Method for preparing amino acid foliar fertilizer from beta-alanine production waste

Abstract

The invention discloses a method for preparing an amino acid foliar fertilizer by using beta-alanine production waste, which comprises the following steps: carrying out ultrasonic pretreatment on the beta-alanine production waste, then adjusting the pH value to 2-3, and hydrolyzing at 80-90 DEG C for 30-40 minutes to obtain an acidolysis solution; and adjusting the pH value of the acidolysis solution to 7.0-8.0, adding a compound enzyme composed of neutral protease, papain and lysozyme into the acidolysis solution, carrying out enzymolysis, filtering, and concentrating to obtain the amino acid foliar fertilizer. According to the method, the beta-alanine production waste is used as a raw material, aiming at the problems of high solid-to-liquid ratio, high system viscosity, low enzymolysis efficiency and high enzyme dosage, ultrasonic pretreatment is adopted and is combined with acidolysis and enzymolysis processes, so that the enzymolysis efficiency is improved, the enzyme dosage is reduced, mycoprotein in the waste is fully hydrolyzed into micromolecular polypeptide and amino acid which are easy to utilize by plants, and the utilization rate of the micromolecular polypeptide and amino acid is increased. When the fertilizer is used as an amino acid foliar fertilizer, the crop yield and quality are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of amino acid production waste treatment, and particularly relates to a method for preparing an amino acid foliar fertilizer by using β-alanine production waste. Background Art

[0002] β-alanine, also known as 3-aminopropionic acid, is a natural β-type amino acid and is one of the important components in muscle peptide substances in muscle tissues. β-alanine has the characteristics of stable chemical properties and non-toxicity, and is widely used in industries such as medicine, food, and chemical engineering.

[0003] At present, the production of β-alanine mainly uses the enzyme conversion method. The obtained enzyme conversion solution contains a certain amount of macromolecular substances such as microbial protein, which is often removed by filtration through a ceramic membrane during the extraction process. The ceramic membrane concentrate produced contains a large amount of microbial protein, which is a single-cell protein and contains rich components such as protein and fat.

[0004] Our company has developed a production process in which acrylic acid is used as a substrate and an aspartase variant is used to catalyze the ammoniation reaction of acrylic acid to produce β-alanine. Every year, a large amount of ceramic membrane concentrate containing microbial protein is generated. The wet microbial volume occupies production space, and stacking will also produce odors, with difficult treatment and high costs, affecting normal production and causing environmental protection problems. To solve the above problems, our company intends to develop an application value for the by-product microbial protein, fully utilize this waste for processing and make it into a fertilizer for plant growth, which is of great significance for strengthening production continuity and reducing environmental protection pressure.

[0005] Since the utilization rate of macromolecular proteins in fertilizers is relatively low, decomposing microbial protein into amino acids and small peptides that are easily absorbed by plants is the key to developing amino acid fertilizers. In the prior art, converting microbial protein into small amino acids or peptides generally uses acid hydrolysis or enzymatic hydrolysis methods. Since strong acid hydrolysis is not specific, amino acids such as tryptophan, serine, and threonine will be destroyed during the acid hydrolysis process, and the obtained amino acids have poor activity; while using a protease with strong specificity for enzymatic hydrolysis, due to the high content of microbial protein in the ceramic membrane concentrate and the high viscosity of the system, there are problems of low enzymatic hydrolysis efficiency and incomplete enzymatic hydrolysis. Therefore, developing a hydrolysis process applicable to the ceramic membrane concentrate of β-alanine enzyme conversion solution and processing it into an amino acid fertilizer is of great significance for our company. Summary of the Invention

[0006] Based on the technical problems existing in the background art, the present invention proposes a method for preparing an amino acid foliar fertilizer by using β-alanine production waste, which adopts ultrasonic pretreatment, combined with acid hydrolysis and enzymatic hydrolysis processes, improves the enzymatic hydrolysis efficiency, reduces the dosage of enzymes, and fully hydrolyzes the microbial protein in the waste into small peptides and amino acids that are easily utilized by plants.

