Biostimulant compound fertilizer containing sea cucumber peptide and preparation method of biostimulant compound fertilizer

Abstract

The invention belongs to the field of agriculture, and particularly discloses a biostimulant compound fertilizer containing sea cucumber peptide and a preparation method thereof, the biostimulant compound fertilizer containing sea cucumber peptide comprises the following components by weight: 5-15 parts of a sea cucumber peptide compound metabolite, 10-20 parts of humic acid, 10-15 parts of potassium fulvate, 40-60 parts of a nitrogen-phosphorus-potassium compound fertilizer, 1-5 parts of trace elements, 5-10 parts of alginic acid, and 5-10 parts of chitosan, the sea cucumber peptide composite metabolite is prepared through microbial fermentation and comprises antibacterial peptide, polyglutamic acid and oligosaccharide. The sea cucumber peptide composite metabolite rich in antibacterial peptide-polyglutamic acid-oligosaccharide is efficiently prepared through a three-stage synergistic enzymolysis process and a microbial symbiotic fermentation technology, a functional synergistic system is formed by combining components such as humic acid and chitosan, the enzymolysis efficiency is remarkably improved, an exogenous enzyme is replaced with a viscera natural enzyme source, the production cost is reduced, and the method is suitable for industrial production. And the enzymolysis efficiency is improved by combining a body wall pretreatment technology.

Description

Technical Field

[0001] The present invention belongs to the field of agriculture, and particularly relates to a bio-stimulating composite fertilizer containing sea cucumber peptides and a preparation method thereof. Background Art

[0002] In the process of modern agricultural development, green, efficient, and sustainable agricultural production methods have become the mainstream trend. Bio-stimulating fertilizers, with their unique advantages, are increasingly widely used in the agricultural field. Bio-stimulants are substances that can improve plant physiological functions, enhance nutrient utilization efficiency, and increase plant stress resistance. Although they do not contain a large amount of nutrients required by plants, they can promote plant healthy growth by regulating plant metabolism and soil microbial communities. In recent years, the market scale of bio-stimulants has been continuously expanding, and their research and development have become a hot topic in the agricultural field.

[0003] As a marine biological resource, sea cucumbers are rich in various bioactive components such as collagen, polypeptides, and active polysaccharides. In the sea cucumber processing industry, a large amount of by-products such as viscera and body walls are generated. If these by-products are directly discarded, it will not only cause waste of resources but also may lead to environmental pollution problems. Therefore, the high-value utilization of sea cucumber by-products and their conversion into functional fertilizers have important economic value and environmental protection significance and have gradually become a research hotspot;

[0004] However, most traditional sea cucumber peptide preparation processes rely on exogenous proteases for hydrolysis. This method requires the additional purchase of a large amount of proteases, significantly increasing production costs, and it is difficult to simultaneously release functional substances such as glycopeptide complexes and polyglutamic acid, resulting in limited effects on crop disease resistance induction and soil improvement, unable to fully meet the requirements of modern agriculture for efficient fertilizers. At the same time, due to the lack of full exploration of the endogenous protease activity naturally present in sea cucumber viscera, the enzymatic hydrolysis process fails to achieve the optimal efficiency. Summary of the Invention

[0005] The purpose of the present invention is to provide a bio-stimulating composite fertilizer containing sea cucumber peptides and a preparation method thereof to solve the problems of high cost, low efficiency, single process, few active ingredients, and difficulty in meeting agricultural requirements proposed in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] A bio-stimulating composite fertilizer containing sea cucumber peptides, comprising the following components in parts by weight: 5 - 15 parts of sea cucumber peptide composite metabolites, 10 - 20 parts of humic acid, 10 - 15 parts of potassium humate, 40 - 60 parts of nitrogen, phosphorus, and potassium compound fertilizer, 1 - 5 parts of trace elements, 5 - 10 parts of alginic acid, 5 - 10 parts of chitosan. The sea cucumber peptide composite metabolites are prepared by microbial fermentation and include antibacterial peptides, polyglutamic acid, and oligosaccharides.

