High-value fruit and vegetable special fertilizer containing fish protein and preparation method thereof

Abstract

This invention discloses a high-value-added fertilizer for fruits and vegetables containing fish protein, which is made from the following raw materials in parts by weight: 40-60 parts of compound microbial inoculant, 10-15 parts of activator, 80-120 parts of diatomaceous earth, 40-60 parts of decomposed microbial residue, 10-20 parts of potassium sulfate, and 10-30 parts of humic acid; the activator is composed of fish protein powder and onion extract in a mass ratio of 3:1; the compound microbial inoculant is composed of *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae*, with a volume ratio of 1:1:1. This fertilizer is based on microbial inoculants, using a combination of three specific advantageous strains for synergistic effects. It is also used in conjunction with an activator formulated from fish protein powder and onion extract in a specific ratio. This provides nutrients to plants while simultaneously enhancing microbial activity, enriching the soil microenvironment, and prolonging the microbial action time, resulting in significant yield increases, improved fruit and vegetable quality, and enhanced soil fertility.

Description

Technical Field

[0001] This invention belongs to the field of bio-organic fertilizer technology, specifically relating to a high-value-added fertilizer for fruits and vegetables containing fish protein and its preparation method. Background Technology

[0002] Chemical fertilizers are the "food" of grains, and their application has effectively increased crop yields, making a significant contribution to my country's consecutive years of bumper harvests. However, in my country's agricultural production, there are problems such as the blind and excessive application of chemical fertilizers, mainly manifested in the following four aspects: First, the amount of chemical fertilizer applied is generally too high. Currently, the average amount of chemical fertilizer applied to grain crops in my country is 21.9 kg per mu (approximately 0.067 hectares), far exceeding the world average of 8 kg per mu. Second, there is a significant imbalance in fertilization. In eastern my country, the lower reaches of the Yangtze River, and the suburbs of large and medium-sized cities, the amount of fertilizer applied is relatively high, while the over-fertilization of fruits, vegetables, and other crops is even more common due to their higher economic value. Third, the utilization rate of nutrient resources is generally low. According to statistics, the total nutrient content of my country's organic fertilizer resources currently reaches more than 70 million tons, but the effective utilization rate is less than 40%. Among them, the nutrient return rate of livestock manure is about 50%, and the nutrient return rate of agricultural straw is about 35%. Although the amount of chemical fertilizer applied in my country is increasing year by year, the fertilizer utilization rate is showing a downward trend. Developed countries have achieved fertilizer utilization rates of 50%–55%, while my country's is only around 35%, with nitrogen, phosphorus, and potassium fertilizers at 30%–35%, 10%–20%, and 30%–50%, respectively. Fourthly, the fertilization structure in agricultural production is severely unbalanced. There is a widespread problem of excessive application of chemical fertilizers and insufficient application of organic fertilizers, as well as excessive application of nitrogen fertilizers and insufficient application of phosphorus and potassium fertilizers, leading to a prominent "three-heavy, three-light" problem in my country's agricultural production. With the continuous improvement of people's living standards and consumption levels, in addition to satisfying daily fruit and vegetable consumption, people have increasingly higher requirements for the safety and quality of fruits and vegetables. How to ensure high yields of fruit and vegetable cash crops while simultaneously improving their quality remains a topic that requires continuous and in-depth research.

[0003] Bio-organic fertilizer refers to a fertilizer that combines the effects of both microbial and organic fertilizers, made from specific functional microorganisms and organic fertilizers primarily derived from animal remains (such as livestock and poultry manure and crop straw) that have undergone harmless treatment and composting. Bio-organic fertilizer has become the best alternative to ordinary chemical fertilizers due to its numerous unique advantages. However, despite its many advantages in improving soil structure, enhancing soil fertility, reducing environmental pollution, and promoting crop growth, bio-organic fertilizer also exhibits some significant drawbacks in practical application. For example, due to differences in soil conditions, the activity of microorganisms in bio-organic fertilizer can be greatly affected in different soil environments, leading to unstable fertilization effects. Furthermore, the nutrient content of bio-organic fertilizer is relatively low. Although its raw materials are mostly organic matter, after fermentation and composting, although it contains abundant organic matter and various nutrients, the effective nutrient content per unit weight is usually lower than that of chemical fertilizers. These drawbacks, to some extent, limit its wider application and promotion. Especially for cash crops such as fruits and vegetables, due to their short growth cycle, large fertilizer requirements, and sensitivity to nutrient needs, there is a greater need for a special bio-organic fertilizer for fruits and vegetables that can provide sufficient nutrients, improve the soil environment, and enhance fruit quality. Summary of the Invention

[0004] The purpose of this invention is to provide a high-value-added fertilizer for fruits and vegetables containing fish protein. This fertilizer is based on microbial fertilizer, and uses a combination of three specific superior strains for synergistic effect. It is also used in conjunction with an activator formulated with fish protein powder and onion extract in a specific ratio. This provides nutrients to plants while also improving microbial activity, enriching the soil microenvironment, and prolonging the action time of microorganisms. It has a significant yield-increasing effect, improves the quality of fruits and vegetables, and enhances soil fertility.

