A kind of wool amino acid water-soluble fertilizer and preparation method thereof

Abstract

The present application provides a wool amino acid water-soluble fertilizer and a preparation method thereof, which are applied in the field of fertilizer technology. A safe and efficient keratin-degrading strain is found to be used in combination with keratinase, thereby achieving a wool fermentation method with high wool degradation efficiency, simple and easy fermentation operation (temperature, pH value and other parameters are not demanding, and the culture medium components are simple), scalable production, rich amino acid types and high yield, and the fermentation liquid can directly produce wool amino acid liquid water-soluble fertilizer, which is applied in agricultural production.

Description

Technical Field

[0001] The present application relates to the technical field of fertilizers, and in particular to a wool amino acid water-soluble fertilizer and a preparation method thereof. Background Art

[0002] With the rapid development of the breeding industry and the prosperity of my country's leather manufacturing industry, the amount of wool waste is increasing. According to authoritative statistics, my country produces more than 4 million tons of keratin waste every year. These wastes have not been effectively utilized due to their processing difficulty and poor degradability. A large amount of untreated waste not only seriously pollutes the environment, but also is a huge waste of resources.

[0003] Amino acids, as the smallest molecules that make up proteins, have significant advantages in fertilizer applications. They are easily absorbed by crops, helping to improve crop disease resistance and crop quality. In particular, free amino acids in fertilizers can be directly absorbed by plants, thereby improving fertilizer utilization efficiency and crop nutrient conversion rates, which is of great significance to plant growth. However, current methods for producing amino acid liquid fertilizers, such as fermenting soybean meal and fish meal, acid hydrolysis of dead livestock and poultry, and enzymatic hydrolysis of feathers and blood, are not only costly, but also pose a risk of secondary pollution, limiting the possibility of their large-scale application.

[0004] Scientific research has proven that microbial and enzyme degradation is an economical and effective method for treating animal keratin. This method not only minimizes the loss of amino acids, but also produces products that are rich in bacteria, a variety of bioactive substances and microbial metabolites, and low in harmful metal ions. Microorganisms degrade keratin by synthesizing keratinase, and the process includes three main steps: denaturation, hydrolysis and transamination. Currently, more than 30 microorganisms are known to have the ability to degrade keratin, mainly including bacteria, fungi and actinomycetes, such as Bacillus licheniformis, thermophilic bacteria, Vibrio bacteria, Streptomyces and Cephalosporium longifolium.

[0005] Wool that has been degraded by microorganisms can retain its rich amino acid components and be used as amino acid fertilizer. Not only will it not accumulate inorganic nitrogen in the soil, but the benefits of nitrogen circulation will be more significant, effectively avoiding the environmental pollution problems that may be caused by traditional chemical fertilizers.

[0006] For example, in CN105733999A, a crude enzyme solution obtained by fermentation culture of a strain of Bacillus subtilis can be used to degrade wool and feathers, and has potential application value in livestock and poultry waste degradation and biological dehairing.

[0007] CN106495839A, wool is soaked in ethanol and then treated with sodium bicarbonate, and then the pretreated wool and fermented bean curd are mixed and fermented to prepare foliar fertilizer.

[0008] "Effects of different microbial agents on wool scrap composting", screening the best microbial agent for wool scrap composting fermentation.

[0009] "Screening and identification of wool keratin-degrading bacteria and optimization of liquid fermentation conditions", three strains with wool keratin-degrading function were screened and obtained.

