Method for preparing amino acid-peptide water-soluble fertilizer from waste animal protein

Abstract

The invention belongs to the field of waste resource utilization, and relates to a method for preparing and producing a liquid amino acid-peptide water-soluble fertilizer by utilizing waste animal protein. Neutral protease and alkaline protease are adopted for compound enzymolysis of animal protein, the protein transfer rate is greatly increased, and the protein transfer rate reaches the maximum value of 76.42%. Then, aminopeptidase is utilized for deep hydrolysis, and finally, inorganic ions are chelated to prepare the amino acid-peptide water-soluble fertilizer. The content of dissolved peptide in the amino acid-peptide water-soluble fertilizer prepared by the invention can reach 116.26 g / L, and the total amount of free amino acid can reach 8235 mg / L. The proportion of small molecule peptide and amino acid is high, and the proportion of oligopeptide with the molecular weight of 180-500 Da can reach 62.1%; and the proportion of components less than 180Da can reach 12.9%. The water-soluble fertilizer prepared by the invention can promote the growth of spinach and enhance the enzyme activity of soil, and is more beneficial to the best balance of spinach absorption and soil improvement compared with a long-peptide peptide fertilizer.

Description

Technical Field

[0001] The present invention belongs to the field of waste resource utilization, and relates to a method for preparing liquid amino acid-peptide water-soluble fertilizer by using waste animal protein. Background Art

[0002] With the growth of meat consumption, the production of waste animal protein has increased sharply, becoming an environmental burden. The production of waste animal protein continues to rise, and the treatment pressure is increasing day by day. Therefore, it is urgent to explore an environmentally friendly and efficient treatment method. Amino acid peptide fertilizer is outstanding in improving the physical and chemical properties of soil, enhancing soil air permeability, water retention and fertilizer retention, and creating a good soil environment for plant growth. At the same time, amino acid peptide fertilizer can promote the growth and development of plant roots, enhance the absorption capacity of plants for nutrients and water in the soil, and significantly improve the drought resistance of plants by regulating the osmotic pressure of plant cells. Amino acids play multiple regulatory roles in the soil ecosystem, and their dynamic changes are jointly regulated by soil microbial activities and soil environment, further affecting the physical and chemical properties and ecological functions of the soil.

[0003] At present, the preparation technologies of amino acid peptide fertilizer are mainly based on the following methods: Chemical hydrolysis method: mainly using acid / alkali treatment, hydrolyzing animal by-products with sulfuric acid or strong alkali, and destroying the protein structure by high temperature and high pressure to generate free amino acids. This method has low cost, but is prone to environmental pollution, and has single amino acid type and low activity (such as CN103804032A, CN109890781A). Enzyme-acid combination: such as CN104003766A uses trypsin for pre-enzyme hydrolysis and then combines with dilute hydrochloric acid hydrolysis to improve the conversion rate, but the steps are complex and the risk of equipment corrosion is high. Single enzyme hydrolysis: using trypsin, pepsin, etc. to decompose fish protein (CN1597626A) or feather keratin (CN109650946A), the conditions are mild but the efficiency is limited, and a long reaction time (0.5 - 1200 hours) is required. Composite enzyme synergy: through the combination of keratinase, neutral protease and other enzymes for hydrolysis. Although this scheme can improve the hydrolysis efficiency to a certain extent, there are still problems such as low content of free amino acids and unreasonable ratio of free amino acids to small peptides. Biological fermentation method: using microorganisms (such as enzyme bacteria, bacillus) or enzymes to co-ferment raw materials such as animal blood (CN105461385A), rice residue (CN106673721A), etc. to generate fertilizers containing amino acids and small peptides. This method is environmentally friendly but has a long cycle and low product concentration, and subsequent concentration is required.

[0004] Therefore, it is necessary to develop a new method to solve the problems existing in the prior art. Summary of the Invention

[0005] To solve the problems existing in the prior art, the present invention proposes a method for preparing amino acid peptide fertilizer using waste animal protein. The waste animal protein is preliminarily hydrolyzed by a composite enzyme to dissolve more protein in the liquid phase and improve the protein transfer rate. Then, an exopeptidase is used to deeply hydrolyze the soluble animal protein to increase the free amino acid content. Finally, trace elements of zinc and iron are chelated to make its nutrition balanced, which is applicable to the field of planting fertilizers. The prepared peptide fertilizer is applied to spinach planting to evaluate its actual application effect of the fertilizer.

