or pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value meets the requirements. Stop adding potassium hydroxide to obtain mixed solution four. The pH value requirement is that the pH value is equal to 7.0;

Step 8: The ultrafiltration equipment concentrates the mixed solution 4. The operating pressure of the ultrafiltration equipment is 0.11Mpa, and the membrane flux of the ultrafiltration membrane is 500L/㎡h. The pressure difference on both sides of the membrane is used as the driving force. The membrane is a filtration medium. Under a certain pressure, when the mixed solution 4 flows through the membrane surface, the many tiny pores densely covered on the surface of the ultrafiltration membrane only allow water and small molecular substances to pass through and become the permeate, while the mixed solution 4 flows through the membrane surface. Substances with a medium volume larger than the micropore size on the membrane surface are trapped on the liquid inlet side of the membrane and become concentrated liquid;

Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. The spray pressure is set to 0.65MPa. The concentrated liquid is atomized to form small droplets. The surface tension and Under the dual action of hot nitrogen, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder.

Example 2

When using the preparation method of this technical solution, the steps are as follows:

Step 1: Prepare a 1% acetic acid solution: Prepare acetic acid and water in a ratio of 1:100, then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution;

Step 2: Dissolve pullulan in the acetic acid solution of step 1. The weight ratio of pullulan to acetic acid solution is 1:20, and stir thoroughly to obtain mixed solution 1;

Step 3: Add hydrogen peroxide into mixed solution one. The weight ratio of mixed solution one and hydrogen peroxide is 1:10, and stir thoroughly to obtain mixed solution two;

Step 4: Set the internal temperature of the reaction kettle to 99°C and the rotation speed to 400r/min. Add mixed solution 2 into the reaction kettle and add acid. The acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid. , the weight ratio of pullulan and acid is 4:100, when the reaction reaches 1 hour, take out 1/10 of the reaction solution;

Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution;

Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed to 200r/min and the time to 5 minutes, and mix it thoroughly to obtain mixed solution three;

Step 7: Use pH test paper or pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value meets the requirements. Stop adding potassium hydroxide to obtain mixed solution four. The pH value requirement is that the pH value is equal to 7.0;

Step 8: The ultrafiltration equipment concentrates the mixed solution 4. The operating pressure of the ultrafiltration equipment is 0.6Mpa, and the membrane flux of the ultrafiltration membrane is 1L/㎡h. The pressure difference on both sides of the membrane is used as the driving force. The membrane is a filtration medium. Under a certain pressure, when the mixed solution 4 flows through the membrane surface, the many tiny pores densely covered on the surface of the ultrafiltration membrane only allow water and small molecular substances to pass through and become the permeate, while the mixed solution 4 flows through the membrane surface. Substances with a medium volume larger than the micropore size on the membrane surface are trapped on the liquid inlet side of the membrane and become concentrated liquid;

Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. The spray pressure is set to 1.2MPa. The concentrated liquid is atomized to form small droplets. The surface tension and Under the dual action of hot nitrogen, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder.

Example 3

When using the preparation method of this technical solution, the steps are as follows:

Step 1: Prepare a 1% acetic acid solution: Prepare acetic acid and water in a ratio of 1:100, then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution;

Step 2: Dissolve pullulan in the acetic acid solution of step 1. The weight ratio of pullulan to acetic acid solution is 1:2, and stir thoroughly to obtain mixed solution 1;

Step 3: Add hydrogen peroxide into mixed solution one. The weight ratio of mixed solution one and hydrogen peroxide is 1:10, and stir thoroughly to obtain mixed solution two;

Step 4: Set the internal temperature of the reaction kettle to 70°C and the rotation speed to 400r/min. Add mixed solution 2 into the reaction kettle and add acid. The acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid. , the weight ratio of pullulan and acid is 1:8, when the reaction reaches 1 hour, take out 1/10 of the reaction solution;

Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution;

Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed to 100r/min and the time to 8 minutes, and mix it thoroughly to obtain mixed solution three;

Step 7: Use pH test paper or pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value meets the requirements. Stop adding potassium hydroxide to obtain mixed solution four. The pH value requirement is that the pH value is equal to 7.0;

Step 8: The ultrafiltration equipment concentrates the mixed solution 4. The operating pressure of the ultrafiltration equipment is 0.3Mpa, and the membrane flux of the ultrafiltration membrane is 200L/㎡h. The pressure difference on both sides of the membrane is used as the driving force. The membrane is a filtration medium. Under a certain pressure, when the mixed solution 4 flows through the membrane surface, the many tiny pores densely covered on the surface of the ultrafiltration membrane only allow water and small molecular substances to pass through and become the permeate, while the mixed solution 4 flows through the membrane surface. Substances with a medium volume larger than the micropore size on the membrane surface are trapped on the liquid inlet side of the membrane and become concentrated liquid;

Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. The spray pressure is set to 0.85MPa. The concentrated liquid is atomized to form small droplets. The surface tension and Under the dual action of hot nitrogen, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder.