[0008] Preferably, the mass fractions of nitrogen, phosphorus, and potassium in the nitrogen-phosphorus-potassium compound fertilizer are 15%-25%, 10%-20%, and 15%-25% respectively.

[0009] Preferably, the trace elements include the following components by mass fraction: zinc 0.1%-1%, iron 0.1%-1%, copper 0.05%-0.5%, manganese 0.05%-0.5%, boron 0.05%-0.5%, molybdenum 0.01%-0.1%.

[0010] According to the preparation method of a bio-stimulating composite fertilizer containing sea cucumber peptides described above, it includes:

[0011] S1. Separate the sea cucumber viscera from the body wall, perform low-temperature crushing on the viscera, and directly use the obtained crushed material as the natural enzyme source for the complex enzymatic hydrolysis reaction. Subject the body wall to ultrasonic plasma synergistic pretreatment.

[0012] S2. Mix the pretreated body wall and the crushed viscera in a mass ratio of 1:0.5 - 1:1, and let the obtained mixture autolyze at 4°C for 12 hours to activate the endogenous protease in the viscera.

[0013] S3. Raise the temperature of the mixture to 45°C, adjust the pH value of the system to 7.5, add exogenous complex protease, and the enzyme addition amount is 0.5%-1% of the total mass of the substrate, and perform hydrolysis reaction for 4 hours.

[0014] S4. Raise the temperature of the mixture to 55°C, add algal polysaccharide enzyme accounting for 0.3%-0.5% of the total mass of the substrate, after enzymatic hydrolysis for 2h, raise the temperature to 100°C to inactivate the enzyme for 10 min.

[0015] S5. Mix the enzymatically hydrolyzed sea cucumber peptide solution, kelp powder, and functional bacterial group in a mass ratio of 5:3:2, and place them in a micro-aerobic fermentation tank for fermentation to obtain a sea cucumber peptide composite metabolite.

[0016] S6. Mix the fermented sea cucumber peptide composite metabolite with humic acid, potassium humate, nitrogen-phosphorus-potassium compound fertilizer, trace elements, alginic acid, and chitosan, dry it, and then granulate and screen it through a granulator to obtain the finished composite fertilizer.

[0017] Preferably, the exogenous complex protease includes trypsin and flavor protease, and the mass ratio is 1:1.

[0018] Preferably, the functional bacterial group is a mixed bacterium agent of Bacillus subtilis and Trichoderma harzianum, and the viable bacteria count is ≥1×10 9 CFU / g, and the mixing ratio is 3:1.

[0019] Preferably, the dissolved oxygen amount in the micro-aerobic fermentation tank is 0.5 - 1.0 mg / L, the temperature is 30 - 35°C, and the fermentation time is 72 hours.

[0020] Preferably, the power of the ultrasonic plasma collaborative pretreatment is 200-300W, the treatment time is 10-15min, the temperature of the cryogenic crushing treatment is -20°C, and the crushing particle size is ≤2mm.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] The present invention efficiently prepares sea cucumber peptide composite metabolites rich in antibacterial peptides, polyglutamic acid, and oligosaccharides through a three-stage collaborative enzymatic hydrolysis process and a microbial symbiotic fermentation technology, forms a functional synergistic system by combining components such as humic acid and chitosan, significantly improves the enzymatic hydrolysis efficiency, uses the natural enzyme source in the viscera to replace exogenous enzymes to reduce production costs, and combines the body wall pretreatment technology to improve the enzymatic hydrolysis efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts. Among them:

[0024] Figure 1 It is a block diagram of the method steps of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In order to make the above objects, features, and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention will be described in detail below with reference to the drawings of the specification.

[0026] Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can also be implemented in other ways different from those described herein. Those skilled in the art can make similar promotions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0027] Secondly, the so-called "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that can be included in at least one implementation manner of the present invention. The "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that mutually excludes other embodiments.