[0005] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A high-value-added fertilizer for fruits and vegetables containing fish protein is made from the following raw materials in parts by weight: 40-60 parts compound microbial inoculant, 10-15 parts activator, 80-120 parts diatomaceous earth, 40-60 parts decomposed microbial residue, 10-20 parts potassium sulfate, and 10-30 parts humic acid; the activator is composed of fish protein powder and onion extract in a 3:1 mass ratio; the compound microbial inoculant consists of *Bacillus desertica*, *Azotobacter molluscica*, and *Pseudomonas aeruginosa*, with a bacterial liquid volume ratio of 1:1:1; the *Bacillus desertica* has the preservation number CGMCC. The strains described herein, with accession number CGMCC No. 1.15888, were purchased from the China General Microbiological Culture Collection Center (CGMCC) on September 20, 2016. The *Azotobacter mollissima* strain has accession number CGMCC No. 1.5340, also purchased from the CGMCC on October 24, 2005. The *Pseudomonas brassicae* strain has accession number CGMCC No. 1.10355, also purchased from the CGMCC on December 31, 2009. All strains selected in this invention can be purchased openly from the CGMCC without further re-preservation.

[0007] Preferably, the composite microbial agent is prepared by the following method: *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae* are thawed and activated respectively. Single colonies are then picked and inoculated into LB liquid medium and cultured at 28-30℃ and 180-200 r / min until OD600≈1.0 to obtain seed culture. The seed culture is then inoculated into LB liquid medium at a rate of 2% and cultured at 28-30℃ and 180-200 r / min. The culture is stopped when the colony activity reaches 1×10⁸ cfu·mL⁻¹. The culture solutions of the three bacteria are mixed in a volume ratio of 1:1:1 to obtain the composite microbial agent.

[0008] The LB liquid culture medium consists of: 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, water as solvent, pH 7.2-7.4, and is sterilized at 121°C.

[0009] Preferably, the fish protein powder is prepared by the following method:

[0010] Step 1: Take the by-products of fish product processing, wash them clean with water, freeze-dry them at low temperature, pulverize them, and pass them through a 100-mesh sieve to obtain solid powder;

[0011] Step 2: Add the solid powder to the degreasing solvent, heat to 50-55℃ and stir magnetically for 1 hour, then centrifuge to separate the solid product. Wash the solid product with deionized water until neutral and dry it for later use.

[0012] Step 3: Mix the solid product obtained in Step 2 with deionized water at a solid-liquid ratio of 1g:10ml to obtain a mixed solution. Inoculate the mixed solution with a compound of Saccharomyces boulardii and Bacillus coagulans. Adjust the temperature to 25-30℃ and the humidity to 70-75%. Ferment for 2-3 days. Filter the fermentation product to obtain the fermentation broth.

[0013] Step 4: Adjust the pH of the fermentation broth obtained in Step 3 to 6.5-7.0, add a complex enzyme containing trypsin, papain and nattokinase to it for 6-8 hours of enzymatic hydrolysis at a temperature of 45-50℃, cool to room temperature after high-temperature inactivation, centrifuge at high speed, and freeze-dry the supernatant to obtain fish protein powder.

[0014] Preferably, the degreasing solvent is composed of a 1 mol / L NaOH solution and sodium dodecyl sulfate in a mass ratio of 10:0.5-1.

[0015] Preferably, the mass ratio of solid powder to degreasing solvent in step 2 is 1:5-6.

[0016] Preferably, in step 3, the mass ratio of *Saccharomyces boulardii* and *Bacillus coagulans* in the compound bacteria is 2:1; and the inoculation amount of the compound bacteria is 5-10% of the mass of the solid product.

[0017] Preferably, the mass ratio of trypsin, papain, and nattokinase in the compound enzyme is (1-3):1:(0.3-0.6); the amount of the compound enzyme is 1.5% of the mass of the solid powder in step 1.

[0018] Preferably, the trypsin has an enzyme activity of 200,000 u / g; the papain has an enzyme activity of 200,000 u / g; and the nattokinase has an enzyme activity of 20,000 u / g.

[0019] Preferably, the effective viable count of the *Saccharomyces boulardii* is 20 billion CFU / g, and the effective viable count of *Bacillus coagulans* is 10 billion CFU / g.

[0020] This invention also provides a method for preparing the above-mentioned high-value-added fruit and vegetable fertilizer containing fish protein, which includes the following steps:

[0021] (1) Prepare fish protein powder and mix it with onion extract in a certain mass ratio to obtain an active agent for later use;

[0022] (2) Preparation of compound microbial inoculants;

[0023] (3) Mix the activator and the compound microbial agent evenly, and incubate at room temperature for 24 hours. Then add diatomaceous earth, decomposed bacterial residue, humic acid and potassium sulfate, mix evenly, dry at low temperature and package to obtain the final product.