[0010] Although the research on microbial degradation of wool is mainly focused on the production of keratinase and protein feed, the complexity of the existing process flow, the harshness of fermentation conditions (strict control of temperature, pH value, culture medium and other parameters), the extended cycle, the limited amount of wool added, and the insufficient amino acid production have limited its promotion in practical applications. Therefore, the development of a safe and efficient application technology for keratin degradation strains and a simple and easy fermentation process is the key to realizing the production of amino acid fertilizers from biodegradable wool. Summary of the invention

[0011] In view of the wool waste treatment problem mentioned in the prior art, as well as the high production cost and secondary pollution risk of amino acid liquid fertilizer, the present invention aims to provide an innovative wool amino acid water-soluble fertilizer and a preparation method thereof. The method not only has high wool degradation efficiency, but also has simple fermentation operation (temperature, pH value and other parameters are not demanding, and the culture medium composition is simple), is easy to scale production, and can produce water-soluble fertilizer with rich amino acid types and high yield.

[0012] The core of the present invention is to screen and apply one or more safe and efficient keratin-degrading strains. The combined use of these strains and keratinase can significantly improve the degradation efficiency of wool protein and convert it into abundant free amino acids. Through a specific fermentation process, these amino acids can be effectively extracted and converted into water-soluble fertilizers for use in agricultural production.

[0013] The embodiments of this specification provide the following technical solutions:

[0014] A wool amino acid water-soluble fertilizer is prepared by the following method: a fermented product containing wool is sterilized at high temperature, a bacterial suspension is inoculated and fermented for a period of time, keratinase is added to continue fermentation, and the obtained fermentation liquid is filtered to obtain the wool amino acid water-soluble fertilizer.

[0015] Furthermore, the fermented product is obtained by adding wool powder into the fermentation medium.

[0016] Furthermore, 5-15 g wool powder is added to every 100 mL fermentation medium; preferably 10 g wool powder.

[0017] Furthermore, the bacterial suspension is a bacterial suspension of Bacillus licheniformis, with a preservation number of CGMCCNO.17458 and or Bacillus subtilis, with a preservation number of CCTCC NO: M 2017495; preferably a bacterial suspension of Bacillus subtilis, with a preservation number of CCTCC NO: M 2017495.

[0018] Furthermore, the fermentation medium is: 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g sodium chloride, and water is added to make the volume to 1 L, with a pH value of 7.0.

[0019] Furthermore, the bacterial suspension concentration was 10 6- 10 10 (CFU / ml); preferably 10 7- 10 9 (CFU / ml); most preferably 10 8 (CFU / ml).

[0020] Furthermore, the inoculation amount is 1-5% by volume of the fermentation product; preferably 1-4%; preferably 1-3%; and most preferably 2%.

[0021] Furthermore, keratinase is added after fermentation for 12-36 hours; preferably 15-35 hours; preferably 20-30 hours.

[0022] Furthermore, the entire fermentation cycle is 72-144 hours; preferably 72-120 hours; most preferably 96 hours.

[0023] Furthermore, 0.005-0.05 g of keratinase is added per gram of wool powder; preferably 0.008-0.03 g; preferably 0.008-0.02 g; preferably 0.01 g.

[0024] Furthermore, the fermentation temperature is 35-40°C, 100-300rpm.

[0025] Furthermore, the amino acid-containing water-soluble fertilizer may further include composite additives such as biochemical potassium humate, boric acid, chelated iron and magnesium sulfate.

[0026] Another aspect of the present application is to provide a method for preparing wool amino acid water-soluble fertilizer. The preparation method comprises sterilizing the fermented material containing wool at high temperature, inoculating the bacterial suspension, fermenting for a period of time, adding keratinase to continue fermentation, and filtering the obtained fermentation liquid to obtain the wool amino acid water-soluble fertilizer.

[0027] Another aspect of the present application is to provide an application of a wool amino acid water-soluble fertilizer, wherein the wool amino acid water-soluble fertilizer is applied as a fertilizer.

[0028] The applicant screened existing wool or feather keratin-degrading strains through experiments (screening was performed on strains that do not have infection problems in industrial applications) and sought to find a safe and efficient keratin-degrading strain to be used in combination with keratinase, thereby achieving a wool fermentation method with high wool degradation efficiency, simple and easy fermentation operation (temperature, pH value and other parameters are not stringent, and the culture medium composition is simple), scalable production, rich amino acid variety and high yield. The fermentation liquid can directly produce wool amino acid liquid water-soluble fertilizer, which is used in agricultural production.

