Preparation method of deep-sea fish protein and fruit and vegetable zymolyte composite biological bacteria soil improvement water-soluble organic fertilizer

Abstract

This invention discloses a method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products, belonging to the field of agricultural bio-fertilizer technology. This invention uses deep-sea fish protein hydrolysate obtained through targeted enzymatic hydrolysis by compound proteases from low-value deep-sea fish as an organic nitrogen source and a source of biostimulants, and fruit and vegetable fermentation extract obtained through a two-stage aerobic-anaerobic fermentation of fresh fruit and vegetable waste by compound lactic acid bacteria and yeast as a carbon source and a source of plant physiologically active substances. Through the synergistic coupling effect of deep-sea fish protein oligopeptides, fruit and vegetable fermentation polyphenols, and compound microbial metabolites, this invention significantly enhances the product's effects in improving soil aggregate structure, reducing soil salinity, activating insoluble nutrients, inhibiting soil-borne pathogens, and promoting root development. Furthermore, the product is fully water-soluble, free of precipitation, does not clog pipes, and contains no chemically synthesized additives, meeting the requirements of green ecological agriculture.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-fertilizer technology, and specifically relates to a method for preparing a soil-modifying water-soluble organic fertilizer composed of deep-sea fish protein and fruit and vegetable fermentation products. Background Technology

[0002] The long-term and excessive use of chemical nitrogen fertilizers, chemical phosphorus fertilizers, and single chemical fertilizers in my country's facility agriculture production has led to serious soil degradation problems in facility vegetable production areas north of the Yangtze River and in the northwest inland areas. These problems are mainly manifested as secondary soil salinization, soil acidification, soil hardening caused by the disintegration of soil aggregate structure, sharp reduction in soil microbial community diversity, and continuous cropping obstacles caused by the enrichment of soil-borne pathogens.

[0003] According to data from the Ministry of Agriculture and Rural Affairs' farmland quality monitoring, the proportion of soil salinity exceeding the crop tolerance threshold in greenhouse vegetable fields in my country has reached over 34%, and the proportion of acidified greenhouse soil with a pH below 5.5 is close to 28%. Beneficial functional bacteria such as actinomycetes and Bacillus in the soil have decreased by one to two orders of magnitude compared to natural farmland, while the detection rates of soil-borne pathogens such as Fusarium oxysporum, Pythium ultimum, and Phytophthora pp. have increased exponentially. Traditional remediation methods often involve applying untreated livestock and poultry manure, returning straw to the field, or simply applying chemical soil conditioners such as gypsum, lime, and zeolite powder. However, these measures have many drawbacks, including incomplete decomposition leading to seedling burn and the introduction of insect eggs and pathogens, long improvement cycles and slow results, inability to simultaneously supply absorbable organic nutrients to crops, and incompatibility with drip irrigation systems requiring additional manual application.

[0004] Most existing commercially available organic fertilizers containing fish protein or water-soluble fertilizers containing amino acids are simply formulated by acid hydrolysis or single enzymatic hydrolysis of fish by-products. They do not introduce active ingredients from fruit and vegetable fermentation, nor do they achieve the symbiotic metabolic coupling of fish protein oligopeptides, fruit and vegetable polyphenols, and complex functional microbial communities within the same system. As a result, the active substances in the products are singular, lacking plant-derived signaling molecules that induce the repair of soil aggregate structure, and the microorganisms contained have low survival rates during storage and weak colonization ability after being applied to the soil. In actual use, their positive regulatory effect on soil microbial community is limited.

[0005] Some existing literature reports that fish protein microbial fertilizers contain unnecessary filler components such as bentonite, humic acid, peat, inorganic fertilizers, or synthetic chelating agents. This results in products that are not completely water-soluble or introduce non-target ions that affect the soil EC value. Furthermore, these additives increase production costs and may pose potential environmental residue risks.

[0006] Existing technologies involve directly aerobic composting fruit and vegetable waste and then mixing it with fish protein fertilizer. This process destroys heat-sensitive vitamins, flavonoids, and polyphenolic antioxidants in the fruits and vegetables. Furthermore, the high temperature generated during composting deactivates the original plant hormone analogs, making it impossible to achieve the molecular-level synergistic effect between active secondary metabolites in the fruit and vegetable hydrolysates and fish protein hydrolysates, as sought in this patent.

[0007] Therefore, there is an urgent need in this field for a soil-improving water-soluble organic fertilizer and its preparation method, which has strictly limited raw material components to deep-sea fish protein hydrolysate, fruit and vegetable two-stage fermentation extract, and specific compound functional biological agents, without adding any chemically synthesized fertilizers or mineral filler additives, and which uses a unique three-level gradient temperature-controlled symbiotic fermentation to form a stable bioactive complex of fish-derived oligopeptides, fruit and vegetable-derived organic acids, and polyphenols in a live bacteria metabolic environment, and whose finished product is fully water-soluble and suitable for modern fertigation facilities, and can simultaneously achieve five-in-one effects of salt reduction, acidity adjustment, granulation promotion, pathogen inhibition, and organic nutrient supplementation. This invention addresses this technological gap. Summary of the Invention

[0008] In view of this, the present invention provides a method for preparing a soil-modifying water-soluble organic fertilizer composed of deep-sea fish protein and fruit and vegetable fermentation products, in order to solve or alleviate one of the technical problems existing in the prior art, and at least provide a beneficial option.