[0024] The application rate of the high-value-added fruit and vegetable fertilizer containing fish protein obtained by this invention is 50-100 kg / mu.

[0025] This invention screened three dominant bacterial strains for combined use. *Bacillus desertica* secretes plant growth hormones and other growth-promoting substances, stimulating plant cell division and elongation, promoting root and above-ground growth, enhancing the plant's ability to absorb nutrients and water, and inhibiting the reproduction of various pathogens. *Azotobacter mollissima* efficiently fixes nitrogen, providing essential nitrogen nutrition for fruit and vegetable growth. This strain also forms a good symbiotic relationship with plant roots, increasing soil nitrogen content through nitrogen fixation, reducing dependence on external nitrogen fertilizers, lowering agricultural production costs, and avoiding environmental pollution caused by excessive chemical nitrogen fertilizer application. *Pseudomonas aeruginosa* produces antibacterial substances such as pyrrolidone carboxylic acids, inhibiting the growth and reproduction of pathogens in the soil, reducing pathogen damage to plant roots, and lowering the incidence of fruit and vegetable diseases. When mixed in equal proportions, these three strains exhibit a good synergistic effect, significantly improving plant disease prevention and growth promotion.

[0026] The specific formulation of the active agent in this invention serves two main purposes. Firstly, it acts as a nutrient that can be rapidly absorbed and utilized by plants, providing nitrogen, various vitamins, minerals, and antioxidants for plant growth. Secondly, it enhances the activity of microorganisms. The small-molecule peptides and free amino acids in fish protein powder provide a rapid source of carbon and nitrogen for microorganisms, accelerating cell proliferation. The nutrients in onion extract can also be utilized by microorganisms, promoting their growth and metabolic activities. Furthermore, the thiosulfinates (such as allicin analogs) and polyphenols in onion extract can scavenge free radicals, alleviate oxidative stress, and prolong the survival period of the microbial agent in the soil. The active substances in onion extract can also activate the quorum sensing system of the bacterial strain, promoting the secretion of lipopeptide antibiotics by *Bacillus desertica*, and enhancing its antagonistic ability against soil-borne pathogens (such as *Fusarium*). The synergistic effect of these two factors improves the overall stability of the fertilizer, making the product less prone to deterioration or inactivation during storage and use.

[0027] The beneficial effects of this invention are as follows: This invention overcomes the bottleneck problems of low activity, single function, weak targeting, and delayed fertilizer effect of traditional bio-organic fertilizers by using a three-dimensional synergistic design of the activator-functional microbial community-carrier system; the use of a specific ratio of fish protein / onion extract not only activates the microbial metabolic network, but also improves the soil environment, promotes plant growth, and enhances plant resistance through the division of labor and cooperation of functional strains. At the same time, beneficial microorganisms form a dominant community around the roots of fruits and vegetables, which can inhibit the growth and reproduction of harmful pathogens, reduce the occurrence of soil-borne diseases, and create favorable conditions for the growth and development of fruits and vegetables, resulting in a significant improvement in both yield and quality. Attached Figure Description

[0028] Figure 1The figure shows the results of the antagonistic verification of the three strains screened in this invention; where 1 is Sonora desert Bacillus, 2 is Azotobacter mollissima, and 3 is Pseudomonas brassicae.

[0029] Figure 2 This invention relates to the effects of different treatment groups on tomato plant height in a field planting experiment.

[0030] Figure 3 This invention investigates the effects of different treatment groups on the stem diameter of tomato plants in a field planting experiment. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments, but is not limited thereto. The *Saccharomyces boulardii* used in this invention was purchased from Shanxi Qixin Biotechnology Co., Ltd., *Bacillus coagulans* from Xi'an Bainiankang Biotechnology Co., Ltd., trypsin from Nanjing Aoduofuni Biotechnology Co., Ltd., papain from Nanning Pangbo Bioengineering Co., Ltd., nattokinase from Shaanxi Jinrun Biotechnology Co., Ltd., and onion extract from Xi'an An'ao Biotechnology Co., Ltd.

[0032] Example 1

[0033] A high-value-added fertilizer for fruits and vegetables containing fish protein is made from the following raw materials in parts by weight: 40 kg of compound microbial inoculant, 10 kg of activator, 80 kg of diatomaceous earth, 40 kg of decomposed inoculant residue, 10 kg of potassium sulfate, and 10 kg of humic acid. The activator is composed of fish protein powder and onion extract in a 3:1 mass ratio. The compound microbial inoculant consists of *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae*, with a bacterial liquid volume ratio of 1:1:1. The *Bacillus desertica* has the accession number CGMCC No. 1.15888, purchased from the China General Microbiological Culture Collection Center, with an original accession date of September 20, 2016. The *Azotobacter mollissima* has the accession number CGMCC No. 1.5340, purchased from the China General Microbiological Culture Collection Center, with an original accession date of October 24, 2005. The *Pseudomonas brassicae* has the accession number CGMCC. No. 1.10355, purchased from the China General Microbiological Culture Collection Center, with the original deposit date being December 31, 2009.