Experimental example

A hydroponic wheat test was carried out. The test variety was Jimai 22. The composite pullulan powder prepared in Example 1, Example 2 and Example 3 was used respectively. The test steps were as follows:

S1: Treatment of seeds: According to the test amount, add an appropriate amount of wheat seeds to the petri dish, then add deionized water to cover the wheat seeds, shake the petri dish slightly to immerse the seeds in water, and wait for 24 hours;

S2: Prepare culture medium: compound pullulan, diluted 500 times;

S3: Clean the beaker and planting basket, and place the planting basket into the beaker;

S4: Use tweezers to place the seeds into beakers, with no less than 10 seeds in each beaker, and set up 3 beakers;

S5: Pour the prepared culture solution into different beakers until the page just covers the wheat seeds;

S6: Wheat grows to the seventh day, harvests the wheat, and measures the data.

Table 1 shows the measurement data of Example 1, Example 2 and Example 3 under experimental examples.

Table 1.

Claims (10)

1. A preparation method of composite pullulan; characterized in that the steps are as follows: Step 1: Prepare 1% acetic acid solution; Step 2: Dissolve pullulan in the acetic acid solution of step 1, and stir thoroughly to obtain mixed solution 1; Step 3: Add hydrogen peroxide into mixed solution one and stir thoroughly to obtain mixed solution two; Step 4: Set the internal temperature and rotation speed of the reaction kettle, add mixed solution 2 into the reaction kettle, and add acid. When the reaction reaches 1 hour, take out 1/10 of the reaction solution; Step 5: Then continue the reaction for 4 hours. When the reaction is completed, take out the reaction solution; Step 6: Pour the reaction solution in step 4 and step 5 into the mixer, set the stirring speed and time, and mix it thoroughly to obtain mixed solution three; Step 7: Use pH test paper or a pH meter to test the pH value of mixed solution three, and then gradually add potassium hydroxide to adjust the pH value of mixed solution three until the pH value is met. Stop adding potassium hydroxide to obtain mixed solution four; Step 8: The ultrafiltration equipment concentrates the mixed solution IV, using the pressure difference on both sides of the membrane as the driving force, and the ultrafiltration membrane as the filter medium. Under a certain pressure, when the mixed solution IV flows through the membrane surface, the ultrafiltration The many tiny pores densely covered on the membrane surface only allow water and small molecular substances to pass through and become the permeate, while the substances in the mixed solution 4 that are larger than the pore size on the membrane surface are trapped on the inlet side of the membrane and become concentrated liquid; Step 9: Use spray drying equipment to dry the concentrated liquid. The concentrated liquid is pressurized by the material pump and enters the spray tower through the pipe and nozzle. Set the spray pressure and the concentrated liquid is atomized to form small droplets. Under the influence of surface tension and hot nitrogen, Under the dual action, the droplets shrink into spherical shapes, the liquid medium is evaporated, and the powder is collected from the bottom of the tower to obtain composite pullulan powder. 2. A kind of preparation method of composite Aureobasidic polysaccharide according to claim 1, characterized in that the specific steps of preparing a 1% acetic acid solution are: preparing acetic acid and water in a ratio of 1:100, Then mix the acetic acid and water thoroughly to obtain a 1% acetic acid solution. 3. A method for preparing composite pullulan according to claim 1, characterized in that the weight ratio of the pullulan to the acetic acid solution is 1:0.5-20. 4. A method for preparing composite aureus pullulan according to claim 1, characterized in that the weight ratio of the mixed solution one and hydrogen peroxide is 1:10. 5. The preparation method of a kind of composite pullulan according to claim 1, characterized in that the acid is any one or more of concentrated hydrochloric acid, concentrated sulfuric acid, nitric acid and phosphoric acid. The weight ratio of randose and acid is 1-4:10-100. 6. The preparation method of composite pullulan according to claim 1, characterized in that the set internal temperature of the reaction kettle is 50-99°C and the rotation speed is 400 r/min. 7. The preparation method of composite pullulan according to claim 1, characterized in that the set stirring speed is 30-200 r/min and the time is 5-10 minutes. 8. The preparation method of composite pullulan according to claim 1, wherein the pH value is required to be equal to 7.0. 9. A kind of preparation method of composite pullulan according to claim 1, characterized in that the operating pressure of the ultrafiltration equipment is 0.11-0.6Mpa, and the membrane flux of the ultrafiltration membrane is 1-0.6Mpa. 500L/㎡h. 10. The preparation method of composite pullulan according to claim 1, characterized in that the spray pressure is 0.65-1.2MPa.