Preparation method of agricultural slow-release polyglutamic acid powder

Abstract

The invention belongs to the technical field of biology, particularly relates to a preparation method of agricultural slow-release polyglutamic acid powder and further relates to application of the agricultural slow-release polyglutamic acid powder in agriculture. The invention relates to a preparation method of agricultural slow-release polyglutamic acid powder. The preparation method comprises the following steps: by taking polyglutamic acid fermentation liquor as a raw material, adding a proper amount of cationic ammonium polyacrylate or sodium polyacrylate; and adding a proper amount of any one or more of polyaluminum sulfate, polyaluminum chloride, polyferric sulfate, polyferric chloride, polyferric chloride sulfate or polymeric phosphate-ferric chloride, stirring to form polyglutamic acid flocculent precipitate, drying, and crushing to prepare the agricultural slow-release polyglutamic acid powder product. The preparation method has the beneficial effects that the preparation method of the polyglutamic acid slow-release powder is low in cost, mild in reaction, low in equipment requirement, high in operation feasibility and excellent in slow-release performance, the utilization rate of crops on polyglutamic acid can be effectively increased, and popularization and application of polyglutamic acid in the field of modern agriculture are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing agricultural slow-release polyglutamic acid powder. Background Art

[0002] Polyglutamic acid (γ-PGA) is a high molecular weight amino acid polymer with good water solubility, high biocompatibility and good safety. Due to its good water retention and chelating ability, it has the functions of retaining fertilizer and water, balancing soil pH, chelating heavy metals, enhancing plant disease resistance / adversity resistance, and promoting growth and production in agricultural production. At present, the preparation of polyglutamic acid is mainly prepared by microbial fermentation such as Bacillus licheniformis and Bacillus subtilis. Its recovery from the fermentation liquid is mostly carried out by organic solvent precipitation method, which uses a large amount of organic solvent, has a low recovery rate, complex equipment and operation, and high production cost. Since the absorption of polyglutamic acid products in the agricultural production process requires a certain period of time, traditional products are prone to loss due to leaching, runoff, microorganisms, etc. during field application due to the lack of sustained release, which greatly reduces its utilization rate and greatly limits the application of polyglutamic acid in agricultural production.

[0003] Invention CN104694590B discloses that after fermenting polyglutamic acid with Bacillus subtilis, the product is precipitated with ethanol and freeze-dried to obtain the product; Invention CN1231575C discloses that after fermentation with Bacillus subtilis X-003, centrifugation is performed, and the supernatant is concentrated and polyglutamic acid is precipitated with 95% ethanol; Invention CN113801856A discloses a method for preparing γ-polyglutamic acid using resting cells of recombinant bacteria expressing polyglutamic acid synthetase and / or polyglutamic acid synthetase, and the reaction solution is centrifuged to remove bacteria, concentrated, precipitated with alcohol or acetone, washed and impurities removed, and dried to obtain a crude γ-polyglutamic acid product. The above patents all use organic solvents to precipitate polyglutamic acid, with a large amount of solvent, low yield, and many impurities such as sugar and protein. In addition, the products are all fully dissolved polyglutamic acid, without a sustained release effect. Invention CN111876347B discloses halotolerant Bacillus licheniformis A-A2-10, its application and application method. The strain can synthesize polyglutamic acid from glutamic acid waste liquid. Although it greatly reduces the cost of polyglutamic acid fermentation, it does not involve a polyglutamic acid recovery scheme.