[0028] As shown in the appended Figure 1 figures:

[0029] Example 1: This example provides a biological stimulant compound fertilizer containing sea cucumber peptides, which includes the following components in parts by weight: 10 parts of sea cucumber peptide complex metabolites, 15 parts of humic acid, 12 parts of potassium fulvate, 50 parts of nitrogen-phosphorus-potassium compound fertilizer (N-P-K mass fraction is 20%-15%-20%), 3 parts of trace elements (0.5% zinc, 0.5% iron, 0.2% copper, 0.2% manganese, 0.2% boron, 0.05% molybdenum), 8 parts of alginic acid, and 8 parts of chitosan.

[0030] A preparation method of a biological stimulant compound fertilizer containing sea cucumber peptides includes:

[0031] S1. Raw material pretreatment: Separate the fresh sea cucumber viscera from the body wall. The viscera are crushed at -20°C to a particle size ≤ 2 mm as the natural enzyme source; the body wall is treated with 250W ultrasonic-plasma for 12 min to destroy the collagen fiber structure.

[0032] S2. Low-temperature autolysis: Mix the body wall and the viscera crushed matter in a mass ratio of 1:0.8 and autolyze at 4°C for 12 hours to activate the endogenous protease.

[0033] S3. Exogenous enzymatic hydrolysis: Raise the temperature to 45°C, adjust the pH = 7.5, add trypsin and flavor protease (1:1), and the enzyme amount is 0.8% of the total substrate mass, and hydrolyze for 4 hours.

[0034] S4. Polysaccharide enzyme synergistic enzymatic hydrolysis: Raise the temperature to 55°C, add 0.4% algal polysaccharide enzyme, inactivate the enzyme at 100°C for 10 min after 2 hours of enzymatic hydrolysis, and centrifuge to obtain the supernatant.

[0035] S5. Microbial fermentation: Mix the enzymatic hydrolysate, kelp powder, and functional flora (Bacillus subtilis: Trichoderma harzianum = 3:1, viable cell count ≥ 1×10 9 CFU / g) in a ratio of 5:3:2, the dissolved oxygen in the micro-aerobic fermentation tank is 0.8 mg / L, and ferment at 32°C for 72 hours to obtain the metabolites containing antibacterial peptides - polyglutamic acid - oligosaccharides.

[0036] S6. Compound preparation: Mix the fermentation product with the remaining components, dry at 55°C until the moisture content ≤ 5%, granulate (particle size 2 - 4 mm), and screen to obtain the finished product.

[0037] Example 2: This example provides a biological stimulant compound fertilizer containing sea cucumber peptides, which includes the following components in parts by weight: 5 parts of sea cucumber peptide complex metabolites, 20 parts of humic acid, 10 parts of potassium fulvate, 60 parts of nitrogen-phosphorus-potassium compound fertilizer (N-P-K mass fraction is 15%-20%-15%), 1 part of trace elements (0.1% zinc, 0.1% iron, 0.05% copper, 0.05% manganese, 0.05% boron, 0.01% molybdenum), 5 parts of alginic acid, and 10 parts of chitosan.

[0038] A preparation method of a biological stimulant compound fertilizer containing sea cucumber peptides, comprising:

[0039] S1. Raw material pretreatment: The viscera are crushed at -20°C to a particle size ≤ 2 mm; the body wall is treated with 200W ultrasonic - plasma for 15 min;

[0040] S2. Low - temperature autolysis: The body wall and the crushed viscera are mixed at a mass ratio of 1:1 and autolyzed at 4°C for 12 hours;

[0041] S3. Exogenous enzymatic hydrolysis: The temperature is raised to 45°C, pH = 7.5, trypsin and flavor protease (1:1) are added, and the enzyme amount is 0.5% of the total mass of the substrate, and hydrolyzed for 4 hours;

