Method for preparing fish protein peptide by biological method and application of fish protein peptide in fertilizer

Abstract

The invention discloses a method for preparing fish protein peptide through a biological method and application of the fish protein peptide in fertilizer, and belongs to the technical field of biology. The content of amino acid in the fish protein biological peptide is remarkably increased by utilizing the synergistic fermentation effect of microorganisms and assisting with biological enzymolysis, and the fish protein biological peptide organic fertilizer is further developed, so that the nutrition of the biological organic fertilizer is improved, the disease resistance and stress resistance of plants are improved, and the growth of the plants is promoted; therefore, an efficient and novel animal protein fertilizer is provided, and iterative updating of the fish protein biological peptide organic fertilizer is realized. The fish protein peptide prepared by the invention has the advantages of high amino acid content, degradability, no toxicity, high biocompatibility, high safety and the like, and provides a high-quality raw material for developing a fish protein peptide fertilizer capable of remarkably improving disease resistance and stress resistance of plants and promoting growth. At present, the fish protein peptide prepared by the method is applied to actual production, and remarkable economic benefits are obtained.

Description

Technical Field

[0001] The invention relates to a method for preparing fish protein peptides by biological method and application thereof in fertilizer, in particular to a method for preparing fish protein biological peptides with high amino acid content by microbial fermentation method and applying the method to the preparation of biological organic fertilizer, belonging to the field of biotechnology. Background Art

[0002] Resource utilization of low-value fish and processed fish waste to prepare fish protein biopeptides provides my country with sufficient bioprotein resources. Fish protein peptide products are rich in nutrition and have been widely used in food, feed, health care, medical and other fields. In particular, fish protein peptides have gradually been used in the field of fertilizers in recent years. For example, patent 2004100356188 discloses a fish protein biopeptide fertilizer and preparation process. The fertilizer is rich in biopeptides and trace elements such as calcium, phosphorus, and zinc, which can effectively improve the disease resistance and stress resistance of plants, thereby improving the quality of crops and promoting plant growth; patent 2024104284171 discloses a highly efficient absorption fish protein fertilizer and its preparation process, which slowly releases the nutrients in the fish protein fertilizer for plant growth, utilizes the free metal elements in the soil, and protects the root system from diseases and pests. Therefore, the use of fish protein peptides in the fertilizer field to develop new, efficient, and environmentally friendly biological organic fertilizers has become the main technical trend of industry development.

[0003] Protein peptides are a general term for different peptides of linear and cyclic structures composed of 20 natural amino acids in proteins in different compositions and arrangements. Studies have shown that low molecular weight peptides are digested faster and absorbed more than free amino acids, and their biological efficacy and nutritional value are higher than free amino acids; and the small peptides produced by protein enzymatic hydrolysis are easy to digest and absorb, promote the absorption of calcium, phosphorus and other trace elements, enhance the activity of macrophages and B cells, enhance immune function, and protect epidermal cells. Fish protein peptide is a type of aquatic protein peptide. It is made from fish tissue and is produced by autolysis, enzymatic hydrolysis, liquid fermentation, or a combination of solid fermentation and liquid enzymatic hydrolysis. Fermentation or enzymatic hydrolysis is the most commonly used method. For example, Patent 2024107361589 discloses a fish protein peptide extraction device and extraction method, Patent 2016109805255 discloses a fish protein peptide preparation method and the obtained fish protein peptide, and Patent 2021113311418 discloses a fish protein peptide and its preparation method. Both use protease enzymatic hydrolysis to prepare fish protein peptides.

[0004] Fish protein fertilizer is a pure natural fertilizer and also a functional fertilizer. It can be used as base fertilizer, topdressing, root irrigation, drip irrigation, and foliar spray. Fish protein fertilizer is rich in organic matter, fish protein, peptides, etc. After being applied to the soil, it greatly increases the content of soil organic matter, can quickly promote the reproduction of soil microorganisms, greatly activates the nutrients in the soil, increases the fertility of the soil, and improves the soil's ability to retain fertilizer and water. The use of fish protein fertilizer can effectively improve the problem of soil compaction, keep the soil fertility fresh, and is suitable for the production of vegetables, fruits, flowers and other crops. Therefore, the green and efficient preparation of fish protein peptides and their use in biofertilizers has become a technical bottleneck in this field. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a method for biologically preparing fish protein peptides and its application in fertilizers, utilizing the synergistic fermentation of microorganisms, assisted by biological enzymatic hydrolysis, to significantly increase the amino acid content in fish protein biological peptides, further develop fish protein biological peptide organic fertilizers, and then improve the nutritional value of biological organic fertilizers, improve the disease resistance and stress resistance of plants, promote plant growth, thereby providing a high-efficiency, new animal protein fertilizer, and realizing the iterative update of fish protein biological peptide organic fertilizers.