[0006] The first aspect of the present invention provides a method for preparing amino acid peptide fertilizer using waste animal protein, comprising the following steps:

[0007] S1. Preliminary hydrolysis: Take the waste animal offal, sieve it, add water to make a slurry, adjust the pH value, add a composite enzyme for enzymatic hydrolysis, inactivate the enzyme, and then centrifuge to collect the liquid phase. After layering, remove the grease layer and the emulsion layer to obtain the preliminary hydrolysis solution.

[0008] S2. Deep hydrolysis: Spray-dry the preliminary hydrolysis solution obtained in step S1 to obtain a preliminary hydrolysis protein powder. After adding water to dissolve it, add aminopeptidase for enzymatic hydrolysis, and inactivate the enzyme to obtain a deep hydrolysis solution.

[0009] S3. Preparation of chelated fertilizer; Add Fe 2+ and Zn 2+ to the deep hydrolysis solution, stir for chelation, and remove the precipitate to obtain a liquid amino acid-peptide water-soluble fertilizer.

[0010] In some embodiments of the present invention, in step S1, the waste animal offal is mixed with water at a ratio of 1:2 - 1:8 to make a slurry; more preferably, the ratio is 1:4.

[0011] Furthermore, in step S1, the enzymatic hydrolysis conditions are: for every 15 g of substrate, the total enzyme activity of the composite enzyme is 10,000 - 15,000 U, wherein the ratio of the enzyme activity addition amounts of alkaline protease and neutral protease is 1:1 - 9:1;

[0012] In some preferred embodiments, the ratio of alkaline protease to neutral protease in the composite enzyme is preferably 7:3.

[0013] Even further, in step S1, the pH is 7 - 11, and the enzymatic hydrolysis temperature is 45 - 55 °C; preferably 50 °C.

[0014] In some embodiments of the present invention, in step S1, the enzymatic hydrolysis time is 8 - 14 h, preferably 10 - 12 h.

[0015] Furthermore, in step S2, the enzymatic hydrolysis conditions are: the substrate concentration is 50 g / L - 100 g / L; more preferably, the substrate concentration is 80 g / L - 100 g / L.

[0016] Further, in step S2, the pH is 6 - 8, preferably 7.

[0017] Furthermore, in step S2, the addition amount of aminopeptidase is 1.25 - 2.50 g / L, the enzymatic hydrolysis temperature is 30 - 45 °C, preferably 40 °C.

[0018] In some embodiments of the present invention, in step S2, the enzymatic hydrolysis time is 1.5 - 3 h, preferably 2 h.

[0019] Further, in step S3, the content of trace elements of zinc and iron ≥ 20 g / L.

[0020] Further, in step S3, the chelation reaction temperature is 40 - 60 °C.

[0021] In one embodiment, S3. Preparation of chelated fertilizer; adding a certain amount of ferrous sulfate heptahydrate and zinc sulfate heptahydrate to the deep hydrolysis solution, stirring and chelating at 50 °C, and filtering off the precipitate generated during the chelation process to obtain the amino acid peptide water-soluble fertilizer.

[0022] The second aspect of the present invention lies in providing an amino acid peptide water-soluble fertilizer prepared by the method described above;

[0023] Further, the proportion of oligopeptides with a molecular weight of 180 - 500 Da in the amino acid peptide water-soluble fertilizer ≥ 60%; and the proportion of components with a molecular weight less than 180 Da ≥ 12%.

[0024] Furthermore, the dissolved peptide content in the amino acid peptide water-soluble fertilizer is 100 - 120 g / L.

[0025] Beneficial effects:

[0026] (1) In the preliminary enzymatic hydrolysis step of the present invention, compound enzymatic hydrolysis with neutral protease and alkaline protease is adopted, the method is simple and feasible, and the protein transfer rate is greatly improved compared with single enzymatic hydrolysis, and the protein transfer rate reaches the highest value of 76.42%.

[0027] (2) In the amino acid-peptide water-soluble fertilizer prepared by the present invention, the content of small molecule peptides and amino acids is high. Among them, the dissolved peptide content can reach 116.26 g / L, and the total amount of free amino acids can reach 8235 mg / L. The proportion of oligopeptides with a molecular weight of 180 - 500 Da can reach more than 60%; and the proportion of components with a molecular weight less than 180 Da can reach 12.9%.