[0034] The compound microbial agent was prepared by the following method: *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae* were thawed and activated respectively, and then single colonies were picked and inoculated into LB liquid medium, and cultured at 30°C and 180 r / min until OD... 600 ≈1.0 yielded seed culture. This seed culture was then inoculated into LB liquid medium at a rate of 2% and cultured at 30°C and 180 rpm. The culture was continued until the colony activity reached 1×10⁻⁶.8 cfu·mL ‑1 After the culture was completed, the culture solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a compound microbial agent.

[0035] The LB liquid culture medium consists of: 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, water as solvent, pH 7.2-7.4, and is sterilized at 121°C.

[0036] The fish protein powder is prepared using the following method:

[0037] Step 1: Take the by-products of fish product processing, wash them clean with water, freeze-dry them at low temperature, pulverize them, and pass them through a 100-mesh sieve to obtain solid powder;

[0038] Step 2: Add the solid powder to the degreasing solvent, heat to 50-55℃ and stir magnetically for 1 hour, then centrifuge to separate the solid product. Wash the solid product with deionized water until neutral and dry it for later use.

[0039] Step 3: Mix the solid product obtained in Step 2 with deionized water at a solid-liquid ratio of 1g:10ml to obtain a mixed solution. Inoculate the mixed solution with a compound of Saccharomyces boulardii and Bacillus coagulans. Adjust the temperature to 30℃ and the humidity to 70%. Ferment for 2 days. Filter the fermentation product to obtain the fermentation liquid.

[0040] Step 4: Adjust the pH of the fermentation broth obtained in Step 3 to 6.5, add a complex enzyme containing trypsin, papain and nattokinase to it for 6 hours of enzymatic hydrolysis at 45℃, cool to room temperature after high-temperature inactivation, separate by high-speed centrifugation, and freeze-dry the supernatant to obtain fish protein powder.

[0041] The degreasing solvent is composed of a 1 mol / L NaOH solution and sodium dodecyl sulfate in a mass ratio of 10:0.5.

[0042] In step 2, the mass ratio of solid powder to degreasing solvent is 1:5.

[0043] In step 3, the mass ratio of Saccharomyces boulardii and Bacillus coagulans in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 5% of the mass of the solid product.

[0044] The mass ratio of trypsin, papain, and nattokinase in the compound enzyme is 1:1:0.3; the amount of the compound enzyme is 1.5% of the mass of the solid powder in step 1.

[0045] The trypsin has an enzyme activity of 200,000 u / g; the papain has an enzyme activity of 200,000 u / g; and the nattokinase has an enzyme activity of 20,000 u / g.

[0046] The effective viable count of *Saccharomyces boulardii* is 20 billion CFU / g, and the effective viable count of *Bacillus coagulans* is 10 billion CFU / g.

[0047] The preparation method of the above-mentioned high-value-added fruit and vegetable fertilizer containing fish protein includes the following steps:

[0048] (1) Prepare fish protein powder and mix it with onion extract in a certain mass ratio to obtain an active agent for later use;

[0049] (2) Preparation of compound microbial inoculants;

[0050] (3) Mix the activator and the compound microbial agent evenly, and incubate at room temperature for 24 hours. Then add diatomaceous earth, decomposed bacterial residue, humic acid and potassium sulfate, mix evenly, dry at low temperature and package to obtain the final product.

[0051] Example 2

[0052] A high-value-added fertilizer for fruits and vegetables containing fish protein is made from the following raw materials in parts by weight: 60 kg of compound microbial inoculant, 15 kg of activator, 120 kg of diatomaceous earth, 60 kg of decomposed inoculant residue, 20 kg of potassium sulfate, and 30 kg of humic acid. The activator is composed of fish protein powder and onion extract in a 3:1 mass ratio. The compound microbial inoculant consists of *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae*, with a bacterial liquid volume ratio of 1:1:1. The *Bacillus desertica* has the accession number CGMCC No. 1.15888, purchased from the China General Microbiological Culture Collection Center, with an original accession date of September 20, 2016. The *Azotobacter mollissima* has the accession number CGMCC No. 1.5340, purchased from the China General Microbiological Culture Collection Center, with an original accession date of October 24, 2005. The *Pseudomonas brassicae* has the accession number CGMCC. No. 1.10355, purchased from the China General Microbiological Culture Collection Center, with the original deposit date being December 31, 2009.

[0053] The compound microbial agent was prepared by the following method: *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae* were thawed and activated respectively, and then single colonies were picked and inoculated into LB liquid medium, and cultured at 28°C and 200 r / min until OD... 600 ≈1.0 yielded seed culture. The seed culture was inoculated into LB liquid medium at a rate of 2% and cultured at 28℃ and 200 rpm. When the colony activity reached 1×10⁻⁶... 8 cfu·mL ‑1 After the culture was completed, the culture solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a compound microbial agent.