[0004] Therefore, developing an efficient and low-cost polyglutamic acid recovery method to produce a new type of agricultural slow-release polyglutamic acid powder is of great significance for reducing the production cost of polyglutamic acid, improving its utilization rate, and extending the fertilizer effect period, which can effectively promote the application and promotion of polyglutamic acid in modern agriculture. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing agricultural slow-release polyglutamic acid powder: using polyglutamic acid fermentation liquid as raw material, adding an appropriate amount of cationic polyacrylate ammonium or sodium polyacrylate, and then adding an appropriate amount of polyaluminum sulfate, polyaluminum chloride, polyferric sulfate, polyferric chloride, polyferric chloride sulfate and polyferric phosphorus chloride, and stirring to form polyglutamic acid flocculation precipitation, and then drying and crushing to prepare agricultural slow-release polyglutamic acid powder products. This method is low in cost, mild in reaction, low in equipment requirements, and high in operational feasibility; in addition, the novel agricultural polyglutamic acid product of the present invention has excellent slow-release performance, can effectively improve the utilization rate of polyglutamic acid by crops, and is conducive to the promotion and application of polyglutamic acid in the field of modern agriculture.

[0006] The specific technical solutions of the present invention are as follows: A method for preparing agricultural slow-release polyglutamic acid powder is characterized in that polyglutamic acid fermentation liquid is used as a raw material, the pH value is adjusted to 5.0-7.0, a proper amount of polymeric organic flocculant is added, and then a proper amount of polymeric inorganic flocculant is further added to form polyglutamic acid flocculation precipitation, and after filtering, drying and crushing, an agricultural slow-release polyglutamic acid powder product is prepared.

[0007] Preferably, the fermentation broth can be prepared by fermenting polyglutamic acid-producing bacteria such as Bacillus licheniformis and Bacillus subtilis using a culture medium composed of sugar, glutamic acid and other components or glutamic acid waste liquid, and the polyglutamic acid content is 0.2-8.0%.

[0008] Preferably, the glutamate waste liquid refers to the liquid obtained after isoelectric crystallization of glutamate from glutamate fermentation liquid and plate-and-frame filtration sterilization during glutamate production, which contains 1.0-3.0% total sugar, 0.3-3.0% glutamate and 0.5-15% ammonium sulfate.

[0009] Preferably, if the polyglutamic acid fermentation broth is prepared by fermenting a culture medium composed of sugar, glutamic acid and other components with polyglutamic acid-producing bacteria such as Bacillus licheniformis and Bacillus subtilis, the organic flocculant is at least one of cationic ammonium polyacrylate or cationic sodium polyacrylate, and the total addition amount is 0.1~1.0‰, and then stirring treatment is required, the stirring temperature is 4~50°C, the stirring speed is 10 rpm~100 rpm, and the time is 0.25~4.0h.

[0010] Preferably, if the polyglutamic acid fermentation liquid is prepared by fermenting glutamic acid waste liquid with polyglutamic acid producing bacteria such as Bacillus licheniformis and Bacillus subtilis, there is no need to add an organic polymer flocculant.

[0011] Preferably, the polyinorganic flocculant is at least one of polyaluminum sulfate, polyaluminum chloride, polyferric sulfate, polyferric chloride, polyferric chloride sulfate and polyferric phosphorus chloride, and the addition amount is 0.1-2.0%. The above flocculants need to be diluted into a 10-30% solution before use. The flocculation reaction process temperature is 4-50°C, the stirring speed is about 10-50rpm, and the time is 0.25-4h; Preferably, the flocculation is followed by centrifugation or plate-and-frame filtration to obtain a wet material of polyglutamate, which is then vacuum dried at a temperature of 50-90° C., crushed and sieved through a 60-150 mesh sieve to obtain a polyglutamate sustained-release powder.

[0012] The beneficial effects of the present invention are as follows: The invention can prepare slow-release polyglutamic acid powder by flocculating polyglutamic acid in fermentation broth and drying at low temperature. Compared with the traditional polyglutamic acid organic solvent precipitation method, the invention has high polyglutamic acid recovery rate, low production cost and good safety.

[0013] The polyglutamic acid sustained-release powder prepared by the invention has excellent sustained-release performance, can effectively reduce the degradation of polyglutamic acid by microorganisms, improve the utilization rate of polyglutamic acid in crops, and enhance the growth-promoting effect of polyglutamic acid in agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a photo of the supernatant of the polyglutamic acid fermentation broth after flocculation and filtration in the present invention.