[0007] A method for preparing amino acid foliar fertilizer by using β-alanine production waste proposed by the present invention is characterized by comprising the following steps:

[0008] S1. Ultrasonically pretreat the β-alanine production waste, then adjust the pH to 2-3, and hydrolyze at 80-90 °C for 30-40 min to obtain an acidolysis solution.

[0009] S2. Adjust the pH of the acidolysis solution to 7.0-8.0, add a composite enzyme thereto for enzymatic hydrolysis, filter, and concentrate to obtain the product.

[0010] The composite enzyme is composed of neutral protease, papain, and lysozyme.

[0011] In the present invention, the β-alanine production waste is mainly microbial protein, and the content of microbial protein can be as high as 20-30%. It occupies production space and accumulates to produce peculiar smell. Due to the existence of the hydration film and double electric layer on the surface of the microbial protein, protein molecules do not aggregate with each other, thus forming a relatively stable colloidal solution. The waste has high viscosity, and direct enzymatic hydrolysis has low efficiency, high enzyme dosage, and high enzymatic hydrolysis cost.

[0012] In the present invention, ultrasonic pretreatment is adopted before acidolysis. Under the cavitation effect and mechanical shearing force of ultrasonic waves, at appropriate ultrasonic power and ultrasonic time, microbial cells are broken, proteins are released from the cells, which is convenient for degradation. At the same time, protein molecules unfold and peptide bonds in the molecules are exposed, which is beneficial to acidolysis at a high solid-liquid ratio.

[0013] Concentrated sulfuric acid is used to adjust the pH of the system. At this time, the system starts to stratify from a viscous gel state, and the viscosity of the acidolysis solution after stratification decreases. Acidolysis degrades proteins into macromolecular polypeptide substances, thereby improving the efficiency of subsequent enzymatic hydrolysis. Through ultrasonic pretreatment, the acidolysis time is shortened.

[0014] Then, through enzymatic hydrolysis treatment, the waste is converted into small molecular polypeptides and amino acids. By controlling the enzymatic hydrolysis time, the enzymatic hydrolysis efficiency is improved, and the problem that macromolecular proteins in the raw materials are not easily utilized by plants is solved.

[0015] Preferably, the β-alanine production waste is a ceramic membrane concentrate obtained by filtering the β-alanine enzyme conversion solution through a ceramic membrane.

[0016] Preferably, the ultrasonic pretreatment is carried out at 150-200 W for 10-15 min.

[0017] It is found through exploration that at low ultrasonic power, the strength of the gel formed by protein water absorption and swelling increases, which is not conducive to subsequent acidolysis; at high ultrasonic power, the solubility decreases instead, and the protein precipitates.

[0018] Preferably, 98% sulfuric acid is used to adjust the pH to 2 - 3.

[0019] Preferably, 20 - 25% ammonia water is used to adjust the pH of the acidolysis solution to 7.0 - 8.0.

[0020] Preferably, the mass ratio of the neutral protease, papain, and lysozyme is 5 - 7:3:1.

[0021] Preferably, the addition amount of the complex enzyme is 3 - 5% of the mass of the acidolysis solution.

[0022] Preferably, the enzymatic hydrolysis temperature is 30 - 40 °C, and the enzymatic hydrolysis time is 3 - 5 h.

[0023] Preferably, in S2, it is concentrated to a solid content of 50 - 60%.

[0024] Beneficial effects: The present invention uses the waste from β-alanine production as raw material. Aiming at the problems of high solid - liquid ratio, large system viscosity, low enzymatic hydrolysis efficiency, and high enzyme dosage in it, ultrasonic pretreatment is adopted, combined with acidolysis and enzymatic hydrolysis processes. First, acidolysis treatment is carried out for a short time and then enzymatic hydrolysis is carried out to improve the enzymatic hydrolysis efficiency, reduce the enzyme dosage, and fully hydrolyze the microbial protein in the waste into small - molecule polypeptides and amino acids that are easily utilized by plants. Moreover, the waste raw material contains a certain amount of amino acids. The enzymatic hydrolysis solution prepared by the above process is concentrated to prepare a high - value amino acid foliar fertilizer for plant cultivation, which can significantly increase the crop yield and improve the crop quality. Specific Embodiments

[0025] Next, the technical solution of the present invention will be described in detail through specific examples.