[0042] S4. Polysaccharide enzyme synergistic enzymatic hydrolysis: The temperature is raised to 55°C, 0.3% algal polysaccharide enzyme is added, and after enzymatic hydrolysis for 2 hours, the enzyme is inactivated, and the supernatant is obtained by centrifugation;

[0043] S5. Microbial fermentation: The enzymatic hydrolysate, kelp powder, and functional flora (Bacillus subtilis: Trichoderma harzianum = 3:1) are mixed at a ratio of 5:3:2, the dissolved oxygen is 0.5 mg / L, and fermented at 30°C for 72 hours;

[0044] S6. Compound preparation: Dried at 50°C until the moisture content ≤ 5%, granulated and screened to obtain the finished product.

[0045] Example 3: This example provides a biological stimulant compound fertilizer containing sea cucumber peptides, including the following components in parts by weight: 15 parts of sea cucumber peptide complex metabolites, 10 parts of humic acid, 15 parts of potassium humate, 40 parts of nitrogen - phosphorus - potassium compound fertilizer (N - P - K mass fraction is 25% - 10% - 25%), 5 parts of trace elements (1% zinc, 1% iron, 0.5% copper, 0.5% manganese, 0.5% boron, 0.1% molybdenum), 10 parts of alginic acid, and 5 parts of chitosan.

[0046] A preparation method of a biological stimulant compound fertilizer containing sea cucumber peptides, comprising:

[0047] S1. Raw material pretreatment: The viscera are crushed at -20°C to a particle size ≤ 2 mm; the body wall is treated with 300W ultrasonic - plasma for 10 min;

[0048] S2. Low - temperature autolysis: The body wall and the crushed viscera are mixed at a mass ratio of 1:0.5 and autolyzed at 4°C for 12 hours;

[0049] S3. Exogenous enzymatic hydrolysis: The temperature is raised to 45°C, pH = 7.5, trypsin and flavor protease (1:1) are added, and the enzyme amount is 1% of the total mass of the substrate, and hydrolyzed for 4 hours;

[0050] S4. Polysaccharide enzyme synergistic enzymatic hydrolysis: The temperature is raised to 55°C, 0.5% algal polysaccharide enzyme is added, and after enzymatic hydrolysis for 2 hours, the enzyme is inactivated, and the supernatant is obtained by centrifugation;

[0051] S5. Mix microbial fermentation enzyme hydrolysate, kelp powder, and functional flora (Bacillus subtilis: Trichoderma harzianum = 3:1) in a ratio of 5:3:2, with a dissolved oxygen content of 1.0 mg / L, and ferment at 35°C for 72 hours;

[0052] S6. Composite preparation: Dry at 60°C until the moisture content is ≤5%, and granulate and screen to obtain the finished product.

[0053] Experimental example: Select the common crop tomato as the experimental object and conduct a planting experiment under the same planting environment (soil type, light, temperature, irrigation conditions).

[0054] Experimental grouping:

[0055] Experimental group 1: Apply the compound fertilizer prepared in Example 1;

[0056] Experimental group 2: Apply the compound fertilizer prepared in Example 2;

[0057] Experimental group 3: Apply the compound fertilizer prepared in Example 3;

[0058] Control group: Apply the fertilizer prepared by the conventional enzymatic hydrolysis process (parts by weight: 10 parts of commercially available sea cucumber peptide powder, 15 parts of humic acid, 12 parts of potassium humate, 50 parts of nitrogen-phosphorus-potassium compound fertilizer, 3 parts of trace elements, 8 parts of alginic acid, 8 parts of chitosan; preparation method: directly mix commercially available sea cucumber peptide powder with other components and granulate);

[0059] Control group: Apply the common ordinary nitrogen-phosphorus-potassium compound fertilizer on the market (N-P-K mass fraction is 15%-15%-15%).