[0028] (4) The water-soluble fertilizer prepared by the present invention is neutral (pH 7.1), has little impact on the soil after application, and can promote the growth of spinach and enhance the soil enzyme activity. Compared with the long peptide segment fertilizer, it is more conducive to the best balance between spinach absorption and soil improvement. Description of the drawings

[0029] The accompanying drawings are used to provide a further understanding of the present application, and constitute a part of the specification. They are used together with the embodiments of the present application to explain the present application, and do not constitute a limitation to the present application. In the accompanying drawings:

[0030] Figure 1 are the hydrolysis protein transfer rate and crude protein quality of neutral protease;

[0031] Figure 2 are the hydrolysis protein transfer rate and crude protein quality of alkaline protease;

[0032] Figure 3 are the hydrolysis protein transfer rate and crude protein quality of compound protease;

[0033] Figure 4 are the hydrolysis protein transfer rate and crude protein quality of compound protease;

[0034] Figure 5 The dissolved peptide content (a) and free amino acid composition (b) in the amino acid-peptide water-soluble fertilizer prepared in Example 4;

[0035] Figure 6 The dissolved peptide content (a) and free amino acid composition (b) in the amino acid-peptide water-soluble fertilizer prepared in Example 5;

[0036] Figure 7 The dissolved peptide content (a) and free amino acid composition (b) in the amino acid-peptide water-soluble fertilizer prepared in Example 6;

[0037] Figure 8 The dissolved peptide content (a) and free amino acid composition (b) in the amino acid-peptide water-soluble fertilizer prepared in Example 7. Detailed embodiments

[0038] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is made in conjunction with the embodiments of the specification.

[0039] In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0040] The test raw material is the powder of meat and bone meal (MBM) after high-temperature harmless treatment of slaughterhouse offal, purchased from a protein feed processing company in Shijiazhuang;

[0041] The test alkaline protease is purchased from Jiangsu Boyang Biotechnology Co., Ltd., with an enzyme activity of 200000 U / g;

[0042] Neutral protease was purchased from Qingdao Weilan Biological Co., Ltd. with an enzyme activity of 500,000 U / g;

[0043] Aminopeptidase was purchased from Qingdao Weilan Biological Co., Ltd. with an enzyme activity of 3000 U / g;

[0044] Commercially available peptide fertilizer: dissolved peptide content 38g / L.

[0045] Test items and methods:

[0046] Determination of protein content in MBM samples: Referring to Jiao Youzhou et al., the Kjeldahl method was used to determine the protein contents in the solid and liquid phases, respectively. The nitrogen conversion factor was 6.25, and the solid phase protein mass S, liquid phase protein mass L and protein transfer rate P were calculated according to the following formulas.

[0047] S=m×d1(1)

[0048] L=(Mm)×d2(2)

[0049]

[0050] Where M represents the weight of the MBM sample / g, m represents the weight of the solid residue remaining after enzymatic hydrolysis / g, d represents the initial protein content of the MBM sample / %, and d1 and d2 represent the solid and liquid phase protein contents / %, respectively.

[0051] Protein electrophoresis peptide analysis: First, weigh an appropriate amount of protein sample and place it into a 1.5mL EP tube. Add protein loading buffer at a 4:1 ratio and mix thoroughly. Then, place the EP tube in a boiling water bath for 10 minutes. After the water bath, centrifuge the sample. The resulting supernatant is the loading sample. Prepare a 5% stacking gel, a 12% separating gel, and electrode buffer according to the established recipe. After preparation, install the electrophoresis instrument and slowly inject the electrode buffer into the inner chamber, followed by an appropriate amount of electrode buffer into the outer chamber. Next, add the protein marker and processed sample to the sample wells in the order shown. Turn on the power supply and set the voltage to 80V for 20 minutes. Then, adjust the voltage to 120V and observe continuously. Stop electrophoresis when the band moves to the bottom of the separating gel. After electrophoresis, stain, destain, and photograph.

[0052] Determination of free amino acid content: refer to NY / T 1975-2010 "Determination of free amino acid content of water-soluble fertilizers".

[0053] Determination of soluble amino acid peptide content: First, dissolve the sample with 15% trichloroacetic acid, stir for 30 min, then filter with filter paper. Take 10 ml of the filtrate for high-temperature digestion and nitrogen distillation. The subsequent steps refer to the Kjeldahl method in GB 5009.5-2016, and calculate the soluble amino acid peptide content T according to the following formula.