[0026] In the following examples, the ceramic membrane concentrate is the ceramic membrane retentate obtained by filtering the β - alanine enzyme fermentation broth through a ceramic membrane; the wet microbial protein is obtained by centrifuging the ceramic membrane concentrate, and its content is 20 - 30%; then the wet microbial protein is dried, and its water content is measured to be 25 - 35%.

[0027] Example 1

[0028] A method for preparing an amino acid foliar fertilizer using the waste from β - alanine production, the steps are as follows:

[0029] 1) Ultrasonically treat the ceramic membrane concentrate (with a wet cell protein content of 27% and a water content of 30% in the wet cell protein) at 200 W for 10 min. Ultrasonic pretreatment is carried out before acid hydrolysis. Under the cavitation effect and mechanical shear force of ultrasonic waves, at an appropriate ultrasonic power and ultrasonic time, the bacterial cells are broken, and the protein is released from the cells, facilitating degradation. At the same time, the protein molecules unfold, and the peptide bonds in the molecules are exposed, which is beneficial for acid hydrolysis at a high solid-liquid ratio. Through exploration, it is found that at a low ultrasonic power, the strength of the gel formed by protein water absorption and swelling increases, which is not conducive to subsequent acid hydrolysis; at a high ultrasonic power, on the contrary, the solubility decreases and the protein precipitates.

[0030] 2) Add 98% sulfuric acid to the ceramic membrane concentrate after ultrasonic pretreatment to adjust the system pH to 2.5, heat up to 85 °C for acid hydrolysis, and the acid hydrolysis time is 30 min. At this time, the ceramic membrane concentrate starts to stratify from a viscous gel state, and the viscosity of the acidified liquid after stratification decreases. Acid hydrolysis degrades the protein into macromolecular polypeptide substances, thereby improving the efficiency of subsequent enzymatic hydrolysis. Through ultrasonic pretreatment, the acid hydrolysis time is shortened.

[0031] 3) Adjust the pH of the acidified liquid to 7.5 with 25% ammonia water, add a composite enzyme (the mass ratio of neutral protease, papain, and lysozyme is 6:3:1) for enzymatic hydrolysis, the addition amount of the enzyme is 4% of the mass of the acidified liquid, the enzymatic hydrolysis temperature is 35 °C, and the enzymatic hydrolysis time is 4 h. Filter to remove the undigested solid residue to obtain an enzymatic hydrolysate, and concentrate it to a solid content of 50% to obtain the amino acid foliar fertilizer.

[0032] Example 2

[0033] A method for preparing an amino acid foliar fertilizer using β-alanine production waste, the steps are as follows:

[0034] 1) Ultrasonically treat the ceramic membrane concentrate (with a wet cell protein content of 27% and a water content of 30% in the wet cell protein) at 150 W for 10 min. Ultrasonic pretreatment is carried out before acid hydrolysis. Under the cavitation effect and mechanical shear force of ultrasonic waves, at an appropriate ultrasonic power and ultrasonic time, the bacterial cells are broken, and the protein is released from the cells, facilitating degradation. At the same time, the protein molecules unfold, and the peptide bonds in the molecules are exposed, which is beneficial for acid hydrolysis at a high solid-liquid ratio. Through exploration, it is found that at a low ultrasonic power, the strength of the gel formed by protein water absorption and swelling increases, which is not conducive to subsequent acid hydrolysis; at a high ultrasonic power, on the contrary, the solubility decreases and the protein precipitates.