[0054] The LB liquid culture medium consists of: 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, water as solvent, pH 7.2-7.4, and is sterilized at 121°C.

[0055] The fish protein powder is prepared using the following method:

[0056] Step 1: Take the by-products of fish product processing, wash them clean with water, freeze-dry them at low temperature, pulverize them, and pass them through a 100-mesh sieve to obtain solid powder;

[0057] Step 2: Add the solid powder to the degreasing solvent, heat to 50-55℃ and stir magnetically for 1 hour, then centrifuge to separate the solid product. Wash the solid product with deionized water until neutral and dry it for later use.

[0058] Step 3: Mix the solid product obtained in Step 2 with deionized water at a solid-liquid ratio of 1g:10ml to obtain a mixed solution. Inoculate the mixed solution with a compound of Saccharomyces boulardii and Bacillus coagulans. Adjust the temperature to 30℃ and the humidity to 75%. Ferment for 2 days. Filter the fermentation product to obtain the fermentation broth.

[0059] Step 4: Adjust the pH of the fermentation broth obtained in Step 3 to 7.0, add a complex enzyme containing trypsin, papain and nattokinase to it for 8 hours of enzymatic hydrolysis at 50°C, cool to room temperature after high-temperature inactivation, separate by high-speed centrifugation, and freeze-dry the supernatant to obtain fish protein powder.

[0060] The degreasing solvent is composed of a 1 mol / L NaOH solution and sodium dodecyl sulfate in a mass ratio of 10:1.

[0061] In step 2, the mass ratio of solid powder to degreasing solvent is 1:6.

[0062] In step 3, the mass ratio of Saccharomyces boulardii and Bacillus coagulans in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 10% of the mass of the solid product.

[0063] The mass ratio of trypsin, papain, and nattokinase in the compound enzyme is 3:1:0.6; the amount of the compound enzyme used is 1.5% of the mass of the solid powder in step 1.

[0064] The trypsin has an enzyme activity of 200,000 u / g; the papain has an enzyme activity of 200,000 u / g; and the nattokinase has an enzyme activity of 20,000 u / g.

[0065] The effective viable count of *Saccharomyces boulardii* is 20 billion CFU / g, and the effective viable count of *Bacillus coagulans* is 10 billion CFU / g.

[0066] The preparation method of the above-mentioned high-value-added fruit and vegetable fertilizer containing fish protein includes the following steps:

[0067] (1) Prepare fish protein powder and mix it with onion extract in a certain mass ratio to obtain an active agent for later use;

[0068] (2) Preparation of compound microbial inoculants;

[0069] (3) Mix the activator and the compound microbial agent evenly, and incubate at room temperature for 24 hours. Then add diatomaceous earth, decomposed bacterial residue, humic acid and potassium sulfate, mix evenly, dry at low temperature and package to obtain the final product.

[0070] Example 3

[0071] A high-value-added fertilizer for fruits and vegetables containing fish protein is made from the following raw materials in parts by weight: 50 kg of compound microbial inoculant, 12 kg of activator, 100 kg of diatomaceous earth, 50 kg of decomposed inoculant residue, 15 kg of potassium sulfate, and 20 kg of humic acid. The activator is composed of fish protein powder and onion extract in a 3:1 mass ratio. The compound microbial inoculant consists of *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae*, with a bacterial liquid volume ratio of 1:1:1. The *Bacillus desertica* has the accession number CGMCC No. 1.15888, purchased from the China General Microbiological Culture Collection Center, with an original accession date of September 20, 2016. The *Azotobacter mollissima* has the accession number CGMCC No. 1.5340, purchased from the China General Microbiological Culture Collection Center, with an original accession date of October 24, 2005. The *Pseudomonas brassicae* has the accession number CGMCC. No. 1.10355, purchased from the China General Microbiological Culture Collection Center, with the original deposit date being December 31, 2009.

[0072] The compound microbial agent was prepared by the following method: *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae* were thawed and activated respectively, and then single colonies were picked and inoculated into LB liquid medium, and cultured at 28°C and 200 r / min until OD... 600 ≈1.0 yielded seed culture. The seed culture was inoculated into LB liquid medium at a rate of 2% and cultured at 30℃ and 200 rpm. When the colony activity reached 1×10⁻⁶... 8 cfu·mL ‑1 After the culture was completed, the culture solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a compound microbial agent.

[0073] The LB liquid culture medium consists of: 10 g / L peptone, 5 g / L sodium chloride, 5 g / L yeast extract, water as solvent, pH 7.2-7.4, and is sterilized at 121°C.