[0054]

[0055] In the formula, △V is the volume of NaOH consumed during enzymatic hydrolysis; 6.25 is the coefficient for converting nitrogen to protein; c is the concentration of the hydrochloric acid standard titration solution, mol / L; 0.014 is the mass of nitrogen equivalent to the hydrochloric acid standard titration solution, g; m is the mass of the sample, g; v is the total volume after dissolving the sample with trichloroacetic acid.

[0056] Determination of degree of hydrolysis: The ratio of ammonia peptide nitrogen to total nitrogen is an important indicator for measuring the degree of hydrolysis of a sample, especially in protein and amino acid analysis. This ratio is usually used to evaluate the degree of protein hydrolysis. Ammonia nitrogen is determined by the formaldehyde titration method, and total nitrogen is determined by the Kjeldahl method.

[0057]

[0058] In the formula: DH is the degree of hydrolysis, %; V0 is the volume of the sodium hydroxide standard solution consumed after adding formaldehyde in the blank test, mL; V1 is the volume of the sodium hydroxide standard solution consumed after adding formaldehyde in the determination of the sample, mL; V2 is the total volume of the hydrolysis solution, mL; V3 is the volume of the hydrolysis solution used for titration, mL; c is the concentration of the sodium hydroxide standard solution, mol / L; m is the total amount of amino nitrogen in the hydrolysis solution (Kjeldahl method), g.

[0059] Determination of peptide molecular weight distribution: The chromatographic column is TSK-gel G2000SWxl (7.8 mm × 30 mm), the mobile phase is acetonitrile: water: trifluoroacetic acid = 40:60:0.1 (v / v), the column temperature: 30 °C, the flow rate: 0.5 mL / min; the detection wavelength: 220 nm; the injection volume: 10 μL. The sample mass concentration is 10 mg / mL, and it is filtered with a 0.45 μm organic filter membrane before injection. Prepare a standard curve by configuring different relative molecular mass peptide standard solutions with the mobile phase into 2 mg / ml, mixing them in equal proportions, and then filtering with an organic filter membrane and injecting. Plot with the logarithm of the relative molecular weight of the peptide standard as the abscissa x and the retention time as the ordinate y, and obtain the calibration curve through data fitting. The corresponding fitting equation is as follows:

[0060] Y = 0.004704418x 3 - 0.2269086x 2 + 3.344785x - 11.50635

[0061] R2 = 0.0000342, R = 0.9999671

[0062] As the molecular weight decreases, the retention time becomes longer. It is divided into eight intervals: > 10000 Da, 5000 - 10000 Da, 3000 - 5000 Da, 2000 - 3000 Da, 1000 - 2000 Da, 500 - 1000 Da, 180 - 500 Da, and < 180 Da. The corresponding retention times are 12.774, 14.227, 15.039, 15.636, 16.632, 17.686, and 20.073 min respectively. Substituting the chromatographic data of the sample into the above calibration curve calculation equation, the molecular weight of the sample can be obtained.

[0063] Neutral protease hydrolysis in Example 1

[0064] (1) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to make a slurry; under the condition of adjusting the initial pH to 7, carry out enzymatic hydrolysis at a temperature of 45 °C for 12.0 h, with a total addition amount of neutral protease of 4000 U. After enzymatic hydrolysis, boil the solution for 15 min to inactivate the enzyme, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolyzate.

[0065] (2) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, mix it with water at a ratio of 1:4 to make a slurry; under the condition of adjusting the initial pH to 7, carry out enzymatic hydrolysis at 35, 40, 45, 50, 55, and 60 °C for 8 h respectively, with a total addition amount of neutral protease of 4000 U. After enzymatic hydrolysis, boil the solution for 15 min to inactivate the enzyme, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolyzate.

[0066] (3) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to make a slurry; under the condition of adjusting the initial pH to 7, at a temperature of 45 °C, add 1000, 2000, 3000, 4000, 5000, and 6000 U of neutral protease respectively, carry out enzymatic hydrolysis for 8 h, after enzymatic hydrolysis, boil the solution for 15 min to inactivate the enzyme, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolyzate.