[0015] Figure 2 This is a photo of the wet polyglutamic acid material after plate and frame filtration described in the present invention.

[0016] Figure 3 This is a photo of the polyglutamic acid sustained-release powder after drying described in the present invention. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in conjunction with specific embodiments. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0018] Example 1 Polyglutamic acid sustained-release powder is prepared by using polyglutamic acid fermentation liquid (polyglutamic acid content of 3.0%) prepared by fermenting glucose, glutamic acid and other components with Bacillus subtilis as a raw material, and the steps are as follows: Step 1, adjusting the pH value of the polyglutamic acid fermentation liquid to 6.2 with liquid ammonia, adding 0.1‰ of cationic ammonium polyacrylate, stirring at room temperature for 3 hours, and stirring at a speed of 30 rpm; Step 2: Prepare 15% polyaluminium chloride solution, slowly add it into the solution in step 1 at room temperature (about 25°C), stir and mix continuously during the addition process, the stirring speed is about 30 rpm, the amount of polyaluminium chloride added is 1.5%, and continue stirring and reacting for 2.5 hours; Step 3: The flocculation treatment liquid in step 2 is subjected to solid-liquid separation in a three-legged centrifuge to obtain a polyglutamic acid wet material; Step 4: The wet material obtained in step 3 is dried in a double cone rotary vacuum dryer at 70° C., and then crushed and passed through an 80-mesh sieve to obtain polyglutamic acid sustained-release powder.

[0019] Example 2

[0020] Polyglutamic acid sustained-release powder is prepared by using polyglutamic acid fermentation liquid (polyglutamic acid content of 2.5%) prepared by fermenting glucose, glutamic acid and other components with Bacillus licheniformis as a raw material, and the steps are as follows: Step 1: The pH value of the fermentation broth was determined to be 6.8, the broth was heated to 30° C., 0.5% cationic sodium polyacrylate was added, and the mixture was stirred for 1 h; Step 2: Prepare 20% polyaluminium sulfate solution, slowly add it to the solution in step 1, stir and mix continuously during the addition process, the stirring speed is about 25 rpm, the amount of polyaluminium sulfate added is 1.4%, and then continue stirring and reacting for 2 hours; Step 3: The flocculation treatment liquid in step 2 is subjected to solid-liquid separation by a plate-and-frame filter to obtain a wet polyglutamate material; Step 4: The wet material obtained in step 3 is dried in a double cone rotary vacuum dryer at 80° C., and then crushed and passed through a 60-mesh sieve to obtain polyglutamic acid sustained-release powder.

[0021] Example 3

[0022] Polyglutamic acid sustained-release powder is prepared using polyglutamic acid fermentation liquid (polyglutamic acid content of 1.0%) prepared by fermenting glutamic acid waste liquid with Bacillus licheniformis as raw material. Glutamic acid waste liquid is the liquid obtained after glutamic acid is extracted from glutamic acid fermentation liquid by isoelectric crystallization and sterilized by plate and frame filtration during glutamic acid production. Its ammonium sulfate content is about 13%. The steps are as follows: Step 1: The pH value of the fermentation liquid was measured to be 6.2, and the fermentation liquid was heated to 40°C.

[0023] Step 2: prepare 17% polyaluminium sulfate ferric solution, slowly add it to the solution in step 1, stir and mix continuously during the addition process, the stirring speed is about 30 rpm, the amount of polyaluminium sulfate added is 1.0%, and then continue stirring and reacting for 3 hours; Step 3: The flocculation treatment liquid in step 2 is subjected to solid-liquid separation by a plate-and-frame filter to obtain a wet polyglutamate material; Step 4: The wet material obtained in step 3 is dried in a double cone rotary vacuum dryer at 85° C., and then crushed and passed through a 100-mesh sieve to obtain polyglutamic acid sustained-release powder.