[0006] The above object of the present invention is achieved through the following technical solutions: A method for preparing fish protein peptides by biological method comprises the following steps: (1) Mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to above 7.0; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry. The enzymatic hydrolysis temperature is 40°C. After the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, and the enzymatic hydrolysis is stopped. The temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain the enzymatic hydrolyzate. (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide.

[0007] Preferably, in step (1), the mixing ratio of aquatic animal protein to water is 1:1.2-1.5.

[0008] Preferably, the fermentation enzyme solution in step (2) is prepared by the following steps: (1) Fermentation medium: 500 kg natural seawater, 7 kg bran, 14 kg soybean meal, 14 kg corn flour, 7 kg Na2HPO4, 0.21 kg KH2PO4, 0.7 kg CaCl2, 0.5 kg defoaming agent, pH 8.4 before sterilization, pH 7.5 after sterilization; (2) Sterilization: 121℃ for 30min; (3) Inoculation: inoculating the activated microbial strain at an inoculation rate of 1%, wherein the microbial strain is marine Pseudoalteromonas SM9913; (4) Temperature-controlled fermentation: Temperature-controlled aerobic fermentation, where the fermentation temperature is 15°C and the ventilation volume is 20-40m 3 / h, the fermentation time is 48h, and the fermentation enzyme liquid is obtained.

[0009] The activation culture medium of the microbial strain SM9913 described in the present invention is prepared with 1% peptone, 0.5% yeast powder, and natural seawater, pH 8.0, and high-pressure steam sterilization at 121°C for 20 minutes.

[0010] The present invention provides a method for preparing fish protein peptides by biological method, wherein the prepared fish protein peptide liquid is used to prepare fish protein peptide powder by spray drying or freeze drying process; Preferably, the spray drying process parameters are: inlet air temperature 200°C, outlet air temperature 100°C, and feed frequency 14 HZ.

[0011] Preferably, 1-2% of protease is added in the fish protein peptide enzymatic hydrolysis process, and the protease is a mixture of one or more of trypsin, pepsin and papain.

[0012] The present invention provides a method for preparing fish protein peptides by biological method. The fish protein peptides are powder or liquid and can be used for the production of fish protein biological organic fertilizer, fish protein urea, water-soluble fertilizer, foliar fertilizer, concentrated fertilizer and the like.

[0013] The present invention provides a method for preparing fish protein peptides by biological method and application thereof in fertilizer, which has the following significant advantages compared with the prior art: (1) The present invention uses low-value aquatic protein (aquatic processing waste, fish skin, etc.) as raw materials, utilizes the synergistic fermentation of microorganisms, and is supplemented by biological enzymatic hydrolysis to significantly increase the amino acid content in fish protein biological peptides, thereby increasing the utilization value of fish protein in fertilizers. The present invention has the advantages of being degradable, non-toxic, highly biocompatible, and highly safe, and lays a raw material foundation for the development of fertilizers that can significantly improve plant disease resistance, stress resistance, and growth promotion; (2) The present invention utilizes microbial fermentation enzyme liquid to degrade fish protein. The fermentation strain SM9913 selected is a natural strain screened from the deep sea, which is easy to culture, not easily contaminated during the fermentation process, and easy to industrialize. At the same time, the protein degrading enzyme produced by microbial fermentation is a composite enzyme system, which degrades polypeptides that are not suitable for plant absorption into active amino acids, which is more conducive to plant absorption, significantly improving the application value of fish protein peptides in fertilizers, and has a significant effect compared with the traditional direct enzymatic hydrolysis method; (3) The present invention utilizes microbial fermentation enzyme liquid to degrade fish protein. During the fermentation process, a large amount of extracellular polysaccharides are produced, which has a significant effect on subsequent concentration and drying, thereby reducing the concentration and drying costs. At the same time, the produced microbial polysaccharides have a significant synergistic effect on improving the application value of fish protein peptides in fertilizers. These are also one of the fundamental reasons for utilizing microbial fermentation enzyme liquid to degrade fish protein. (4) The present invention utilizes microbial fermentation complex enzyme liquid and protease to synergistically degrade fish skin protein. Through the synergistic effect between the two, the amino acid content in the fish protein peptide is significantly increased. The experimental results show that the synergistic effect between the two has achieved the technical effect of "1+1>2"; (5) The present invention provides a method for preparing fish protein peptides by biological methods. The fish protein peptides prepared by this method have been applied to actual production and have achieved commercial success. At present, the enterprise standard Q / 370201HDS193-2023 "Fish Protein Peptide" has been registered and used in the production of fertilizers, achieving significant economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The diagram is a rendering of the fish protein peptide hydrolysate of the present invention, wherein the left picture is the supernatant after centrifugation of the hydrolysate, the middle picture is the hydrolysate before centrifugation, and the right picture is the residue after centrifugation of the hydrolysate. DETAILED DESCRIPTION