[0067] The experimental results of neutral protease hydrolysis are as Figure 1 shown: As Figure 1 (a) shows, when the enzymatic hydrolysis time is 8 h, the protein transfer rate reaches 54.37%. As Figure 1 (b) shows, when the enzymatic hydrolysis temperature is 45 °C, the solid-phase protein is at a lower value of 3.88 g, and the liquid-phase protein is at a higher value of 4.97 g. At this time, the protein transfer rate reaches 54.41%. As Figure 1As shown in (c), when the enzyme addition amount is 5000 U, the solid-phase protein mass is 3.75 g, and the liquid-phase protein mass is 5.11 g. At this time, the protein transfer rate is 55.95%.

[0068] Example 2 Hydrolysis with Alkaline Protease

[0069] (1) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; under the condition of adjusting the initial pH to 11, enzymatically hydrolyze at 50 °C for 12.0 h, with a total addition amount of alkaline protease of 8000 U. After enzymatic hydrolysis, boil the solution for 15 min to inactivate the enzyme, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolysis solution.

[0070] (2) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, mix it with water at a ratio of 1:4 to adjust the slurry; under the condition of adjusting the initial pH to 11, enzymatically hydrolyze at 40, 45, 50, 55, 60, and 65 °C for 10 h respectively, with a total addition amount of alkaline protease of 8000 U. After enzymatic hydrolysis, boil the solution for 15 min to inactivate the enzyme, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolysis solution.

[0071] (3) After sieving the meat and bone meal after high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; under the condition of adjusting the initial pH to 11 and at a temperature of 55 °C, add 2000, 4000, 6000, 8000, 10000, and 12000 U of alkaline protease respectively, enzymatically hydrolyze for 10 h, boil the solution for 15 min to inactivate the enzyme after enzymatic hydrolysis, cool it, centrifuge to collect the liquid phase, and remove the grease layer and emulsion layer after stratification to obtain the hydrolysis solution.

[0072] The experimental results of hydrolysis with alkaline protease are as Figure 2 shown: As Figure 2 shown in (a), when the enzymatic hydrolysis time is 10 h, the protein transfer rate reaches 64.03%; as the time continues to extend, the protein transfer rate does not increase significantly. As Figure 2 shown in (b), when the temperature is fixed at 55 °C, the protein transfer rate reaches 67.8%. As Figure 2 shown in (c), when the enzyme addition amount is 8000 U, at this time, the solid-phase and liquid-phase protein masses are 2.60 g and 6.29 g respectively, and the protein transfer rate reaches the highest; at an enzymatic hydrolysis time of 10 h, a temperature of 55 °C, and an enzyme addition amount of 8000 U, the protein transfer rate is 67.8%.

[0073] Example 3 Hydrolysis with Composite Enzyme

[0074] (1) Complex enzyme hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; adjust the initial pH = 9; set different volume ratios of the two enzymes, and combine alkaline protease with proportions of (10, 30, 50, 70, 90%) to form a complex enzyme. The total enzyme amount of the complex enzyme is 10,000 U, and hydrolyze for 10 h at a temperature of 50 °C. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution; measure the protein mass in the solid and liquid phases and calculate the protein transfer rate.

[0075] (2) Complex enzyme hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; set different pH values (7, 8, 9, 10, and 11); combine alkaline protease with a proportion of 70% to form a complex enzyme. The total enzyme amount of the complex enzyme is 10,000 U, and hydrolyze for 10 h at a temperature of 50 °C. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0076] (3) Complex enzyme hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; set the initial pH = 10; combine alkaline protease with a proportion of 70% to form a complex enzyme. The total enzyme amount of the complex enzyme is 10,000 U. Under the condition of a temperature of 50 °C, set different hydrolysis times (6.0, 8.0, 10.0, 12.0, and 14.0 h). After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0077] (4) Complex enzyme hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; set the initial pH = 10; combine alkaline protease with a proportion of 70% to form a complex enzyme. The total enzyme amount of the complex enzyme is 10,000 U. Hydrolyze for 10 h at temperatures of (40, 45, 50, 55, and 60 °C) respectively. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0078] (5) Complex enzyme hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through a 60-mesh sieve, take 15 g of the substrate and mix it with water at a ratio of 1:4 to adjust the slurry; set the initial pH = 10; combine alkaline protease with a proportion of 70% to form a complex enzyme. With different total enzyme addition amounts (4000, 8000, 10000, 12000, and 15000 U), hydrolyze for 10 h at a temperature of 50 °C. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0079] The results of the above five groups of experiments are as Figure 3 、 Figure 4As shown, as the proportion of alkaline protease increases, the solid-phase protein first decreases and then increases, while the change of liquid-phase protein is opposite. When the proportion of alkaline protease reaches 50%, the protein transfer rate is 66.96%. When the proportion of alkaline protease continues to increase to 70%, the protein transfer rate optimally reaches 73.23%.