[0029] The wool amino acid water-soluble fertilizer and preparation method thereof proposed by the present invention have the following significant beneficial effects based on a series of scientific experiments and data analysis:

[0030] 1) Improvement of keratin degradation efficiency: Fermentation with keratinase and specific strains can significantly increase the content of soluble protein. As can be seen from Table 1, most strains combined with keratinase have higher soluble protein content than keratinase or strains alone. Among them, the combination of bacteria 4 (Bacillus subtilis, deposit number CCTCC NO: M 2017495) and keratinase performed best in increasing the soluble protein content, reaching 602.03±23.21μg / mL, and Bacillus licheniformis, deposit number CGMCCNO.17458, also performed well, indicating that these two strains may be an efficient wool keratin degradation strain. The soluble protein content of keratinase alone was the lowest (312.03±12.54μg / mL), indicating that the activity of keratinase may be enhanced by the strain. There were differences in the soluble protein content among bacterial species, which may be related to the keratinase activity of different bacterial species or its synergistic effect with keratinase.

[0031] 2) Richness of amino acid species and increased yield: Keratin in wool is effectively broken down into a variety of free amino acids (17 free amino acids), including serine (Ser), glutamic acid (Glu), and arginine (Arg), which are essential for plant growth. The concentrations of serine (Ser), glutamic acid (Glu), and arginine (Arg) are relatively high, at 0.135 mg / mL, 0.236 mg / mL, and 0.153 mg / mL, respectively, indicating that these amino acids are released in large amounts during the fermentation process. The rich composition and high content of these amino acids not only prove the high efficiency of this method in amino acid output, but also provide comprehensive nutrients for crops.

[0032] 3) Significant effect on promoting crop growth. Amino acid water-soluble fertilizer can promote the growth of Chinese cabbage. The plant height and fresh weight of Chinese cabbage treated with amino acid water-soluble fertilizer were significantly higher than those of the control group (CK) (Table 3). The plant height and fresh weight of Chinese cabbage in treatment 1 (amino acid water-soluble fertilizer diluted 2 times) and treatment 2 (amino acid water-soluble fertilizer diluted 3 times) were higher than those in the control group, but the effect of treatment 1 was slightly better than that of treatment 2. This may indicate that there is a certain dose-effect relationship between the concentration of amino acid water-soluble fertilizer and the crop growth response. Amino acid water-soluble fertilizer can not only increase the biomass of crops, but also, because it contains a variety of essential amino acids, it may have a positive effect on improving crop nutrient absorption and enhancing crop disease resistance. Since amino acid water-soluble fertilizer is derived from biodegradable wool, the use of this fertilizer may reduce the burden on the environment and may have lower ecological risks compared with traditional chemical fertilizers. The data in Table 3 suggest the potential of amino acid water-soluble fertilizer in agricultural applications.

[0033] 4) Environmental friendliness and ecological benefits: The method of the present invention produces amino acid water-soluble fertilizer by biodegrading wool waste, which not only reduces the burden on the environment, but also has lower ecological risks compared with traditional chemical fertilizers. This environmentally friendly fertilizer production method is in line with the current development trend of sustainable development and green chemistry.

[0034] 5) Large-scale production and economic benefits: The fermentation process of the present invention is simple and easy (no strict temperature, pH value and other parameter requirements are required), low cost, and easy to mass produce. It is expected to reduce the market cost of amino acid water-soluble fertilizers and increase their popularity in agricultural production, thereby bringing significant economic benefits.

[0035] 6) Enhancement of crop nutrient absorption and disease resistance: Since the amino acid water-soluble fertilizer prepared by the present invention contains a variety of essential amino acids, the presence of these amino acids helps to improve the nutrient absorption capacity and disease resistance of crops, thereby improving the overall growth quality and yield of crops. DETAILED DESCRIPTION

[0036] The following describes the implementation methods of the present application through specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the contents disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. The present application can also be implemented or applied through other different specific implementation methods, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present application. It should be noted that, in the absence of conflict, the following embodiments and the features in the embodiments can be combined with each other. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present application.