[0035] 2) Add 98% sulfuric acid to the concentrated ceramic membrane solution after ultrasonic pretreatment to adjust the pH of the system to 2, heat it up to 80 °C for acid hydrolysis, and the acid hydrolysis time is 30 min. At this time, the concentrated ceramic membrane solution starts to stratify from the viscous gel state, and the viscosity of the acidified solution after stratification decreases. Acid hydrolysis degrades proteins into macromolecular polypeptide substances, thereby improving the efficiency of subsequent enzymatic hydrolysis. Through ultrasonic pretreatment, the acid hydrolysis time is shortened.

[0036] 3) Adjust the pH of the acidified solution to 7.0 with 25% ammonia water, add a composite enzyme (the mass ratio of neutral protease, papain, and lysozyme is 5:3:1) for enzymatic hydrolysis, the addition amount of the enzyme is 3% of the mass of the acidified solution, the enzymatic hydrolysis temperature is 30 °C, and the enzymatic hydrolysis time is 3 h. Filter to remove the undigested solid residue to obtain an enzymatic hydrolysate, and concentrate it to a solid content of 50% to obtain the amino acid foliar fertilizer.

[0037] Example 3

[0038] A method for preparing amino acid foliar fertilizer using β-alanine production waste, the steps are as follows:

[0039] 1) Ultrasonically treat the concentrated ceramic membrane solution (the content of wet bacterial protein in the concentrated ceramic membrane solution is 27%, and the water content of the wet bacterial protein is 30%; the content of β-alanine in the concentrated ceramic membrane solution is 3.6 g / L) at 200 W for 15 min. Pretreatment with ultrasound before acid hydrolysis. Under the cavitation effect and mechanical shear force of ultrasound, with appropriate ultrasonic power and ultrasonic time, bacterial cells are broken, proteins are released from the cells for easy degradation. At the same time, protein molecules unfold and peptide bonds in the molecules are exposed, which is beneficial for acid hydrolysis at a high solid-liquid ratio. It is found through exploration that at a low ultrasonic power, the strength of the gel formed by protein swelling with water increases, which is not conducive to subsequent acid hydrolysis; at a high ultrasonic power, the solubility decreases instead, and the protein precipitates.

[0040] 2) Add 98% sulfuric acid to the concentrated ceramic membrane solution after ultrasonic pretreatment to adjust the pH of the system to 3, heat it up to 90 °C for acid hydrolysis, and the acid hydrolysis time is 40 min. At this time, the concentrated ceramic membrane solution starts to stratify from the viscous gel state, and the viscosity of the acidified solution after stratification decreases. Acid hydrolysis degrades proteins into macromolecular polypeptide substances, thereby improving the efficiency of subsequent enzymatic hydrolysis. Through ultrasonic pretreatment, the acid hydrolysis time is shortened.

[0041] 3) Adjust the pH of the acidified solution to 8.0 with 25% ammonia water, add a composite enzyme (the mass ratio of neutral protease, papain, and lysozyme is 7:3:1) for enzymatic hydrolysis, the addition amount of the enzyme is 5% of the mass of the acidified solution, the enzymatic hydrolysis temperature is 40 °C, and the enzymatic hydrolysis time is 5 h. Filter to remove the undigested solid residue to obtain an enzymatic hydrolysate, and concentrate it to a solid content of 50% to obtain the amino acid foliar fertilizer.

[0042] Comparative Example 1

[0043] Compared with Example 1, the only difference is that it does not contain the ultrasonic pretreatment step in step 1).

[0044] Comparative Example 2

[0045] Compared with Example 1, the only difference is that it does not contain the acid hydrolysis step in step 2).

[0046] Test Example

[0047] 1. Detect the enzymolysis solutions prepared in Examples 1-3 and Comparative Examples 1-2.