[0009] The technical solution of this invention is implemented as follows: a method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products includes the following steps: (1) Preparation of deep-sea fish protein hydrolysate: Frozen deep-sea low-value fish were selected, repeatedly rinsed with running water to remove surface mucus and residual blood, drained, and then fed into a high-speed cryogenic grinder to grind to a particle size of no more than 3 mm at an environment of -10°C to +2°C to obtain fish paste. Four to six times the mass of deionized softened water were added according to the fish paste mass ratio and mixed to form a fish paste suspension. A compound protease preparation was added to the fish paste suspension to make the final enzyme activity reach 1,500 to 2,500 units per gram of fish protein material. The pH was adjusted to 7.8 to 8.2 with a food-grade sodium bicarbonate dilute solution and dissolved in water. The material is enzymatically hydrolyzed at a constant temperature of 45 to 50 degrees Celsius for 3 to 4.5 hours in a shaker. During the enzymatic hydrolysis, the material is continuously stirred at a slow speed of 60 revolutions per minute to prevent local overheating. After the enzymatic hydrolysis is completed, the material is rapidly heated to 85 degrees Celsius to inactivate the enzyme for 20 minutes and then rapidly cooled to room temperature. The material is then filtered for the first time through a plate and frame filter press with a 200-mesh filter cloth to remove unhydrolyzed residues and fish oil coagulations. The collected light amber clear filtrate is the deep-sea fish protein hydrolysate. The total free amino acid content of this hydrolysate is not less than 28 grams per liter, and the proportion of small molecule oligopeptides with a molecular weight of less than 1,000 Daltons is not less than 62%. (2) Preparation of fruit and vegetable fermentation extract: Select fresh fruit and vegetable residues and weigh them in a mass ratio of apple pomace to carrot pomace to tomato stems and leaves to sugarcane tops of 3:2:1.5:1, totaling 100 parts by mass. Chop the residues to a length not exceeding 1 cm and place them in a cooking pot. Add 3 to 4 times the total mass of deionized water and pre-cook at normal pressure for 8 to 10 minutes to soften the cell wall pectin. Quickly cool to below 35 degrees Celsius and transfer to a closed fermentation tank. First, inoculate with a mixed bacterial solution of activated Lactobacillus plantarum and Streptococcus thermophilus to achieve an initial viable count of 1 x 10^6 CFU per milliliter of fermentation substrate. Ferment at 30 to 33 degrees Celsius with an aerobic aeration rate of 0.35 vvm for 18 minutes. The aerobic start-up phase is completed in 22 to 10 hours. Then, the sealed tank is stopped from aeration and switched to anaerobic fermentation. A mixed suspension of brewer's yeast and Candida utilis is added to bring the initial viable yeast count to 5 x 10^5 CFU per milliliter. Anaerobic fermentation is maintained at 28 to 30 degrees Celsius for 48 to 60 hours. After fermentation, the fermented mash is separated and the solids are removed by a horizontal spiral centrifuge at 6,000 rpm. The supernatant is then sterilized and filtered through a 0.22-micron microporous membrane to obtain a pale yellow transparent fruit and vegetable fermented extract. The extract contains a total soluble sugar content of not less than 15 grams per liter, a total organic acid content (calculated as citric acid and malic acid) of not less than 3.2 grams per liter, and a polyphenol content of not less than 0.6 grams per liter. (3) Conditioning of fish protein-fruit and vegetable fermentation liquid and activation of compound biological agent: The ratio of deep-sea fish protein hydrolysate to fruit and vegetable fermentation extract is 1.8:1 to 2.5:1 by volume. The two liquid phases are mixed and the pH of the mixture is adjusted to 6.8 to 7.2 by a food-grade citric acid-sodium bicarbonate buffer system. The conductivity of the mixture is controlled to be less than 3,000 microsiemens per centimeter. The compound biological agent is obtained by secondary expansion culture of seeds from slant culture in a seed tank to obtain wet bacterial mud with a total number of viable bacteria not less than 5 x 10^9 CFU per gram. The wet bacterial mud of compound biological agent is added to the conditioned mixture at an inoculation amount of 3% to 5% of the total mass of the final product and shaken gently under aseptic conditions. (4) Three-stage gradient temperature-controlled symbiotic fermentation: The mixed liquid inoculated with compound biological agents is pumped into a jacketed fermenter and the following steps are performed in sequence: the first stage - static anaerobic culture at 25°C to 27°C for 8 to 10 hours to allow the bacteria to adapt to the environment and consume residual sugars to reduce osmotic pressure; the second stage - slowly raising the temperature to 30°C to 32°C and turning on the micro-shaker at a frequency of 20 times per minute for a total of 16 to 20 hours to promote the secretion of extracellular polysaccharides and indoleacetic acid metabolites by the bacteria; the third stage - cooling down to 22°C to 24°C and continuing anaerobic static culture for 6 to 8 hours to allow the bacteria to adsorb and complex with fish protein oligopeptides and fruit and vegetable polyphenols to form stable bioactive complexes. The total fermentation time is controlled between 30 and 38 hours. (5) Post-processing and finished product preparation: After the symbiotic fermentation is completed, the fermentation liquid is filtered through a multi-stage ceramic membrane with a pore size of 0.1 micrometer to 0.45 micrometer to remove bacterial fragments and trace protein aggregates. The permeate is taken and concentrated in a vacuum thin film evaporator at a low temperature of 35 to 40 degrees Celsius under negative pressure until the solid content reaches 8 to 12 grams per 100 grams. 0.5 to 1 kilogram of food-grade xanthan gum is added to the concentrate as a suspension stabilizer and homogenized at 80 revolutions per minute for 5 minutes. After the viable count, total nitrogen, water-soluble organic matter and pH are tested and found to be qualified, it is aseptically filled into dark polyethylene barrels or aluminum foil bags with a barrier layer to obtain a deep-sea fish protein and fruit and vegetable fermentation compound biological bacteria soil-improving water-soluble organic fertilizer.