[0024] Example 4

[0025] Polyglutamic acid sustained-release powder is prepared using polyglutamic acid fermentation liquid (polyglutamic acid content of 0.9%) prepared by fermenting glutamic acid waste liquid with Bacillus licheniformis as raw material. Glutamic acid waste liquid is the liquid obtained after glutamic acid is extracted from glutamic acid fermentation liquid by isoelectric crystallization and sterilized by plate and frame filtration during glutamic acid production. Its ammonium sulfate content is about 11%. The steps are as follows: Step 1, adjusting the pH value of the fermentation liquid to 5.5; Step 2: Prepare 30% polyferric sulfate solution, slowly add it to the solution in step 1, stir and mix continuously during the addition process, the stirring speed is about 50 rpm, the amount of polyaluminum sulfate added is 0.8%, and then continue stirring and reacting for 1 hour; Step 3: The flocculation treatment liquid in step 2 is subjected to solid-liquid separation by a plate-and-frame filter to obtain a wet polyglutamate material; Step 4: The wet material obtained in step 3 is dried in a double-cone rotary vacuum dryer at 90° C., and then crushed and passed through an 80-mesh sieve to prepare polyglutamic acid sustained-release powder.

[0026] Comparative Example 1 The polyglutamic acid alcohol extraction process commonly used in the industry is used as comparative example 1. The raw materials are consistent with those in Example 1.

[0027] The data of the polyglutamic acid alcohol extraction process commonly used in the existing industry are compared with those in Examples 1 to 4 of the present invention as follows: Table 1 Comparison of polyglutamic acid recovery rates of Examples 1 to 4 and Comparative Example 1 project Comparative Example 1 Example 1 Example 2 Example 3 Example 4 Process Name Alcohol extraction Flocculation Flocculation Flocculation Flocculation Required Reagents Ethanol Soluble salt, flocculant Soluble salt, flocculant Soluble salt, flocculant Soluble salt, flocculant Required Equipment Sealed extraction equipment, ethanol recovery equipment General mixing equipment General mixing equipment General mixing equipment General mixing equipment Security Difference good good good good Recovery rate of polyglutamic acid (%) 85% 95% 98% 97% 99% <![CDATA[Release rate a > 100% 43% 45% 55% 57% Note: a Release rate refers to the release rate in water immersion test, i.e. the release rate of polyglutamic acid after the polyglutamic acid powder product is immersed in water for 30 days.

[0028] From the data in Table 1 above, it can be seen that under the scheme in Comparative Example 1, the yield of polyglutamic acid is only 85%, while the recovery rate of polyglutamic acid in Examples 1 to 4 of the present invention reaches more than 85%. Compared with the yield of polyglutamic acid, the method for extracting polyglutamic acid in the present invention is significantly better than the treatment method in Comparative Example 1.

[0029] In Comparative Example 1, ethanol is used as an alcohol extraction reagent, and the amount of ethanol used is about 4 times the volume of the fermentation liquid. The amount is large, the cost is high, and professional equipment is required for ethanol recovery. The working conditions are high, and there is a large safety risk in production. The cost of treating a single ton of fermentation liquid is as high as about 500 yuan (calculated according to the ethanol loss rate of 2.0%). The core process of the present invention only requires the addition of soluble salts and flocculants in ordinary stirring equipment for flocculation reaction, with low equipment requirements and high safety. The extraction cost of a single ton of conventional polyglutamic acid fermentation liquid is only about 50 yuan, while the extraction cost of polyglutamic acid fermentation liquid prepared from glutamic acid waste liquid (without the need to add additional soluble salts) is only about 15 yuan. Compared with the economic benefits and production safety, the present invention brings huge economic benefits and safety, which is significantly better than the treatment method of Comparative Example 1.

[0030] The polyglutamic acid powder product produced in Comparative Example 1 is very soluble and is easily lost through leaching, runoff, microbial degradation, etc. during field application. The polyglutamic acid powder product prepared in the present invention has a good sustained release property, which can significantly reduce the loss of polyglutamic acid in the soil and increase the utilization efficiency of polyglutamic acid by crops.