[0060] Experimental method: Set 3 replicated planting areas in each group, plant 30 tomato plants in each planting area, operate according to the conventional planting management method, record the data at different growth stages, measure the yield and quality at the harvest stage, and collect soil samples for analysis at the same time.

[0061] Experimental results and analysis:

[0062] The comparison of crop growth indicators is as follows in the table:

[0063]

[0064]

[0065] Result: The compound fertilizer of the present application (Experimental groups 1-3) significantly promotes the growth of tomato plants, and the plant height, stem diameter, number of leaves, and number of flowers are all significantly better than those of the control group and the control group. The active ingredients produced by the three-stage enzymatic hydrolysis and microbial symbiotic fermentation process can effectively stimulate the growth and development of crops, while the conventional enzymatic hydrolysis process and ordinary compound fertilizers are difficult to achieve the same effect.

[0066] The comparison of crop yields and qualities is as follows in the table:

[0067]

[0068] Results: The yield per tomato plant in the experimental group increased by about 50% compared to the control group and by about 40% compared to the comparative ratio group. At the same time, the contents of vitamin C and soluble sugar in the fruits increased significantly, indicating that the fertilizer of this application can not only increase the yield but also effectively improve the fruit quality, which benefits from the synergistic effect of active substances such as antibacterial peptides, polyglutamic acid, and oligosaccharides in the sea cucumber peptide complex metabolites.

[0069] The comparison of soil improvement effects is as follows in the table:

[0070]

[0071] Results: In the experimental group applying the compound fertilizer of this application, the content of soil organic matter and the number of microorganisms increased significantly, and the soil pH value tended to be neutral, effectively improving the soil structure and fertility. While the soil improvement effects of the comparative ratio group and the control group were poor, indicating that the components such as humic acid and alginic acid in the fertilizer of this application and the sea cucumber peptide complex metabolites have a synergistic effect, which is of great significance for improving the soil ecological environment.

[0072] Conclusion: Through multi-dimensional experimental comparisons, it can be seen that the sea cucumber peptide-containing bio-stimulant compound fertilizer of this application shows significant advantages in promoting crop growth, increasing yield and quality, improving soil, and environmental protection, and uses waste sea cucumbers for reuse to reduce costs.

[0073] Importantly, it should be noted that the construction and arrangement of the present application shown in multiple different exemplary embodiments are merely illustrative. Although only a few embodiments are described in detail in this disclosure, those who refer to this disclosure should easily understand that many modifications are possible without materially departing from the novel teachings and advantages of the subject matter described in this application (e.g., changes in the dimensions, scales, structures, shapes and proportions of various elements, as well as parameter values (such as temperature, pressure, etc.), installation arrangements, use of materials, colors, orientations, etc.). For example, an element shown as integrally formed may be composed of multiple parts or elements, the positions of the elements may be inverted or otherwise changed, and the nature, number or position of discrete elements may be altered or changed. Accordingly, all such modifications are intended to be included within the scope of the present invention. The order or sequence of any process or method steps may be changed or re-ordered according to alternative embodiments. In the claims, any "means-plus-function" clauses are intended to cover the structures that perform the recited function described herein, and not only structural equivalents but also equivalent structures. Other substitutions, modifications, changes and omissions may be made in the design, operating conditions and arrangement of the exemplary embodiments without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments, but extends to various modifications that still fall within the scope of the appended claims.

[0074] In addition, in order to provide a concise description of the exemplary embodiments, all features of the actual embodiments may not be described (i.e., those features that are not relevant to the currently contemplated best mode of carrying out the present invention or those features that are not relevant to the implementation of the present invention).

[0075] It should be understood that in the development of any actual implementation, as in any engineering or design project, numerous specific implementation decisions may be made. Such development efforts may be complex and time-consuming, but for those of ordinary skill in the art who benefit from this disclosure, without undue experimentation, such development efforts will be a routine task of design, manufacturing and production.

[0076] 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 claims of the present invention.