[0037] Additionally, in the following description, specific details are provided to facilitate a thorough understanding of the examples. However, it will be understood by those skilled in the art that the examples can be practiced without these specific details.

[0038] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0039] Source of bacteria: The following bacteria were purchased or already available in the laboratory.

[0040] Bacteria 1: Bacillus licheniformis, deposit number CCTCC NO. M 2011319

[0041] Bacteria 2: Bacillus licheniformis, deposit number CGMCC NO. 17458

[0042] Bacteria 3: Bacillus subtilis, deposit number CGMCC NO. 12086

[0043] Bacteria 4: Bacillus subtilis, deposit number CCTCC NO: M 2017495

[0044] Bacteria 5: Bacillus subtilis, deposit number CCTCC NO:M 2017387

[0045] Bacteria 6: Lysobacter sp., deposit number CGMCC NO. 16072

[0046] Bacteria 7: Bacillus cereus, deposit number CGMCC NO.14221

[0047] Bacteria 8: Stenotrophomonas maltophilia, deposit number CGMCCNO.2231.

[0048] Keratinase: purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S25743.

[0049] Wool powder: Rinse the discarded wool that has not been treated with acid or alkali with tap water, 1×10 5 Pa was sterilized for 20 min, dried at 105 °C, crushed in a pulverizer and ground in a mortar to make wool powder.

[0050] The formula of the fermentation medium is: 0.5 g of potassium dihydrogen phosphate, 1 g of dipotassium hydrogen phosphate, 1 g of sodium chloride, add water to make up to 1 L, pH value 7.0.

[0051] Fermentation: Add 10 g wool powder to 100 mL fermentation medium.

[0052] Preparation of bacterial suspension: Use culture medium to activate the culture medium according to the conventional method, and resuspend each bacterial cell to a concentration of 1×10 8 (CFU / mL).

[0053] Wool fermentation steps:

[0054] The bioreactor was disinfected, and then the fermentation product was added. After high temperature sterilization, different groups of bacterial suspensions (10 8 CFU / ml), 37℃, 180rpm, add keratinase after 24 hours of fermentation (0.01g keratinase per gram of wool powder), and take samples after 96 hours to determine the content of soluble protein and free amino acids in the fermentation liquid. Each experiment was carried out in 3 parallel groups.

[0055] The soluble protein content was determined by Coomassie brilliant blue colorimetry, with bovine serum albumin as the standard protein and a measurement wavelength of 595 nm, and the soluble protein content (μg / mL) was calculated.

[0056] The decomposition liquid was obtained by filtration, and 5 ml of the decomposition liquid was centrifuged at 4°C and 8000 r / min for 15 min. 10% sulfosalicylic acid (10 g sulfosalicylic acid was dissolved in 100 mL distilled water) was uniformly mixed with the supernatant at a volume ratio of 4:1, and the mixture was stored in a refrigerator at 4°C for 1 h. The supernatant was centrifuged and filtered through a 0.22 μm filter membrane, diluted to an appropriate multiple, and then filtered through a filter membrane. The free amino acid content was detected by an amino acid analyzer.

