Method for preparing amino acid water-soluble fertilizer from waste animal blood and application of amino acid water-soluble fertilizer

Abstract

The invention belongs to the field of fertilizers, and particularly relates to a method for preparing an amino acid water-soluble fertilizer from waste animal blood and application of the amino acid water-soluble fertilizer. Waste animal blood and a microbial agent are combined and fermented to prepare the waste animal blood microbial agent, and the microbial agent is composed of bacillus pumilus, bacillus subtilis, aspergillus oryzae and pichia pastoris. The bacillus pumilus, the bacillus subtilis, the aspergillus oryzae and the pichia pastoris are combined for use, so that the degradation rate of blood can be remarkably increased, the breakage of peptide bonds in macromolecular proteins is effectively promoted, and more types of polypeptides are generated; as an amino acid water-soluble fertilizer, the amino acid water-soluble fertilizer can be more easily utilized by plants, so that the amino acid water-soluble fertilizer participates in multiple life processes such as plant growth, development and stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of fertilizers, specifically relating to a method for preparing amino acid water-soluble fertilizer using waste animal blood and its application. Background Technology

[0002] In recent years, with the rapid development of the livestock industry, global livestock and poultry production has been increasing. During processing, apart from a small amount of blood resources being utilized, most slaughter blood is not effectively utilized and is directly discharged into the environment, causing water and soil pollution, foul odors, and animal diseases. Furthermore, slaughter blood contains a large amount of protein resources, but due to technical issues, its utilization rate is low, resulting in a waste of protein resources. Currently, the main directions for the resource utilization of livestock and poultry slaughter blood are fivefold: feed utilization, energy utilization, food utilization, pharmaceutical utilization, and fertilizer utilization.

[0003] Blood fertilization refers to the process of producing fertilizer from blood through hydrolysis or bio-fermentation. Because blood proteins have large molecular weights, they cannot be directly absorbed by plants. Only through hydrolysis or bio-fermentation can they be broken down into smaller peptides or amino acids for plant utilization. Common methods include chemical hydrolysis and bio-fermentation. Using biodegradation processes such as fermentation or enzymatic methods to produce amino acid fertilizers avoids environmental pollution, preserves the types of amino acids in the degradation products, protects beneficial amino acids for plants, results in higher oligopeptide content, and generates fewer harmful substances during degradation. Therefore, using microorganisms to degrade waste blood to produce water-soluble fertilizers containing amino acids can achieve comprehensive resource recycling while protecting the environment. Many researchers have isolated different microorganisms from various matrices, including Aspergillus oryzae and Bacillus, which degrade blood to varying degrees. Compared to the raw material before fermentation, the blood fermentation broth shows increased amino acid content and soluble protein, with a richer variety of amino acids. However, due to the complex and sophisticated spatial structures of hemoglobin and fibrin in blood, single-bacterial formulations are difficult to completely degrade and utilize blood. Therefore, extensive experiments are needed to screen out multiple combinations of microorganisms that can effectively degrade bovine blood, and the synergistic effect between different bacterial species is necessary to achieve full degradation and comprehensive utilization of blood proteins.

[0004] This study found that combining Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris can significantly improve the degradation rate of blood, effectively promote the breaking of peptide bonds in macromolecular proteins, and generate a variety of peptides; there are no related reports in the existing technology. Summary of the Invention

[0005] This invention provides a method for preparing amino acid water-soluble fertilizer using waste animal blood and its application. The specific technical solution is as follows: A method for preparing amino acid water-soluble fertilizer using waste animal blood involves fermenting the waste animal blood with a microbial agent. The microbial agent consists of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris. The animal blood can be bovine, pig, chicken, sheep, etc.

[0006] Preferably, the mass ratio of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris is 1-5:1-5:1-5:1-5. Specifically, the mass ratio of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris is 1:1:1:1.

[0007] Preferably, the concentrations of *Bacillus pumilus*, *Bacillus subtilis*, *Aspergillus oryzae*, and *Pichia pastoris* are 5-15 billion / g, 1-10 billion / g, and 1-10 billion / g, respectively. Specifically, the concentrations are 10 billion / g for *Bacillus pumilus*, 10 billion / g for *Bacillus subtilis*, 5 billion / g for *Aspergillus oryzae*, and 5 billion / g for *Pichia pastoris*.

[0008] The amino acid water-soluble fertilizer provided by this invention is used for plant applications, especially for promoting plant growth, development, and stress resistance.