Claims

1. A biological stimulant compound fertilizer containing sea cucumber peptides, characterized in that, Comprising the following components in parts by weight: 5-15 parts of sea cucumber peptide complex metabolites, 10-20 parts of humic acid, 10-15 parts of potassium fulvate, 40-60 parts of nitrogen-phosphorus-potassium compound fertilizer, 1-5 parts of trace elements, 5-10 parts of alginic acid, 5-10 parts of chitosan, and the sea cucumber peptide complex metabolites are prepared by microbial fermentation and include antibacterial peptides, polyglutamic acid, and oligosaccharides.

2. The bio-stimulating composite fertilizer containing sea cucumber peptides according to claim 1, characterized in that, In the nitrogen-phosphorus-potassium compound fertilizer, the mass fractions of nitrogen, phosphorus, and potassium are 15%-25%, 10%-20%, and 15%-25% respectively.

3. A bio-stimulating composite fertilizer containing sea cucumber peptides according to claim 1, characterized in that, The trace elements include the following components in mass fractions: zinc 0.1%-1%, iron 0.1%-1%, copper 0.05%-0.5%, manganese 0.05%-0.5%, boron 0.05%-0.5%, and molybdenum 0.01%-0.1%.

4. A method for preparing a bio-stimulating compound fertilizer containing sea cucumber peptides according to any one of claims 1-3, characterized in that, Including: S1. Separate the sea cucumber viscera from the body wall, perform low-temperature crushing treatment on the viscera, and directly use the obtained crushed material as the natural enzyme source for the complex enzymatic hydrolysis reaction. Subject the body wall to ultrasonic plasma synergistic pretreatment. S2. Mix the pretreated body wall and the crushed viscera material in a mass ratio of 1:0.5-1:1, and let the obtained mixture autolyze at 4°C for 12 hours to activate the endogenous protease in the viscera. S3. Heat the mixture to 45°C, adjust the pH value of the system to 7.5, add exogenous complex protease, and the enzyme addition amount is 0.5%-1% of the total mass of the substrate, and perform hydrolysis reaction for 4 hours. S4. Heat the mixture to 55°C, add alginate lyase accounting for 0.3%-0.5% of the total mass of the substrate, and after enzymatic hydrolysis for 2 hours, heat to 100°C to inactivate the enzyme for 10 minutes. S5. Mix the enzymatically hydrolyzed sea cucumber peptide liquid, kelp powder, and functional flora in a mass ratio of 5:3:2, and place them in a micro-aerobic fermentation tank for fermentation to obtain sea cucumber peptide complex metabolites. S6. Mix the fermented sea cucumber peptide complex metabolites with humic acid, potassium fulvate, nitrogen-phosphorus-potassium compound fertilizer, trace elements, alginic acid, and chitosan, dry them, granulate them with a granulator, and sieve them to obtain the finished compound fertilizer.

5. The preparation method of a biological stimulant compound fertilizer containing sea cucumber peptides according to claim 4, characterized in that, The exogenous complex protease includes trypsin and flavor protease, and the mass ratio is 1:

1.

6. The preparation method of a bio-stimulating composite fertilizer containing sea cucumber peptides according to claim 4, characterized in that, The functional flora is a mixed microbial agent of Bacillus subtilis and Trichoderma harzianum, and the viable count of each is ≥ 1×10 9 CFU / g, and the mixing ratio is 3:

1.

7. The preparation method of a biological stimulant compound fertilizer containing sea cucumber peptides according to claim 4, characterized in that, The dissolved oxygen content of the micro-aerobic fermentation tank is 0.5-1.0 mg / L, the temperature is 30-35°C, and the fermentation time is 72 hours.

8. The preparation method of a bio-stimulating composite fertilizer containing sea cucumber peptides according to claim 4, characterized in that, The power of the ultrasonic plasma synergistic pretreatment is 200-300 W, the treatment time is 10-15 min, the temperature of the low-temperature crushing treatment is -20°C, and the crushing particle size is ≤2 mm.

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