[0057] Table 1 The content of soluble protein in fermentation broth of different groups

[0058] Group Soluble protein content (μg / mL) Bacteria 1+keratinase 450.76±18.32 Bacteria 2+ keratinase 520.43±32.45 Bacteria 3+ keratinase 500.12±26.87 Bacterial 4+ keratinase 602.03±23.21 Bacterial 5+ keratinase 480.87±15.64 Bacteria 6+keratinase 492.12±25.57 Bacteria 7+keratinase 460.31±20.85 Bacteria 8+keratinase 440.87±22.74 Keratinase alone 312.03±12.54 Solitary bacteria 1 410.76±20.45 Solitary bacteria 2 442.54±23.13 Single bacteria 3 450.87±29.64 Solitary bacteria 4 480.74±26.49 Single bacteria 5 430.87±30.65 Single bacteria 6 450.98±36.16 Solitary bacteria 7 400.43±21.45 Solitary bacteria 8 396.84±17.47

[0059] It can be concluded from Table 1 that the combination of keratinase and specific strains for fermentation can significantly increase the content of soluble protein. As can be seen from Table 1, most strains combined with keratinase have higher soluble protein content than keratinase alone or the strain itself. The combination of strain 4 (Bacillus subtilis, CCTCC NO: M2017495) and keratinase performed best in increasing the soluble protein content, with a content of 602.03±23.21 μg / mL, indicating that strain 4 may be an efficient keratin-degrading strain. The soluble protein content of keratinase alone was the lowest (312.03±12.54 μg / mL), indicating that the activity of keratinase may be enhanced by the strain. There are differences in the soluble protein content between strains, which may be related to the keratinase activity of different strains or its synergistic effect with keratinase.

[0060] Table 2 Composition and content of free amino acids in fermentation broth of different groups

[0061] type Concentration (mg / mL) Ser 0.135 Glu 0.236 Aspartic acid Asp 0.112 Valine 0.111 Glycine 0.082 Alanine Ala 0.106 Threonine 0.112 Tyrosine 0.101 Phenylalanine 0.088 Lysine 0.097 His 0.073 Arginine 0.153 Cysteine ​​cys 0.192 Isoleucine Ile 0.088 Leucine 0.125 MethionineMet 0.074 Proline Pro 0.125

[0062] It can be concluded from Table 2 that the keratin in wool is effectively decomposed into a variety of free amino acids (17 free amino acids) through the fermentation process. Amino acids are essential for plant growth and development. The concentrations of serine (Ser), glutamic acid (Glu) and arginine (Arg) are relatively high, 0.135 mg / mL, 0.236 mg / mL and 0.153 mg / mL, respectively, which indicates that these amino acids are released in large amounts during the fermentation process. The composition and content of amino acids show that the amino acid water-soluble fertilizer produced by microbial fermentation contains a variety of essential amino acids, which has a positive effect on improving crop nutrient absorption and enhancing crop disease resistance, and has the potential to be used as an amino acid water-soluble fertilizer.

[0063] Based on the above results, strain 4 was used to prepare wool amino acid water-soluble fertilizer.

[0064] The bioreactor was disinfected, the fermentation product was added, and after high temperature sterilization, the bacterial solution of Bacillus 4 was inoculated at a volume ratio of 2% (10 8 CFU / ml), 37℃, 180rpm, add keratinase after 24 hours of fermentation (0.01g keratinase per gram of wool powder). After 96 hours, the fermentation liquid is filtered through an 80-mesh sieve to obtain wool amino acid water-soluble fertilizer.

[0065] Remove the topsoil, take the soil from the 10cm tillage layer, dry it in the sun, crush it and mix it evenly, and then divide it into flower pots. Fertilize after the pakchoy seeds germinate and grow 2-3 true leaves. Fertilize 45ml per pot for each treatment, and CK is fertilized with the same amount of water. Fertilize 4 times in total, with an interval of 7 days each time. Take samples 40 days after sowing to measure the plant height and fresh weight of pakchoy.

[0066] There are three treatments in total, namely:

[0067] Clear water (CK).

[0068] Treatment 1: The amino acid water-soluble fertilizer obtained in Example 2 was diluted 2 times;

[0069] Treatment 2: The amino acid water-soluble fertilizer obtained in Example 2 was diluted 3 times.