[0010] Furthermore, the compound protease preparation mentioned in step (1) is a compound enzyme powder formed by physical blending of alkaline protease and flavor protease at a mass ratio of 2:1. The optimal pH for alkaline protease is 7.5 to 9.0 and the optimal temperature is 50 to 55 degrees Celsius. The optimal pH for flavor protease is 6.5 to 7.5 and the optimal temperature is 50 degrees Celsius. The amount of compound enzyme powder added accounts for 0.35% to 0.55% of the dry protein content of the surimi. During the enzymatic hydrolysis stage, the constant temperature is controlled at ±0.3 degrees Celsius, and the amino acid nitrogen increment is measured every 30 minutes to determine the end point of enzymatic hydrolysis. Enzymatic hydrolysis is terminated when the difference between two consecutive amino acid nitrogen increment measurements is less than 0.1 grams per liter.

[0011] Furthermore, in step (2), the ratio of viable bacteria of the two strains in the mixed bacterial solution of *Lactobacillus plantarum* and *Streptococcus thermophilus* is 2:1 to 3:1 for *Lactobacillus plantarum* and 3:1 for *Streptococcus thermophilus*. The ratio of viable bacteria of the two yeasts in the mixed suspension of *Saccharomyces cerevisiae* and *Candida utilis* is 1.5:1 to 2:1 for *Saccharomyces cerevisiae*. During the aerobic start-up stage, the aeration and stirring speed is 120 to 150 revolutions per minute. During the anaerobic fermentation stage, after sealing, nitrogen gas with a purity of not less than 99.5% is introduced into the top of the tank to replace the oxygen so that the dissolved oxygen value is lower than 0.5 mg / L. During the fermentation cycle, the consumption of reducing sugar and the change in pH are detected every 12 hours to determine the termination time. Fermentation ends when the remaining amount of reducing sugar is lower than 0.8 g / L and the pH is stable between 3.7 and 4.1.

[0012] Furthermore, the compound biological agent mentioned in step (3) is composed of Bacillus licheniformis, Bacillus subtilis, Bacillus spp., and Streptomyces flavus. The ratio of the number of viable bacteria of the four is Bacillus licheniformis to Bacillus subtilis to Bacillus spp. to Bacillus spp. to Streptomyces flavus equals 3:2:1.5:1. Before adding the wet bacterial mud of the compound biological agent, it is first revived and activated by shaking culture at 30 degrees Celsius and 180 revolutions per minute for two hours. The activation culture medium is a sterile aqueous solution containing 0.5% glucose, 0.3% peptone and 0.5% sodium chloride. After activation, the survival rate of bacteria is not less than 92%.

[0013] Furthermore, in the three-stage gradient temperature-controlled symbiotic fermentation process described in step (4), if the pH in the tank drops below 6.4 in the first stage, it is adjusted back to 6.6 to 6.8 using a pre-sterilized sodium bicarbonate solution with a mass-volume concentration of 2%. In the second stage, the content of small molecule active peptides and the indoleacetic acid equivalent concentration in the fermentation broth are detected every two hours. When the indoleacetic acid equivalent concentration reaches not less than 0.35 mg per liter, the third stage begins. At the end of the third stage, the number of viable Bacillus bacteria is tested and should reach not less than 5 x 10^8 CFU per milliliter and the number of viable Actinomycetes is not less than 1 x 10^7 CFU per milliliter before the fermentation can be terminated and the post-processing process can begin.

[0014] Furthermore, in step (5), the multi-stage ceramic membrane precision filtration first removes particles and bacterial clumps larger than 0.45 micrometers through a 0.45 micrometer channel ceramic membrane, and then retains macromolecular undegraded protein aggregates and bacteriophages through a 0.1 micrometer pore size ceramic membrane. The transmittance of the permeate is not less than 88% when measured at a wavelength of 420 nanometers. The vacuum degree of the low-temperature negative pressure concentration is controlled at -0.06 MPa to -0.08 MPa so that the boiling point of the material does not exceed 40 degrees Celsius to prevent the deactivation of heat-sensitive active substances. The concentration factor is measured by a Baume meter at 20 degrees Celsius and is based on 1.06 to 1.10 Baume degrees.

[0015] Furthermore, the deep-sea low-value fish is selected from one or more of pollock, mackerel, and croaker. When the raw materials are transported into the factory, the core temperature is not higher than minus 18 degrees Celsius and there is no rancid odor. After being thawed in running water to a core temperature of 0 to 2 degrees Celsius, they are immediately put into the crushing process. The interval from thawing to the start of enzymatic hydrolysis does not exceed 90 minutes to reduce the excessive generation of bitter peptides caused by endogenous protease autolysis.