[0080] As Figure 4 shown, when the pH is 9 - 10, the enzymolysis time is not less than 10 h, the temperature is 45 - 55 °C, and the total enzyme addition amount is 10000 - 15000 U, the protein transfer rate is relatively high. Among them, when the volume ratio of alkaline protease to neutral protease is 7:3, the total addition amount of the composite enzyme is 12000 U, and the enzymolysis is carried out for 10 h at pH = 10 and a temperature of 50 °C, at this time the protein transfer rate reaches the highest value of 76.42%.

[0081] Compared with single-enzyme hydrolysis, the hydrolysis effect of the composite enzyme in this application is significantly better than that of single-enzyme hydrolysis, and the conditions are milder.

[0082] Example 4

[0083] (1) Preliminary hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through 60 meshes, mix and adjust the slurry with water at a ratio of 1:4; adjust the initial pH = 9; combine alkaline protease with a proportion of 70% to form a composite enzyme, with a total addition amount of the composite enzyme of 12000 U, and hydrolyze for 10 h at a temperature of 50 °C. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0084] (2) Deep hydrolysis: Spray-dry the preliminary hydrolysis solution to obtain the preliminary hydrolyzed protein powder. After dissolving it in water, add aminopeptidase for enzymolysis, with a substrate concentration of 250 g / L. Enzymolyze for 150 min by adding 1.5 g / L aminopeptidase at 40 °C under the conditions of pH being 5, 6, 7, 8, 9, and 10 respectively; obtain the deep hydrolysis solution after inactivating the enzyme;

[0085] (3) Preparation of chelated fertilizer, the same as in Example 3

[0086] As Figure 5 shown: when the pH is 7 - 9, the content of free amino acids is relatively high; when the pH is 8, the content of dissolved peptides is the highest, and at this time the contents of leucine and isoleucine are also relatively high.

[0087] Example 5

[0088] (1) Preliminary hydrolysis: After sieving the meat and bone meal treated by high-temperature harmless treatment through 60 meshes, mix and adjust the slurry with water at a ratio of 1:4; adjust the initial pH = 9; combine alkaline protease with a proportion of 70% to form a composite enzyme, with a total addition amount of the composite enzyme of 12000 U, and hydrolyze for 10 h at a temperature of 50 °C. After inactivating the enzyme, centrifuge to collect the liquid phase, and remove the grease layer and the emulsion layer after stratification to obtain the preliminary hydrolysis solution.

[0089] (2) Deep hydrolysis: The preliminarily hydrolyzed solution was spray-dried to obtain preliminarily hydrolyzed protein powder. After dissolving in water, aminopeptidase was added for enzymatic hydrolysis. The substrate concentration was 250 g / L, and under the condition of pH 8, at enzymatic hydrolysis temperatures (25, 30, 35, 40, 45, and 50 °C), 1 g / L aminopeptidase was added for enzymatic hydrolysis for 150 min; after inactivating the enzyme, a deeply hydrolyzed solution was obtained.

[0090] (3) The preparation steps of the chelated fertilizer were the same as those in Example 3.

[0091] As Figure 6 (a) shows: IC represents the sample solution before deep enzymatic hydrolysis. At this time, the soluble amino acid peptide content was only 85.48 g / L, and the total amino acid content was 4014 mg / L. After adding aminopeptidase for hydrolysis, both the soluble peptide content and the amino acid content increased to varying degrees. With the increase in temperature, it showed a trend of first rising and then falling. However, after exceeding 40 °C, the enzyme activity might be affected, resulting in a sharp decline in the soluble peptide content. From Figure 6 (b), it can be seen that at 40 °C, the total amino acid content was 7940 mg / L. Among them, the phenylalanine content was the highest, reaching 1230 mg / L, about 2.17 times that of the phenylalanine content in the IC group. The glutamic acid content was 691 mg / L, about 2.06 times that of the glutamic acid content in the IC group.