[0048] Detection of free amino acid content: determined by an automatic amino acid analyzer;

[0049] Detection of small molecule polypeptide content: determined by IEC amino acid analysis method. The sample is pretreated with trichloroacetic acid to remove precipitated macromolecular proteins, and peptide amino acids and free amino acids are extracted. Then, all are hydrolyzed with hydrochloric acid solution to obtain acid-soluble proteins, and the acid-soluble protein content is determined by IEC amino acid analysis method. The difference between the acid-soluble protein content and the total free amino acid content is the small molecule polypeptide content;

[0050] Determination of the molecular weight of small molecule polypeptides: determined by high performance gel filtration chromatography. Using porous filler as the stationary phase, separation is carried out according to the difference in the relative molecular weights of sample components. Detection is carried out under the condition of the ultraviolet absorption wavelength of the peptide bond at 220 mm. Use the special data processing software for gel chromatography to determine the relative molecular weight distribution (i.e., GPC software) to process the chromatogram and its data, and calculate the relative molecular weight size and distribution range of the peptide.

[0051] The experimental results are shown in Table 1.

[0052] Table 1 Relevant data of the enzymolysis solution

[0053] Polypeptide hydrolysis rate / % Polypeptide molecular weight / Da Proportion of polypeptides with molecular weight < 1500 Da / % Amino acid hydrolysis rate / % Example 1 41.5 200-5000 52.2 4.3 Example 2 39.6 200-5000 51.6 3.7 Example 3 37.5 200-5000 48.9 4.2 Comparative Example 1 25.2 200-5000 39.3 3.4 Comparative Example 2 19.7 200-5000 35.2 2.5

[0054] 2. Soybean planting

[0055] Conduct experiments on 2 experimental fields for planting soybeans, divided into an experimental group and a control group. The area of the experimental fields in both groups is 1 mu;

[0056] Spray foliar fertilizer during the flowering and pod-setting stages of soybeans. Among them, the experimental group uses the amino acid foliar fertilizer prepared in Example 1, with a dosage of 1000 g / mu and sprayed 800 times on the leaves; the control group only sprays an equal amount of clear water; the daily management of the experimental group and the control group is the same. After planting, count the yield of soybeans and detect the crude protein content of soybeans. The results are shown in Table 2.

[0057] Table 2

[0058] Yield kg / mu Crude protein content mg / kg Experimental group 273 46.3 Control group 191 41.2

[0059] As can be seen from Table 2, the amino acid foliar fertilizer prepared by the present invention can significantly increase the yield of crops and the crop quality is better.

[0060] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention.

Claims

1. A method for preparing amino acid foliar fertilizer by using β-alanine production waste, which is characterized in that It includes the following steps: S1. Ultrasonically pretreat the β-alanine production waste, then adjust the pH to 2-3, and hydrolyze at 80-90°C for 30-40 min to obtain an acid hydrolysis solution; S2. Adjust the pH of the acid hydrolysis solution to 7.0-8.0, add a composite enzyme thereto for enzymatic hydrolysis, filter, and concentrate to obtain the product; The composite enzyme is composed of neutral protease, papain, and lysozyme.

2. The method according to claim 1, characterized in that, The β-alanine production waste is the ceramic membrane concentrate obtained by filtering the β-alanine enzyme conversion solution through a ceramic membrane.

3. The method according to claim 1 or 2, characterized in that The ultrasonic pretreatment is carried out by ultrasonic treatment at 150-200 W for 10-15 min.

4. The method according to any one of claims 1-3, characterized in that Adjust the pH to 2-3 with 98% sulfuric acid.

5. The method according to any one of claims 1-4, characterized in that Adjust the pH of the acid hydrolysis solution to 7.0-8.0 with 20-25% ammonia water.

6. The method according to any one of claims 1-5, characterized in that The mass ratio of the neutral protease, papain, and lysozyme is 5-7:3:

1.

7. The method according to any one of claims 1-6, characterized in that, The addition amount of the composite enzyme is 3-5% of the mass of the acid hydrolysis solution.

8. The method according to any one of claims 1-7, characterized in that The enzymatic hydrolysis temperature is 30-40°C, and the enzymatic hydrolysis time is 3-5 h.

9. The method according to any one of claims 1 to 8, characterized in that, In S2, concentrate to a solid content of 50-60%.