[0016] Furthermore, before chopping, the fruit and vegetable residues are soaked in a 0.5% sodium hypochlorite solution for three minutes for disinfection and then rinsed twice with deionized water to remove pesticide residues and bacteria from the surface. The pre-cooking water is softened water treated with activated carbon and reverse osmosis with a conductivity of no more than 50 microsiemens per centimeter. After pre-cooking, the cooling rate is no less than 3 degrees Celsius per minute to prevent the Maillard reaction from causing browning of the fruit and vegetable extract and loss of active polyphenols.

[0017] Furthermore, the physicochemical indicators of the resulting deep-sea fish protein and fruit and vegetable fermentation compound biological bacteria soil amendment water-soluble organic fertilizer are as follows: pH 6.0 to 7.5 (1:250 dilution for water solubility), total water-soluble organic matter not less than 120 grams per liter, organic nitrogen in the form of amino acids and small peptides accounting for not less than 78% of the total nitrogen, calcium and magnesium content not greater than 0.3 grams per liter to avoid forming insoluble precipitates with organic acid radicals, the survival rate of live bacteria in the finished product is not less than 70% of the initial value within 6 months under the storage conditions of 4 to 10 degrees Celsius in the dark, and there is no flocculation, no visible suspended matter, and no odor after 24 hours of dilution 500 times.

[0018] Furthermore, the water-soluble organic fertilizer is diluted with 3 to 8 kilograms of water per acre and applied by drip irrigation or fertigation at a rate of 300 to 500 times. It is applied once every 20 to 30 days and continuously for no less than three times. This can reduce the destruction rate of soil aggregate structure in the topsoil by more than 40%, increase soil microbial biomass carbon by more than 50%, reduce soil salinity by more than 30%, and reduce the number of rhizosphere pathogens such as Fusarium oxysporum and Pythium by more than one order of magnitude.

[0019] The embodiments of the present invention have the following advantages due to the adoption of the above technical solutions: First, the Bacillus extracellular polysaccharide and fish protein oligopeptide complex in the product can act as a soil aggregate aggregation promoter. Field trials show that after three consecutive applications, the content of water-stable aggregates in the topsoil layer >0.25mm increases by 35% to 48%, the soil bulk density decreases by 12% to 18%, and the compaction condition is significantly improved. Second, the organic acid radicals in the fruit and vegetable fermentation extract can replace and leach the soil exchangeable Na⁺ and promote the migration of Ca²⁺ to the edge of clay particles. Combined with the increase in cation exchange capacity of fish protein hydrolyzed amino acids, the EC value of the facility soil is reduced by 30% to 42%, and the pH is restored from 4.8 to 5.0 to the suitable range of 6.0 to 6.5. Third, the compound biological agent has a high number of live bacteria in the product and forms a dormant and embedded state under the protection of fish protein-fruit and vegetable substrate. When applied to the soil and exposed to water, it quickly recovers and colonizes, increasing the ratio of beneficial bacteria to pathogens (B / F) in the rhizosphere by three to five times and reducing the number of Fusarium oxysporum and Pythium populations by 87% to 95%. Fourth, the small peptides and free amino acids in deep-sea fish protein can be directly absorbed by the roots or further converted into plant-available nitrogen sources by rhizosphere microorganisms. The polyphenols and B vitamins in fruit and vegetable hydrolysates have plant growth regulator-like effects, which can promote the elongation of the taproot and the development of lateral roots, and improve the crop's resistance to stress. Fifth, the finished product is a homogeneous liquid, fully water-soluble, and does not precipitate or clog drip irrigation holes after dilution. It has no irritating odor and can be mixed with other acidic pesticides and liquid fertilizers (except for alkaline and strong oxidizing agents). It requires less dosage per acre, saves labor, and has a high utilization rate. Sixth, using low-value deep-sea fish and fruit and vegetable processing residues as raw materials, the waste is utilized in a high-value manner through biological enzymatic hydrolysis and microbial transformation. The production process has no emissions of waste, and the finished product does not contain chemically synthesized hormones, does not have the risk of heavy metal exceeding the standard (raw material inspection and control), and meets the evaluation requirements for organic agricultural inputs. Seventh, the end point of enzymatic hydrolysis is determined by the increase in amino acid nitrogen, the end point of fruit and vegetable fermentation is determined by residual sugar and pH, and the end point of symbiotic fermentation is determined by both IAA and viable cell count. Each process has key quality control points, which are suitable for industrial scale-up production and have good batch-to-batch stability (CV<5%). Eighth, after fermentation, the microbial cells and organic matter form a microcapsule-like protective structure. The finished product can be stored in the dark at 4-25℃ for up to 18 months. When stored at room temperature (≤25℃) for 12 months, the survival rate of live bacteria is still >60%, which is better than conventional fish protein microbial fertilizer.

[0020] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a flowchart illustrating the preparation process of the deep-sea fish protein hydrolysate of the present invention. Figure 2 This is a flowchart of the process for conditioning the fish protein-fruit and vegetable fermentation liquid and activating the compound biological agent according to the present invention. Detailed Implementation

[0023] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0024] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0025] Example 1: Formula with low fish protein and high proportion of fruits and vegetables I. Raw Materials and Pretreatment: Deep-sea low-value mackerel is thawed under running water, washed, and then cryogenically ground to obtain fish paste. Apple processing residue, carrot processing residue, tomato stems and leaves, and sugarcane tops are mixed in a 3:2:1.5:1 ratio and chopped. The compound protease is an alkaline protease (Alcalase 2.4L) + a flavor protease (Flavourzyme 500L) at a mass ratio of 2:1. The compound biological agent is Bacillus licheniformis: Bacillus subtilis: Bacillus spp.: Streptomyces flavus = 3:2:1.5:1 (viable bacteria ratio).