[0015] The fermentation enzyme solution in the following examples was prepared by the following steps: (1) Activation of fermentation strains: Marine Pseudoalteromonas SM9913 was inoculated for activation culture, wherein the activation culture medium was composed of 1% peptone, 0.5% yeast powder, and natural seawater, pH 8.0, and sterilized by high-pressure steam at 121°C for 20 min; (2) Fermentation medium: 500 kg natural seawater, 7 kg bran, 14 kg soybean meal, 14 kg corn flour, 7 kg Na2HPO4, 0.21 kg KH2PO4, 0.7 kg CaCl2, 0.5 kg defoaming agent, pH 8.4 before sterilization, pH 7.5 after sterilization; (3) Sterilization: 121℃ for 30min; (4) Inoculation: Inoculate the activated microbial strains at a rate of 1%; (5) Temperature-controlled fermentation: Temperature-controlled aerobic fermentation, where the fermentation temperature is 15°C and the ventilation volume is 20-40m 3 / h, the fermentation time is 48h, and the fermentation enzyme liquid is obtained.

[0016] After testing, the enzyme activity of the fermentation enzyme solution was about 500U / mL. Example

[0017] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry. The enzymatic hydrolysis temperature is 40°C. After the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, and the enzymatic hydrolysis is stopped. The temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain the enzymatic hydrolyzate. (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide. Example

[0018] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry, and 1.5% of composite protease is added; the enzymatic hydrolysis temperature is 40°C; after the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, the enzymatic hydrolysis is stopped, the temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain an enzymatic hydrolyzate; (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide.

[0019] The composite protease consists of 60% trypsin and 40% papain. Example

[0020] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry, and 1.5% trypsin is added; the enzymatic hydrolysis temperature is 40°C; after the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, the enzymatic hydrolysis is stopped, the temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain an enzymatic hydrolyzate; (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide. Example

[0021] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Adding fermentation enzyme liquid to the mixed slurry and stirring evenly, the amount of fermentation enzyme liquid added is 40% of the mixed slurry, and 1.5% papain is added; the enzymatic hydrolysis temperature is 40°C; after the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, the enzymatic hydrolysis is stopped, the temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain an enzymatic hydrolyzate; (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide. Example

[0022] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry, and 1.5% of composite protease is added; the enzymatic hydrolysis temperature is 40°C; after the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, the enzymatic hydrolysis is stopped, the temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain an enzymatic hydrolyzate; (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide.

[0023] The composite protease consists of 50% trypsin and 50% papain. Example

[0024] A method for preparing fish protein peptides by biological method, the specific process steps include: (1) mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to be above 7.0, wherein the mixing ratio of aquatic animal protein to water is 1.5; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry, and 1.5% of composite protease is added; the enzymatic hydrolysis temperature is 40°C; after the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, the enzymatic hydrolysis is stopped, the temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain an enzymatic hydrolyzate; (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide.

[0025] The composite protease consists of 40% trypsin and 60% papain.