[0009] Compared with the prior art, the present invention has the following beneficial effects: The synergistic effect between different bacterial species is used to achieve full degradation and comprehensive utilization of blood proteins. Combining Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris can significantly improve the degradation rate of blood proteins and effectively promote the breaking of peptide bonds in macromolecular proteins. Detailed Implementation

[0010] The specific embodiments of the present invention will be further explained below with reference to specific examples.

[0011] Example 1: Selection of Microbial Inoculants Take a 1000mL Erlenmeyer flask, add 300mL of fresh bovine blood and 30g of microbial inoculant, and incubate in a shaker at 45℃ for 96h. Take an appropriate amount of sample and determine the degree of hydrolysis of blood proteins and the statistical peptide length.

[0012] Test method: Degradation rate determination (Reference: Ma Xianying, Screening of strains degrading bovine blood proteins and study on their enzyme production characteristics): The amino nitrogen content of the degradation solution was determined by the formaldehyde value method, and the total nitrogen content of the substrate was determined by the Kjeldahl method. Then, the substrate degradation rate was calculated according to the following formula: DH = A / N × 100%, where DH - degradation degree of degradation solution (%), A - amino acid nitrogen content in degradation solution (mg), and N - total nitrogen content of substrate (mg). Peptide detection (Reference: Ma Xianying, Screening of strains degrading bovine blood proteins and study on their enzyme production characteristics): Weigh 10 mg of lyophilized blood protein fermentation broth, add 500 μL of 100 mM NH4HCO3 solution, shake for 20 min, centrifuge at 12000 r / min, 4℃ for 20 min, and take 200 μL of the supernatant for peptide reductive alkylation. The reductively alkylated sample is centrifuged at 12000 r / min for 20 min using a 10 KD ultrafiltration tube, and the filtrate is collected and dried. Take the dried sample, add 20 μL for desalting and dry again. The dried peptide is reconstituted with 20 μL of loading buffer (2% acetonitrile and 0.1% formic acid mixture) and added to a sample vial for peptide determination. Chromatographic column: C18 reversed-phase analytical column; ion source: nanoESI. Test results: Blood degradation rate test results Table 1: Effects of Microbial Inoculant Type on Bovine Blood Fermentation Degradation

[0013] Note: Bacillus pumilus (10 billion / g), Bacillus subtilis (10 billion / g), Aspergillus oryzae (5 billion / g), Pichia pastoris (5 billion / g) The ability of different bacterial strains to degrade blood proteins is determined by the quantity and activity of their own proteases. Hemoglobin and fibrin in livestock and poultry blood have complex spatial structures and complex physicochemical factors. Furthermore, the utilization capacity of existing single bacterial strains of blood proteins is limited, resulting in incomplete hydrolysis by proteases. Table 1 shows that optimizing the bacterial strain formulation to prepare a compound bacterial agent can effectively improve the ability of the strains to utilize blood. Combining Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris can significantly improve the degradation rate of bovine blood by the microbial agent.

[0014] Table 2: Statistics on peptide lengths before and after bovine blood protein degradation

[0015] Table 2 shows that bovine blood contains abundant macromolecular peptides, with a significant number of peptides longer than 9. After the addition of microbial strains, these macromolecular peptides in the blood are broken down into smaller peptides and even amino acids. In particular, treatment 1, which combines *Bacillus pumilus*, *Bacillus subtilis*, *Aspergillus oryzae*, and *Pichia pastoris*, effectively promotes the breaking of peptide bonds in macromolecular proteins, resulting in a greater variety of polypeptides. Furthermore, as an amino acid water-soluble fertilizer, it is more readily utilized by plants, thus participating in many life processes such as plant growth, development, and stress resistance.

Claims

1. A method for preparing amino acid water-soluble fertilizer using waste animal blood, characterized in that, The microbial agent is prepared by fermenting waste animal blood with a combination of microbial agents, which consists of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris.

2. The method according to claim 1, characterized in that, The mass ratio of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae and Pichia pastoris is 1-5:1-5:1-5:1-5.

3. The method according to claim 2, characterized in that, The mass ratio of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris is 1:1:1:

1.

4. The method according to any one of claims 1-3, characterized in that, The concentrations of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris were 50-150 billion / g, 50-150 billion / g, 10-100 billion / g, and 10-100 billion / g, respectively.

5. The method according to claim 4, characterized in that, The concentrations of Bacillus pumilus, Bacillus subtilis, Aspergillus oryzae, and Pichia pastoris were 10 billion / g, 10 billion / g, 5 billion / g, and 5 billion / g, respectively.

6. The use of the amino acid water-soluble fertilizer prepared according to any one of claims 1-5 for its intended function.

7. The use according to claim 6, characterized in that, It is used to promote plant growth, development, and stress resistance.

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