[0074] The fish protein powder is prepared using the following method:

[0075] Step 1: Take the by-products of fish product processing, wash them clean with water, freeze-dry them at low temperature, pulverize them, and pass them through a 100-mesh sieve to obtain solid powder;

[0076] Step 2: Add the solid powder to the degreasing solvent, heat to 50-55℃ and stir magnetically for 1 hour, then centrifuge to separate the solid product. Wash the solid product with deionized water until neutral and dry it for later use.

[0077] Step 3: Mix the solid product obtained in Step 2 with deionized water at a solid-liquid ratio of 1g:10ml to obtain a mixed solution. Inoculate the mixed solution with a compound of Saccharomyces boulardii and Bacillus coagulans. Adjust the temperature to 28℃ and the humidity to 75%. Ferment for 3 days. Filter the fermentation product to obtain the fermentation liquid.

[0078] Step 4: Adjust the pH of the fermentation broth obtained in Step 3 to 6.8, add a complex enzyme containing trypsin, papain and nattokinase to it for 7 hours of enzymatic hydrolysis at 50°C, cool to room temperature after high-temperature inactivation, separate by high-speed centrifugation, and freeze-dry the supernatant to obtain fish protein powder.

[0079] The degreasing solvent is composed of a 1 mol / L NaOH solution and sodium dodecyl sulfate in a mass ratio of 10:0.8.

[0080] In step 2, the mass ratio of solid powder to degreasing solvent is 1:5.

[0081] In step 3, the mass ratio of Saccharomyces boulardii and Bacillus coagulans in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 8% of the mass of the solid product.

[0082] The mass ratio of trypsin, papain, and nattokinase in the compound enzyme is 2:1:0.5; the amount of the compound enzyme used is 1.5% of the mass of the solid powder in step 1.

[0083] The trypsin has an enzyme activity of 200,000 u / g; the papain has an enzyme activity of 200,000 u / g; and the nattokinase has an enzyme activity of 20,000 u / g.

[0084] The effective viable count of *Saccharomyces boulardii* is 20 billion CFU / g, and the effective viable count of *Bacillus coagulans* is 10 billion CFU / g.

[0085] The preparation method of the above-mentioned high-value-added fruit and vegetable fertilizer containing fish protein includes the following steps:

[0086] (1) Prepare fish protein powder and mix it with onion extract in a certain mass ratio to obtain an active agent for later use;

[0087] (2) Preparation of compound microbial inoculants;

[0088] (3) Mix the activator and the compound microbial agent evenly, and incubate at room temperature for 24 hours. Then add diatomaceous earth, decomposed bacterial residue, humic acid and potassium sulfate, mix evenly, dry at low temperature and package to obtain the final product.

[0089] Comparative Examples 1-9

[0090] Based on the high-value-added fruit and vegetable fertilizer containing fish protein described in Example 3, the proportion of functional microbial strains in the compound microbial agent was changed, and comparative examples 1-9 were set up. Except for the different composition of the compound microbial agent, everything else was the same as in Example 3. Among them, Bacillus desertica (A), Azotobacter mollissima (B), and Pseudomonas rapeseed (C) were used, as shown in Table 1.

[0091] Table 1. Composition and proportion of functional strains in different comparative studies

[0092]

[0093] Comparative Example 10

[0094] A high-value-added fertilizer for fruits and vegetables containing fish protein has the same raw material composition and preparation method as in Example 3, except that it does not contain an active agent.

[0095] Comparative Example 11

[0096] A high-value-added fertilizer for fruits and vegetables containing fish protein has the same raw material composition and preparation method as in Example 3, except that the active agent is only fish protein powder.

[0097] Comparative Example 12

[0098] A high-value-added fertilizer for fruits and vegetables containing fish protein has the same raw material composition and preparation method as in Example 3, except that the active agent is only onion extract.

[0099] Comparative Example 13

[0100] A high-value-added fertilizer for fruits and vegetables containing fish protein has the same raw material composition and preparation method as in Example 3, except that the active agent is composed of fish protein powder and onion extract in a mass ratio of 4:1.

[0101] Comparative Example 14

[0102] A high-value-added fertilizer for fruits and vegetables containing fish protein is basically the same as that in Example 3 in terms of raw material composition and preparation method, except that the active agent is composed of fish protein powder and onion extract in a 1:1 mass ratio. Performance testing.

[0103] The antagonistic effects between the three strains screened in this invention were verified by streak plating. The results are as follows: Figure 1 As shown, the strains grew well at the cross intersection and did not produce any antagonistic effects.

[0104] Phosphate-solubilizing ability test: The composite microbial agents obtained in Example 3 and Comparative Examples 1-9 were adjusted with sterile water to make the bacterial suspensions of all strains uniform (all 1×10⁻⁶). 8 CFU·mL ‑1 20 μL of bacterial suspension was inoculated into the center of a PVK solid plate and incubated upside down in a 30°C incubator for 5 days. Each experimental group was treated three times. After 5 days, the colony diameter (D) and the diameter of the phosphate-solubilizing zone (d) were measured to calculate the phosphate solubilizing index (PSI). The amount of phosphate solubilized by the strain was determined using the molybdenum antimony colorimetric method.