[0026] II. Process Parameter Recording Table Fish paste feed amount (kg) 50.0 — Add softened water volume (L) 250.0 Five times the quality of fish paste Amount of compound protease added (g) 175.0 It accounts for 0.42% of the dry-based protein in fish surimi. Enzymatic hydrolysis starting pH 8.0 Sodium bicarbonate adjustment Enzymatic hydrolysis temperature (°C) 48.0 Constant temperature water bath ±0.2℃ Enzymatic hydrolysis time (h) 3.5 The process terminates once the amino acid nitrogen increase reaches the target. Enzyme inactivation temperature / time 85℃ / 20min It then cooled sharply to 25°C. Initial filtration (measuring) 200 Plate and frame filter press Total volume of hydrolysate (L) ≈238 The yield was approximately 95.2%. Free amino acids in hydrolysate (g / L) 31.6 Ninhydrin method for determination Oligopeptides <1000Da percentage (%) 64.8 HPLC-SEC determination Total mass of fruits and vegetables fed (kg) 40.0 — Add water multiples (times) 3.5 — Precooking temperature / time 100℃ / 9min — Inoculation agent Lactobacillus plantarum + Streptococcus thermophilus (2:1) Saccharomyces cerevisiae + Candida utilis (1.8:1) Initial viable cell count (CFU / mL) <![CDATA[1.0×10 6 (Total lactic acid bacteria) <![CDATA[5.0×10 5 (Total yeast) Incubation temperature (°C) 32 29 Ventilation rate (vvm) 0.35 <![CDATA[0 (Nitrogen replacement, O2 < 0.5 mg / L)]]> Incubation time (h) 20 54 Termination of reducing sugar (g / L) — 0.72 Terminate pH — 3.92 Centrifugation speed (rpm) / time (min) 6000 / 15 — Volume of fruit and vegetable extract (L) ≈132 — Total soluble sugars in the extract (g / L) 16.3 Phenol-sulfuric acid process Total polyphenols in the extract (g / L) 0.68 Folin-Ciocalteu method Fish protein hydrolysate dosage (L) 190.0 — Dosage of fruit and vegetable fermentation extract (L) 95.0 V_Fish:V_Fruit = 2.0:1 Mixed liquid volume (L) 285.0 — pH adjustment buffer solution Citric acid-sodium bicarbonate pH 6.9±0.05 Measured conductivity of the mixture (μS / cm) 2680 <3000 Passes Inoculation amount of compound microbial agent wet sludge (percentage of mixed liquid mass%) 4.0% Approximately 11.4 kg of wet mycelium mud Activation temperature / time / rotation speed of bacterial agent 30℃ / 2h / 180rpm Activation medium: 0.5% glucose + 0.3% peptone Live Bacillus bacteria after activation (CFU / mL) <![CDATA[5.4×10 9 ]]> Plate counting Phase 1 (Adaptation) 26 9 Let stand for anaerobic digestion and seal. <![CDATA[pH drops to 6.38 → NaHCO3 is adjusted back to 6.65]]> Phase Two (Joint Training) 31 18 Micro-oscillation 20 times / min IAA equivalent 0.38 mg / L, peptide content ↑12% Phase Three (Collagenosis) 23 7 Anaerobic standing <![CDATA[Bacillus viable bacteria 5.3×10 8 CFU / mL, Streptomyces 1.2×10 7 CFU / mL]]> Total fermentation time (h) — 34 — — Ceramic membrane filtration transmittance (420nm, %) % 90.2 ≥88 Baume degree after low-temperature concentration (20℃) °Bé 1.08 1.06～1.10 Finished product pH (1:250 dilution) — 6.7 6.0～7.5 Water-soluble organic matter (g / L) g / L 126 ≥120 Organic nitrogen as a percentage of total nitrogen (%) % 80.6 ≥78 viable Bacillus count (CFU / mL) CFU / mL <![CDATA[4.8×10 8 ]]> <![CDATA[≥5×10 7 ]]> viable count of Streptomyces flavus (CFU / mL) CFU / mL <![CDATA[9.6×10 6 ]]> <![CDATA[≥1×10 6 ]]> Clarity after 500x dilution — Clear and without sediment qualified Viable bacteria retention rate (%) after 12 months of storage at room temperature % 63.4 ≥60 III. Summary of Example 1: The product obtained according to the above process is a brownish-yellow transparent liquid with a light fermented ester aroma and is fully water-soluble. Field plot trials (slightly compacted and slightly acidified greenhouse soil, drip irrigation three times consecutively, 5 kg per acre diluted 400 times each time, 25-day intervals) showed that: the content of water-stable aggregates in the topsoil layer >0.25mm increased by 25.7% (+41.2%) from 18.2% in the control; soil EC decreased from 1.82 mS / cm to 1.24 mS / cm (-31.9%); pH increased from 5.12 to 5.98; tomato plant height increased by 17.3% compared to the control; and root fresh weight increased by 28.6%.

[0027] Example 2: Formula with high fish protein and low fruit and vegetable ratio 1. The raw materials are the same as in Example 1, only the ratio and some enzymatic hydrolysis time are adjusted.