[0026] Determined by conventional acid hydrolysis method specified in GB / T18246-2019;

[0027] Amino acid content;

[0028] Table 1 Amino acid content of fish protein peptides in different examples Group measurement indicators Amino acid salt content (%) Example 1 72.15 Example 2 82.32 Example 3 76.08 Example 4 73.11 Example 5 77.29 Example 6 75.17 The above experimental results show that the content of amino acids in fish protein peptides in Example 2 is the highest, reaching 82.32%, which is the best embodiment of the present invention. The results of Example 1 show that the amino acid content in the fish protein peptides reaches 72.15% by degrading the fish protein peptides through microbial fermentation enzyme liquid; the results of Examples 2-6 show that in the present invention, the composite protease can significantly increase the amino acid content in the fish protein peptides, among which the addition of trypsin and papain composite enzymes has the best effect, and the addition of papain alone has little effect on the amino acid content. The experimental results show that in the system of microbial enzymatic degradation of fish protein peptides of the present invention, trypsin and papain have a significant promoting effect, and can achieve the technical effect of "1+1>2" under a specific ratio.

[0029] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein with equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing fish protein peptides by biological method, characterized in that The steps include: (1) Mixing aquatic animal protein with water, grinding and stirring to obtain a mixed slurry, and adjusting the pH value to above 7.0; (2) Add fermentation enzyme liquid to the mixed slurry and stir evenly. The amount of fermentation enzyme liquid added is 40% of the mixed slurry. The enzymatic hydrolysis temperature is 40°C. After the enzymatic hydrolysis time is 4 hours, the liquid becomes thinner, and the enzymatic hydrolysis is stopped. The temperature is raised to 90°C, the enzyme is inactivated for 15 minutes, and then the temperature is naturally lowered to obtain the enzymatic hydrolyzate. (3) centrifuging the enzymatic hydrolysate using a butterfly centrifuge to remove solids from the enzymatic hydrolysate to obtain a centrifugal supernatant of the fish protein polypeptide liquid; (4) Concentrate the centrifuged supernatant until the solid content is more than 20% to obtain fish protein peptide.

2. The method for preparing fish protein peptides by biological method according to claim 1, characterized in that: The mixing ratio of aquatic animal protein to water is 1:1.2~1.

5.

3. The method for preparing fish protein peptides by biological method according to claim 1, characterized in that The fermentation enzyme solution is prepared by the following steps: (1) Fermentation medium: 500 kg natural seawater, 7 kg bran, 14 kg soybean meal, 14 kg corn flour, 7 kg Na2HPO4, 0.21 kg KH2PO4, 0.7 kg CaCl2, 0.5 kg defoaming agent, pH 8.4 before sterilization, pH 7.5 after sterilization; (2) Sterilization: 121℃ for 30min; (3) Inoculation: inoculating the activated microbial strain at an inoculation rate of 1%, wherein the microbial strain is marine Pseudoalteromonas SM9913; (4) Temperature-controlled fermentation: Temperature-controlled aerobic fermentation, where the fermentation temperature is 15°C and the ventilation volume is 20-40m 3 / h, the fermentation time is 48h, and the fermentation enzyme liquid is obtained.

4. The method for preparing fish protein peptides by biological method according to claim 3, characterized in that The activation culture medium of the microbial strain SM9913 is prepared with 1% peptone, 0.5% yeast powder and natural seawater, pH 8.0, and high-pressure steam sterilization at 121°C for 20 minutes.

5. The method for preparing fish protein peptides by biological method according to claim 1, characterized in that The fish protein peptide is prepared into fish protein peptide powder by spray drying or freeze drying process.

6. The method for preparing fish protein peptides by biological method according to claim 5, characterized in that The spray drying process parameters are: inlet air temperature 200°C, outlet air temperature 100°C, and feed frequency 14 HZ.

7. The method for preparing fish protein peptides by biological method according to claim 1, characterized in that: 1-2% of protease is added in the enzymatic hydrolysis process of fish protein peptides, and the protease is a mixture of one or more of trypsin, pepsin and papain.

8. Fish protein peptide prepared by the method of claim 1; 9. Use of the fish protein peptide according to claim 8 in biological organic fertilizer, urea, water-soluble fertilizer, foliar fertilizer and concentrated fertilizer.

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