[0105] PSI = (D + d) / D

[0106] In the formula, PSI is the phosphorus solubility index; D is the colony diameter (cm); d is the diameter of the phosphorus-solubilizing bacterial zone (cm); and PVK solid medium consists of: glucose 10.00 g, (NH4)2SO4 0.50 g, NaCl 0.30 g, KCl 0.30 g, MgSO4·7H2O 0.30 g, FeSO4·7H2O 0.03 g, MnSO4·H2O 0.03 g, Ca3(PO4) 5.00 g, and agar 15.00 g.

[0107] Table 2 Phosphorus dissolution test results

[0108]

[0109] As can be seen from Table 2 above, the phosphorus-solubilizing effect is best when the three strains of the present invention are mixed in equal proportions. Changing the proportion or composition of the strains will disrupt the microecological balance and weaken the phosphorus-solubilizing effect.

[0110] Field planting experiment 1

[0111] The tested crop variety was cherry tomato, "Hongfei No. 6".

[0112] Experimental Methods: The experiment consisted of 18 treatment groups: a blank control group (no fertilizer), Example 1-3 groups, and Comparative Examples 1-14 groups. Except for the blank control group, each treatment group received 50 kg / mu of fertilizer. Tomato seeds were first treated with seedling cultivation. Healthy and uniform seedlings were selected and planted on raised beds approximately 0.3 m high, with a plant spacing of 0.5 m. Each plot was 10.0 m². 2(1.0 m × 10.0 m), each treatment was repeated 3 times, randomized block design, and each plot was separated from the adjacent plot by field ridges (50 cm wide and 20 cm high).

[0113] Statistical analysis: After maturity, 5 tomato plants were randomly selected from each treatment group to measure and record their plant height and stem diameter. The results are as follows: Figure 2-3 As shown in the table; harvesting begins after the fruit matures, and the yield of each plot is recorded in batches by plot size and weighed. Quality determination: During the peak fruiting period of tomatoes, approximately 500g of two bunches of tomatoes of similar maturity and free from pests and diseases are collected from each plot and brought back to the laboratory. The vitamin C content of the fruit is determined by the 2,6-dichlorophenolindophenol titration method, the soluble sugar content is determined by the sulfuric acid-anthrone colorimetric method, the titratable acid content is determined by the acid-base neutralization titration method, and the nitrate content is determined by ultraviolet spectrophotometry. The sugar-acid ratio is calculated as soluble sugar content / titratable acid content. The results are shown in Table 3.

[0114] Table 3. Results of tomato yield and quality in different treatment groups

[0115]

[0116] As can be seen from the results in Table 3 above, the tomato yields obtained from Examples 1-3 of the present invention were significantly higher than those of the blank control group and Comparative Examples 1-14, and the vitamin C content and sugar-acid ratio were also optimal, indicating that the present invention has significant effects on increasing yield and improving tomato quality. In Comparative Examples 1-9, the strain ratio was unbalanced, resulting in lower yields and quality compared to the Example groups. Data from Comparative Examples 10-14 show that the tomato yield and quality also deteriorated when the activator was absent or its ratio was imbalanced. This indicates that the synergistic effect of the activator (fish protein powder + onion extract 3:1) on microbial activity and nutrient release is indispensable.

[0117] Field planting experiment 2

[0118] The tested crop variety was strawberry, named "Hongyan".

[0119] Experimental Methods: The experiment consisted of 18 treatment groups: a blank control group (no fertilizer), Example 1-3 groups, and Comparative Examples 1-14 groups. Except for the blank control group, each treatment group received 50 kg / mu of fertilizer. Each treatment was replicated three times, and each plot was 89 m². 2 Protective rows were set up around each plot. The strawberries were planted on September 11, 2022, using double-row cultivation with a plant spacing of 20 cm × 25 cm, and 1,000 strawberry seedlings were planted in each plot. Except for the fertilization, other field management measures (such as flower and fruit thinning, old leaf removal, and disease and pest control) were consistent in all treatments.

[0120] Statistical analysis: Ten fruits with 90% skin coloring were randomly selected from each plot. The weight of each fruit was measured, and the sugar content was determined using a PAL-1 digital handheld miniature refractometer. The average value of the results was taken. Fruits were harvested as they ripened, and the marketable fruit weight was calculated by weighing them by plot. The total yield was accumulated and converted to the yield per acre. Specific results are shown in Table 4.

[0121] Table 4. Effects of different treatment groups on strawberry yield and quality.

[0122]

[0123] As can be seen from the results in Table 4 above, the average strawberry yield of Examples 1-3 of the present invention was 1822 kg / mu, with a single fruit weight greater than 46.3 g and a sugar content greater than 14.9%, all significantly better than the blank control and comparative examples. This indicates that the combination of the functional bacterial groups, activators, and carriers (diatomaceous earth, etc.) selected in the present invention significantly improves strawberry yield and quality. The performance of all comparative examples was lower than that of the examples, proving the irreplaceable nature of the strain ratio, activator ratio, and preparation method of the present invention.