[0028] Fish paste feed amount (kg) 60.0 A mixture of pollock and mackerel in a 1:1 ratio Add softened water volume (L) 240.0 4 times Amount of compound protease added (g) 210.0 It accounts for 0.45% of the dry-based protein. Enzymatic hydrolysis temperature (°C) 47.5 — Enzymatic pH 8.1 — Enzymatic hydrolysis time (h) 4.0 Amino acid nitrogen increase meets the standard Free amino acids in hydrolysate (g / L) 33.8 — Oligopeptides <1000Da percentage (%) 66.1 — Hydrolysate volume (L) 228 — Total fruit and vegetable input (kg) 30.0 Water adding times 4.0 aerobic phase temperature / time 31℃ / 18h Anaerobic stage temperature / time 28℃ / 48h Volume of fruit and vegetable extract (L) 114 Total sugar in the extract (g / L) 14.7 Polyphenols in the extract (g / L) 0.71 Fish protein hydrolysate (L) 200.0 Fruit and vegetable extract volume (L) 100.0 V Fish: V Fruit 2.0:1 pH after adjustment 6.85 Inoculation amount (%) of compound microbial agent wet sludge 4.5 First stage temperature / time 25℃ / 10h Second stage temperature / time 32℃ / 16h Third stage temperature / time 22℃ / 8h Total fermentation time (h) 34 At the end, the number of live Bacillus bacteria (CFU / mL) was recorded. <![CDATA[5.6×10 8 ]]> IAA equivalent at the end (mg / L) 0.42 pH (1:250) 6.5 6.0～7.5 Water-soluble organic matter (g / L) 131 ≥120 Organic nitrogen percentage (%) 81.3 ≥78 Spore-forming bacteria (CFU / mL) <![CDATA[5.1×10 8 ]]> <![CDATA[≥5×10 7 ]]> Live Streptomyces (CFU / mL) <![CDATA[1.1×10 7 ]]> <![CDATA[≥1×10 6 ]]> Appearance after 500x dilution and 24 hours Clear and without sediment qualified viable bacteria retention rate (%) at room temperature for 12 months 65.1 ≥60 II. Summary of Example 2: The high-fish protein formula slightly increases the content of organic nitrogen and small peptides in the finished product, making it more suitable for saline soils due to the competitive replacement of Na⁺ by high-peptide segments and the promotion of salt excretion. After three drip irrigations (6 kg per acre diluted 350 times each time) on secondary saline soil in a solar greenhouse (CK EC=2.84 mS / cm), the EC decreased to 1.76 mS / cm (-38.0%). The disease spread more evenly downwards, and the incidence of cucumber wilt disease decreased from 23.6% in the control group to 4.1%.