[0092] Example 6

[0093] The experimental steps (1) and (3) were the same as those in Example 3, and the difference was in step (2);

[0094] (2) Deep hydrolysis: The preliminarily hydrolyzed solution was spray-dried to obtain preliminarily hydrolyzed protein powder. After dissolving in water, aminopeptidase was added for enzymatic hydrolysis. The substrate concentration was 250 g / L, and under the condition of pH 8, at the enzymatic hydrolysis temperature (40 °C), with different enzyme addition concentrations (0.25, 0.50, 1.0, 1.50, and 2.50 g / L), the corresponding enzyme activity addition amounts were 30 - 300 U, and aminopeptidase was used for enzymatic hydrolysis for 150 min; after inactivating the enzyme, a deeply hydrolyzed solution was obtained.

[0095] As Figure 7 shown: When the enzyme concentration was 1 - 1.5 g / L, it had a better effect.

[0096] Example 7

[0097] The experimental steps (1) and (3) were the same as those in Example 3, and the difference was in step (2)

[0098] (2) Deep hydrolysis: The preliminarily hydrolyzed solution was spray-dried to obtain preliminarily hydrolyzed protein powder. After dissolving it in water, aminopeptidase was added for enzymatic hydrolysis. The substrate concentration was 250 g / L. Under the condition of pH 8, different reaction times (30, 60, 90, 120, 150, and 180 min) were set. 1 g / L of aminopeptidase was added and the enzymatic hydrolysis was carried out at 40 °C. After inactivating the enzyme, a deeply hydrolyzed solution was obtained.

[0099] As Figure 8 shown: The dissolved peptide content and free amino acid composition were measured. The results are as Figure 8 (a) shows that the dissolved peptide content reached the highest (116.26 g / L) at 120 min.

[0100] When hydrolyzed with 1 g / L of aminopeptidase at an enzymatic hydrolysis time of 120 min, a temperature of 40 °C, and a pH of 8, the total amounts of dissolved peptides and free amino acids reached 116.26 g / L and 8235 mg / L, respectively. The final pH of the water-soluble fertilizer was 7.1. Peptides with a molecular weight of 180 - 500 Da accounted for 62.1%, and components with a molecular weight less than 180 Da accounted for 12.9%. The total phosphorus content was 0.372 g / L, and the total potassium content was 0.557 g / L. The contents of trace elements iron, manganese, zinc, copper, and boron were 22.075 mg / L, <0.001 mg / L, 3.289 mg / L, 0.202 mg / L, and 0.798 mg / L, respectively.

[0101] Experimental example - Spinach potted plant experiment

[0102] The experiment was carried out in a constant-temperature room, and spinach was planted by soil cultivation. Conventional base fertilizer (N:P:K = 10:10:15) was applied before transplanting the seedlings. After the transplantation was stable, the plants were fixed, and the planting days were 60 days.

[0103] The chelate solution (Animal protein deep enzymatic hydrolysis solution peptide chelate fertilizer, ADPCF) prepared with an enzymatic hydrolysis time of 120 min in Example 7 was compared with a commercial animal peptide fertilizer (CAPF) in a certain city. They were respectively diluted to a concentration of 300 mg / L for spraying. CK was used as a water control group. Spraying was carried out once every 7 days, 100 mL each time.

[0104] The results are shown in Table 1-2 below; in terms of spinach quality, compared with the CAPF group, the maximum leaf length of the ADPCF group is 0.4 cm more, and the plant height is about 1 cm higher. In addition, the soluble protein content of the ADPCF group is about 0.85 mg / g higher than that of the CAPF group; the short peptide molecular weight in the ADPCF group is small and can be quickly absorbed by plants through the peptide transport carrier on the cell membrane, enabling plants to obtain the raw materials required for protein synthesis in a shorter time, thus promoting the synthesis of soluble protein.

[0105] In terms of soil nutrients, the organic matter content and nitrate nitrogen content of the ADPCF group are significantly higher than those of the CAPF group. This indicates that zinc and iron in the ADPCF group of the present invention form a more stable structure with small peptide segments, promoting the mineralization process of minerals and providing a nitrogen source after the release of minerals, thereby increasing the nitrate content in spinach. In contrast, the effect of long peptide segments in the CAPF group is slightly insufficient. The alkaline protease activity of the ADPCF group is higher; in summary, applying the ADPCF fertilizer of the present invention has a positive impact on the growth of spinach and soil properties.