[0031] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing agricultural sustained-release polyglutamic acid powder, characterized in that: The polyglutamic acid fermentation liquid is used as raw material, the pH value is adjusted to 5.0-7.0, a proper amount of polymer organic flocculant is added, and then a proper amount of polymer inorganic flocculant is further added to form polyglutamic acid flocculation precipitation, and after filtering, drying and crushing, an agricultural slow-release polyglutamic acid powder product is prepared.

2. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The polyglutamic acid fermentation liquid is prepared by fermenting polyglutamic acid producing bacteria such as bacillus licheniformis and bacillus subtilis with sugar, glutamic acid or glutamic acid waste liquid as raw materials, and the polyglutamic acid content is between 0.2 and 8.0%.

3. The method for preparing a sustained-release polyglutamic acid powder suitable for agricultural use according to claim 2, characterized in that: The glutamic acid waste liquid refers to the liquid obtained after glutamic acid is extracted from the glutamic acid fermentation liquid by isoelectric crystallization and sterilized by plate-frame filtration during the glutamic acid production process, which contains 1.0-3.0% total sugar, 0.3-3.0% glutamic acid and 0.5-15.0% ammonium sulfate, and sodium polyacrylate has been added during the extraction of bacterial protein. If the polyglutamic acid fermentation liquid is prepared by fermenting glutamic acid waste liquid with polyglutamic acid producing bacteria such as Bacillus licheniformis and Bacillus subtilis, it is not necessary to add an organic polymer flocculant.

4. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The pH value of the polyglutamic acid fermentation liquid is adjusted to 5.0-7.0 by using sulfuric acid or liquid ammonia.

5. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: If the polyglutamic acid fermentation liquid is prepared by fermenting polyglutamic acid producing bacteria such as Bacillus licheniformis and Bacillus subtilis using a culture medium composed of sugar, glutamic acid and other components, the organic flocculant is at least one of cationic ammonium polyacrylate or cationic sodium polyacrylate, and the total addition amount is 0.1-1.0‰, and then stirring is performed, the stirring temperature is 4-50°C, the stirring speed is 10-100rpm, and the stirring time is 0.25-4h.

6. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The poly-inorganic flocculant is one or a mixture of polyaluminium sulfate, polyaluminium chloride, polyferric sulfate, polyferric chloride, polyferric chloride sulfate and polyferric phosphorus chloride, and the addition amount is 0.1-2.0%. The flocculation reaction process temperature is 4-50°C, the stirring speed is about 10-100 rpm, the time is 0.25-4h, and the flocculated liquid is filtered by plate and frame or centrifuged to obtain the flocculation product.

7. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The flocculation product is dried by vacuum drying at a temperature of 50-90°C.

8. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The product after reduced pressure drying is crushed to 60-150 mesh and then sieved to obtain a powdery product.

9. The method for preparing the agricultural sustained-release polyglutamic acid powder according to claim 1, characterized in that: The following steps are involved: S1, using liquid ammonia or sulfuric acid to adjust the pH of the fermentation liquid with a polyglutamic acid content of 0.2-8% to 5.0-7.0; S2, according to the characteristics of the fermentation liquid in step S1, at least one of cationic ammonium polyacrylate or cationic sodium polyacrylate is added in an amount of 0-1‰, the temperature is 4-50°C, the stirring speed is about 10-50rpm, and the time is 0.25-4h; S3, adding at least one of polyaluminium sulfate, polyaluminium chloride, polyferric sulfate, polyferric chloride, polyferric chloride sulfate and polyferric phosphorus chloride to the solution obtained in step S2, with the addition amount being 0.1-2.0%, wherein the above flocculants need to be diluted into a 10-30% solution before use, the flocculation reaction process temperature is 4-50°C, the stirring speed is about 10-50rpm, and the time is 0.25-4h; S4, centrifuging or plate-and-frame filtering the flocculation solution obtained in step S3 to obtain a flocculation product; S5, vacuum drying the flocculation product obtained in step S4 at a temperature of 50 to 90° C.; S6, crushing the dried product obtained in S5, and sifting it through a 60-150 mesh sieve to obtain a powdery product.

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