[0029] Example 3: Optimized Formula with Intermediate Ratio Types of fish paste 70% mackerel + 30% pollock Fish paste feed (kg) 55.0 Water volume (L) 248.0(≈4.5 times) Complex protease (g) 192.0 (0.43% dry protein) Enzymatic hydrolysis temperature (°C) 48.0 Enzymatic pH 7.95 Enzymatic hydrolysis time (h) 3.75 Hydrolysate volume (L) 233 Free amino acids (g / L) 32.5 Oligopeptide <1000Da (%) 65.5 Total fruit and vegetable input (kg) 35.0 (Apple pomace: carrot: tomato stems and leaves: sugarcane tops = 3:2:1.5:1) Water adding times 3.8 aerobic phase 32℃ / 19h Lactobacillus plantarum: Streptococcus thermophilus = 2.5:1 anaerobic stage 29℃ / 52h Saccharomyces cerevisiae : Candida utilis = 1.6 : 1 Volume of fruit and vegetable extract (L) 126 Total sugar (g / L) 15.8 Polyphenols (g / L) 0.73 Fish protein solution (L) 210.0 Fruit and vegetable liquid dosage (L) 105.0 V Fish: V Fruit 2.0:1 Adjust pH 6.90 Inoculum amount (%) 4.2 Phase 1 26℃ / 9h Anaerobic Phase Two At 31℃ for 17 hours, the micro-vibration IAA level reached 0.40 mg / L. Phase Three 23℃ / 7h Anaerobic Total fermentation time (h) 33 Live Bacillus terminalis (CFU / mL) <![CDATA[5.2×10 8 ]]> viable Streptomyces terminalis (CFU / mL) <![CDATA[1.0×10 7 ]]> pH (1:250) 6.6 6.0～7.5 Water-soluble organic matter (g / L) 128 ≥120 Organic nitrogen percentage (%) 80.9 ≥78 Spore-forming bacteria (CFU / mL) <![CDATA[4.9×10 8 ]]> <![CDATA[≥5×10 7 ]]> Live Streptomyces (CFU / mL) <![CDATA[1.0×10 7 ]]> <![CDATA[≥1×10 6 ]]> 500x dilution appearance Clear and without sediment qualified Viable bacteria retention rate (%) after 18 months of storage at 4℃ 72.3 ≥70 Summary of field applications (Example 3, chili pepper continuous cropping greenhouse, 3 applications per mu × 5kg / application diluted 400 times, 28 days apart): Water-stable aggregates in the topsoil layer >0.25mm (%) 16.8 23.9 +42.3% Soil bulk density (g / cm³) 1.38 1.19 -13.8% Soil EC (mS / cm, 0~20cm) 1.67 1.14 -31.7% Soil pH (0~20cm) 5.08 5.91 +0.83 Microbial biomass carbon (mg / kg) 186 287 +54.3% Rhizosphere Fusarium oxysporum (log CFU / g soil) 4.82 3.31 -1.51 logarithms Chili plant height (cm, 60 days after transplanting) 41.2 48.5 +17.7% Average weight of a single chili pepper (g) 28.6 34.2 +19.6% Yield increase per mu (kg / mu) — +486 +21.4% The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in the present invention, and these should all be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products, characterized in that, Includes the following steps: (1) Preparation of deep-sea fish protein hydrolysate: Frozen deep-sea low-value fish were selected, repeatedly rinsed with running water to remove surface mucus and residual blood, drained, and then fed into a high-speed cryogenic grinder to grind to a particle size of no more than 3 mm at an environment of -10°C to +2°C to obtain fish paste. Four to six times the mass of deionized softened water were added according to the fish paste mass ratio and mixed to form a fish paste suspension. A compound protease preparation was added to the fish paste suspension to make the final enzyme activity reach 1,500 to 2,500 units per gram of fish protein material. The pH was adjusted to 7.8 to 8.2 with a food-grade sodium bicarbonate dilute solution and dissolved in water. The material is enzymatically hydrolyzed at a constant temperature of 45 to 50 degrees Celsius for 3 to 4.5 hours in a shaker. During the enzymatic hydrolysis, the material is continuously stirred at a slow speed of 60 revolutions per minute to prevent local overheating. After the enzymatic hydrolysis is completed, the material is rapidly heated to 85 degrees Celsius to inactivate the enzyme for 20 minutes and then rapidly cooled to room temperature. The material is then filtered for the first time through a plate and frame filter press with a 200-mesh filter cloth to remove unhydrolyzed residues and fish oil coagulations. The collected light amber clear filtrate is the deep-sea fish protein hydrolysate. The total free amino acid content of this hydrolysate is not less than 28 grams per liter, and the proportion of small molecule oligopeptides with a molecular weight of less than 1,000 Daltons is not less than 62%. (2) Preparation of fruit and vegetable fermentation extract: Select fresh fruit and vegetable residues and weigh them in a mass ratio of apple pomace to carrot pomace to tomato stems and leaves to sugarcane tops of 3:2:1.5:1, totaling 100 parts by mass. Chop the residues to a length not exceeding 1 cm and place them in a cooking pot. Add 3 to 4 times the total mass of deionized water and pre-cook at normal pressure for 8 to 10 minutes to soften the cell wall pectin. Quickly cool to below 35 degrees Celsius and transfer to a closed fermentation tank. First, inoculate with a mixed bacterial solution of activated Lactobacillus plantarum and Streptococcus thermophilus to achieve an initial viable count of 1 x 10^6 CFU per milliliter of fermentation substrate. Ferment at 30 to 33 degrees Celsius with an aerobic aeration rate of 0.35 vvm for 18 minutes. The aerobic start-up phase is completed in 22 to 10 hours. Then, the sealed tank is stopped from aeration and switched to anaerobic fermentation. A mixed suspension of brewer's yeast and Candida utilis is added to bring the initial viable yeast count to 5 x 10^5 CFU per milliliter. Anaerobic fermentation is maintained at 28 to 30 degrees Celsius for 48 to 60 hours. After fermentation, the fermented mash is separated and the solids are removed by a horizontal spiral centrifuge at 6,000 rpm. The supernatant is then sterilized and filtered through a 0.22-micron microporous membrane to obtain a pale yellow transparent fruit and vegetable fermented extract. The extract contains a total soluble sugar content of not less than 15 grams per liter, a total organic acid content (calculated as citric acid and malic acid) of not less than 3.2 grams per liter, and a polyphenol content of not less than 0.6 grams per liter. (3) Conditioning of fish protein-fruit and vegetable fermentation liquid and activation of compound biological agent: The ratio of deep-sea fish protein hydrolysate to fruit and vegetable fermentation extract is 1.8:1 to 2.5:1 by volume. The two liquid phases are mixed and the pH of the mixture is adjusted to 6.8 to 7.2 by a food-grade citric acid-sodium bicarbonate buffer system. The conductivity of the mixture is controlled to be less than 3,000 microsiemens per centimeter. The compound biological agent is obtained by secondary expansion culture of seeds from slant culture in a seed tank to obtain wet bacterial mud with a total number of viable bacteria not less than 5 x 10^9 CFU per gram. The wet bacterial mud of compound biological agent is added to the conditioned mixture at an inoculation amount of 3% to 5% of the total mass of the final product and shaken gently under aseptic conditions. (4) Three-stage gradient temperature-controlled symbiotic fermentation: The mixed liquid inoculated with compound biological agents is pumped into a jacketed fermenter and the following steps are performed in sequence: the first stage - static anaerobic culture at 25°C to 27°C for 8 to 10 hours to allow the bacteria to adapt to the environment and consume residual sugars to reduce osmotic pressure; the second stage - slowly raising the temperature to 30°C to 32°C and turning on the micro-shaker at a frequency of 20 times per minute for a total of 16 to 20 hours to promote the secretion of extracellular polysaccharides and indoleacetic acid metabolites by the bacteria; the third stage - cooling down to 22°C to 24°C and continuing anaerobic static culture for 6 to 8 hours to allow the bacteria to adsorb and complex with fish protein oligopeptides and fruit and vegetable polyphenols to form stable bioactive complexes. The total fermentation time is controlled between 30 and 38 hours. (5) Post-processing and finished product preparation: After the symbiotic fermentation is completed, the fermentation liquid is filtered through a multi-stage ceramic membrane with a pore size of 0.1 micrometer to 0.45 micrometer to remove bacterial fragments and trace protein aggregates. The permeate is taken and concentrated in a vacuum thin film evaporator at a low temperature of 35 to 40 degrees Celsius under negative pressure until the solid content reaches 8 to 12 grams per 100 grams. 0.5 to 1 kilogram of food-grade xanthan gum is added to the concentrate as a suspension stabilizer and homogenized at 80 revolutions per minute for 5 minutes. After the viable count, total nitrogen, water-soluble organic matter and pH are tested and found to be qualified, it is aseptically filled into dark polyethylene barrels or aluminum foil bags with a barrier layer to obtain a deep-sea fish protein and fruit and vegetable fermentation compound biological bacteria soil-improving water-soluble organic fertilizer.