[0106] The basic properties of the commercially available peptide fertilizer are as follows: pH = 4.9, dissolved peptide content 38 g / L, the proportion of peptides with a molecular weight of 180 - 500 Da ≥ 35%; and the proportion of components with a molecular weight less than 180 Da ≥ 7%, total phosphorus content 0.031 g / L, total potassium content 0.231 g / L; the contents of trace elements iron, manganese, zinc, copper, and boron are 0.215 mg / L, <0.001 mg / L, <0.001 mg / L, 0.140 mg / L, and 1.939 mg / L respectively.

[0107] Table 1 Changes in the growth morphology and nutrient index content of spinach after applying the amino acid - peptide fertilizer of the present invention

[0108]

[0109] Table 2 Effects on soil nutrients and soil enzyme activities after applying the amino acid - peptide fertilizer of the present invention

[0110]

[0111] In summary, the present invention relates to a method for preparing a liquid amino acid-peptide water-soluble fertilizer using animal processing waste. First, a composite enzymatic hydrolysis with neutral protease and alkaline protease is adopted to significantly improve the protein transfer rate, and the protein transfer rate reaches the highest value of 76.42%. The content of dissolved peptides in the amino acid-peptide water-soluble fertilizer prepared by the present invention can reach 116.26 g / L, and the total amount of free amino acids can reach 8235 mg / L. The water-soluble fertilizer prepared by the present invention has a high proportion of small molecule peptides and amino acids, among which the proportion of oligopeptides with a molecular weight of 180-500 Da can reach 62.1%; the proportion of components with a molecular weight less than 180 Da can reach 12.9%. The water-soluble fertilizer prepared by the present invention can promote the growth of spinach and enhance the soil enzyme activity, and is more conducive to the best balance between spinach absorption and soil improvement compared with the long peptide segment fertilizer.

[0112] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the present invention.

Claims

1. A method for preparing amino acid peptide water-soluble fertilizer using waste animal protein, characterized in that, It includes the following steps: S1. Preliminary hydrolysis: Take the waste animal offal, sieve it, add water to make a slurry, adjust the pH value, add a complex enzyme for enzymatic hydrolysis, inactivate the enzyme, then centrifuge to collect the liquid phase, and remove the oil layer and the emulsion layer after stratification to obtain the preliminary hydrolyzate; S2. Deep hydrolysis: Spray-dry the preliminary hydrolyzate obtained in step S1 to obtain the preliminary hydrolyzed protein powder, dissolve it in water, add aminopeptidase for enzymatic hydrolysis, and obtain the deep hydrolyzate after inactivating the enzyme; S3. Chelated fertilizer preparation; add Fe 2+ and Zn 2+ into the deep hydrolysis solution, stir for chelation, and remove the precipitate to obtain the liquid amino acid-peptide water-soluble fertilizer.

2. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, characterized in that, In step S1, the waste animal offal is mixed with water at a ratio of 1:2 - 1:8 to make a slurry.

3. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, wherein, In step S1, for every 15 g of substrate, the total enzyme activity of the complex enzyme added is 10,000 - 15,000 U, and the ratio of the added enzyme activities of alkaline protease and neutral protease is 1:1 - 9:

1.

4. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, characterized in that, In step S1, the pH is 7 - 11, the enzymatic hydrolysis temperature is 45 - 55 °C; the enzymatic hydrolysis time is 8 - 14 h.

5. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, characterized in that, In step S2, the enzymatic hydrolysis conditions are: the substrate concentration is 50 g / L - 100 g / L; the pH is 6 - 8.

6. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, characterized in that, In step S2, the addition amount of aminopeptidase is 1.25 - 2.50 g / L; the enzymatic hydrolysis temperature is 30 - 45 °C; the enzymatic hydrolysis time is 1.5 - 3 h.

7. The method for preparing amino acid peptide water-soluble fertilizer by using recycled waste animal protein according to claim 1, wherein In step S3, the content of trace elements of zinc and iron is ≥ 20 g / L.

8. The amino acid peptide water-soluble fertilizer prepared by the method according to any one of claims 1-7, characterized in that, The proportion of oligopeptides with a molecular weight of 180 - 500 Da is ≥ 60%; and the proportion of components with a molecular weight less than 180 Da is ≥ 12%.

9. The amino acid peptide water-soluble fertilizer according to claim 8, characterized in that The content of dissolved peptides is 100 - 120 g / L.

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