[0070] Table 3 Effects of wool amino acid water-soluble fertilizer on the production of Chinese cabbage

[0071] Treatment Group Plant height / cm Fresh weight / g CK 8.52±0.53 1.26±0.21 Process 1 16.76±0.58 6.18±0.63 Process 2 15.65±0.85 5.85±0.72

[0072] From Table 3 above, it can be concluded that the plant height and fresh weight of pakchoi treated with amino acid water-soluble fertilizer were significantly higher than those of the control group (CK), indicating that amino acid water-soluble fertilizer can promote the growth of pakchoi. The plant height and fresh weight of pakchoi in treatment 1 (amino acid water-soluble fertilizer diluted 2 times) and treatment 2 (amino acid water-soluble fertilizer diluted 3 times) were higher than those in the control group, but the effect of treatment 1 was slightly better than that of treatment 2. This may indicate that there is a certain dose-effect relationship between the concentration of amino acid water-soluble fertilizer and crop growth response. Amino acid water-soluble fertilizer can not only increase crop biomass, but also, because it contains a variety of essential amino acids, it may have a positive effect on improving crop nutrient absorption and enhancing crop disease resistance. Since amino acid water-soluble fertilizer is derived from biodegradable wool, the use of this fertilizer may reduce the burden on the environment and may have lower ecological risks compared with traditional chemical fertilizers. The data in Table 3 suggest the potential of amino acid water-soluble fertilizer in agricultural applications.

[0073] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. A wool amino acid water-soluble fertilizer, characterized in that: The water-soluble fertilizer is prepared by the following method: a fermented product containing wool is sterilized at high temperature and inoculated with 10 7- 10 9 After a period of fermentation of a bacterial suspension with a concentration of 100 CFU / ml, keratinase is added to continue the fermentation. After the fermentation liquid is filtered, wool amino acid water-soluble fertilizer is obtained; The fermented product is a fermentation medium in which wool powder is added; Add 5-15g wool powder per 100mL fermentation medium; The fermentation medium is: 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g sodium chloride, add water to make up to 1 L, pH value 7.0; The bacterial suspension is a bacterial suspension of Bacillus subtilis, with a preservation number of CCTCC NO: M 2017495; The inoculation amount of the bacterial suspension is 1-4% of the volume ratio of the fermentation product; After the inoculated bacterial suspension has been fermented for 12-36 hours, keratinase is added; Add 0.008-0.03g of keratinase per gram of wool powder; The fermentation temperature of the inoculated bacterial suspension is 35-40°C, 100-300rpm; The keratinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S25743.

2. A wool amino acid water-soluble fertilizer according to claim 1, characterized in that: Biochemical potassium fulvic acid, boric acid, chelated iron and or magnesium sulfate additives are further added.

3. The method for preparing a wool amino acid water-soluble fertilizer according to claim 1, characterized in that: The preparation method is as follows: the fermented material containing wool is sterilized at high temperature and inoculated with 10 7- 10 9 After the bacterial suspension (CFU / ml) is fermented for a period of time, keratinase is added to continue the fermentation, and the obtained fermentation liquid is filtered to obtain wool amino acid water-soluble fertilizer; The fermented product is a fermentation medium in which wool powder is added. Add 5-15g wool powder per 100mL fermentation medium; The fermentation medium is: 0.5 g potassium dihydrogen phosphate, 1 g dipotassium hydrogen phosphate, 1 g sodium chloride, add water to make up to 1 L, pH value 7.0; The bacterial suspension is a bacterial suspension of Bacillus subtilis, with a preservation number of CCTCC NO: M 2017495; The inoculation amount of the bacterial suspension is 1-4% of the volume ratio of the fermentation product; After the inoculated bacterial suspension has been fermented for 12-36 hours, keratinase is added; Add 0.008-0.03g of keratinase per gram of wool powder; The fermentation temperature of the inoculated bacterial suspension is 35-40°C, 100-300rpm; The keratinase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number S25743.

4. The use of a wool amino acid water-soluble fertilizer according to claim 1, characterized in that: The wool amino acid water-soluble fertilizer is used as fertilizer.