[0124] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A fertilizer specifically for fruits and vegetables containing fish protein, characterized in that, It is made from the following raw materials in parts by weight: 40-60 parts of compound microbial inoculant, 10-15 parts of activator, 80-120 parts of diatomaceous earth, 40-60 parts of decomposed fungal residue, 10-20 parts of potassium sulfate, and 10-30 parts of humic acid; the activator is composed of fish protein powder and onion extract in a mass ratio of 3:1; the compound microbial inoculant is composed of *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae*, with a volume ratio of 1:1:1; the preservation number of *Bacillus desertica* is CGMCC No. 1.15888; the preservation number of *Azotobacter mollissima* is CGMCC No. 1.5340; and the preservation number of *Pseudomonas brassicae* is CGMCC No. 1.10355. The compound microbial agent was prepared by the following method: *Bacillus desertica*, *Azotobacter mollissima*, and *Pseudomonas brassicae* were thawed and activated respectively, and then single colonies were picked and inoculated into LB liquid medium, and cultured at 28-30℃ and 180-200 r / min until OD... 600 ≈1.0 yielded seed culture. The seed culture was inoculated into LB liquid medium at a rate of 2% and cultured at 28-30℃ and 180-200 rpm. The culture was continued until the colony activity reached 1×10⁻⁶. 8 cfu·mL ‑1 After the culture was completed, the culture solutions of the three bacteria were mixed in a volume ratio of 1:1:1 to obtain a compound microbial inoculant. The fish protein powder is prepared using the following method: Step 1: Take the by-products of fish product processing, wash them clean with water, freeze-dry them at low temperature, pulverize them, and pass them through a 100-mesh sieve to obtain solid powder; Step 2: Add the solid powder to the degreasing solvent, heat to 50-55℃ and stir magnetically for 1 hour, then centrifuge to separate the solid product. Wash the solid product with deionized water until neutral and dry it for later use. Step 3: Mix the solid product obtained in Step 2 with deionized water at a solid-liquid ratio of 1g:10ml to obtain a mixed solution. Inoculate the mixed solution with a compound of Saccharomyces boulardii and Bacillus coagulans. Adjust the temperature to 25-30℃ and the humidity to 70-75%. Ferment for 2-3 days. Filter the fermentation product to obtain the fermentation broth. Step 4: Adjust the pH of the fermentation broth obtained in Step 3 to 6.5-7.0, add a complex enzyme containing trypsin, papain and nattokinase to it for 6-8 hours of enzymatic hydrolysis at a temperature of 45-50℃, cool to room temperature after high-temperature inactivation, centrifuge at high speed, and freeze-dry the supernatant to obtain fish protein powder.

2. The fish protein-containing fertilizer for fruits and vegetables according to claim 1, characterized in that, The degreasing solvent is composed of a 1 mol / L NaOH solution and sodium dodecyl sulfate in a mass ratio of 10:0.5-1.

3. The fish protein-containing fertilizer for fruits and vegetables according to claim 1, characterized in that, In step 2, the mass ratio of solid powder to degreasing solvent is 1:5-6.

4. The fish protein-containing fertilizer for fruits and vegetables according to claim 1, characterized in that, In step 3, the mass ratio of Saccharomyces boulardii and Bacillus coagulans in the compound bacteria is 2:1; the inoculation amount of the compound bacteria is 5-10% of the mass of the solid product.

5. The fish protein-containing fertilizer for fruits and vegetables according to claim 1, characterized in that, The mass ratio of trypsin, papain, and nattokinase in the compound enzyme is (1-3):1:(0.3-0.6); the amount of the compound enzyme is 1.5% of the mass of the solid powder in step 1.

6. The fish protein-containing fertilizer for fruits and vegetables according to claim 5, characterized in that, The trypsin has an enzyme activity of 200,000 u / g; the papain has an enzyme activity of 200,000 u / g; and the nattokinase has an enzyme activity of 20,000 u / g.

7. The fish protein-containing fertilizer for fruits and vegetables according to claim 4, characterized in that, The effective viable count of *Saccharomyces boulardii* is 20 billion CFU / g, and the effective viable count of *Bacillus coagulans* is 10 billion CFU / g.

8. A method for preparing a special fertilizer for fruits and vegetables containing fish protein according to any one of claims 1-7, characterized in that, It includes the following steps: (1) Prepare fish protein powder and mix it with onion extract in a certain mass ratio to obtain an active agent for later use; (2) Preparation of compound microbial inoculants; (3) Mix the activator and the compound microbial agent evenly, and incubate at room temperature for 24 hours. Then add diatomaceous earth, decomposed bacterial residue, humic acid and potassium sulfate, mix evenly, dry at low temperature and package to obtain the final product.

Images