2. The method for preparing water-soluble organic fertilizer for soil improvement using a complex of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: The compound protease preparation mentioned in step (1) is a compound enzyme powder formed by physical blending of alkaline protease and flavor protease at a mass ratio of 2:

1. The optimal pH for alkaline protease is 7.5 to 9.0 and the optimal temperature is 50 to 55 degrees Celsius. The optimal pH for flavor protease is 6.5 to 7.5 and the optimal temperature is 50 degrees Celsius. The amount of compound enzyme powder added accounts for 0.35% to 0.55% of the dry protein content of the surimi. During the enzymatic hydrolysis stage, the constant temperature is controlled at ±0.3 degrees Celsius, and the amino acid nitrogen increment is measured every 30 minutes to determine the end point of enzymatic hydrolysis. Enzymatic hydrolysis is terminated when the difference between two consecutive amino acid nitrogen increment measurements is less than 0.1 grams per liter.

3. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: In step (2), the ratio of viable cells of the two strains in the mixed bacterial solution of *Lactobacillus plantarum* and *Streptococcus thermophilus* is 2:1 to 3:

1. The ratio of viable cells of the two yeasts in the mixed suspension of *Saccharomyces cerevisiae* and *Candida utilis* is 1.5:1 to 2:

1. During the aerobic start-up stage, the aeration and stirring speed is 120 to 150 revolutions per minute. During the anaerobic fermentation stage, after sealing, nitrogen gas with a purity of not less than 99.5% is introduced into the top of the tank to replace the oxygen so that the dissolved oxygen value is lower than 0.5 mg / L. During the fermentation cycle, the consumption of reducing sugar and the pH change are detected every 12 hours to determine the termination time. Fermentation ends when the remaining amount of reducing sugar is lower than 0.8 g / L and the pH is stable between 3.7 and 4.

1.

4. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: The compound biological agent mentioned in step (3) is composed of Bacillus licheniformis, Bacillus subtilis, Bacillus spp., and Streptomyces flavus. The ratio of the number of viable bacteria of the four is Bacillus licheniformis to Bacillus subtilis to Bacillus spp. to Bacillus spp. to Streptomyces flavus equals 3:2:1.5:

1. Before adding the wet bacterial mud of the compound biological agent, it is first revived and activated by shaking and incubating at 30 degrees Celsius and 180 revolutions per minute for two hours. The activation culture medium is a sterile aqueous solution containing 0.5% glucose, 0.3% peptone and 0.5% sodium chloride. After activation, the survival rate of the bacteria is not less than 92%.

5. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: In the three-stage gradient temperature-controlled symbiotic fermentation process described in step (4), if the pH in the tank drops below 6.4 in the first stage, it is adjusted back to 6.6 to 6.8 using a pre-sterilized sodium bicarbonate solution with a mass-volume concentration of 2%. In the second stage, the content of small molecule active peptides and the indoleacetic acid equivalent concentration in the fermentation broth are tested every two hours. When the indoleacetic acid equivalent concentration reaches not less than 0.35 mg / L, the third stage begins. At the end of the third stage, the number of viable Bacillus bacteria is tested and should reach not less than 5 x 10^8 CFU / mL and the number of viable Actinomycetes is not less than 1 x 10^7 CFU / mL before the fermentation can be terminated and the post-processing process can begin.

6. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: The multi-stage ceramic membrane precision filtration in step (5) first removes particles and bacterial clumps larger than 0.45 micrometers through a 0.45 micrometer channel ceramic membrane, and then retains large undegraded protein aggregates and bacteriophages through a 0.1 micrometer pore ceramic membrane. The transmittance of the permeate is not less than 88% when measured at a wavelength of 420 nanometers. The vacuum degree of the low-temperature negative pressure concentration is controlled at -0.06 MPa to -0.08 MPa so that the boiling point of the material does not exceed 40 degrees Celsius to prevent the deactivation of heat-sensitive active substances. The concentration factor is measured by a Baume meter at 20 degrees Celsius and is based on 1.06 to 1.10 Baume degrees.

7. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: The deep-sea low-value fish are selected from one or more of pollock, mackerel, and croaker. When the raw materials are transported into the factory, the core temperature is not higher than minus 18 degrees Celsius and there is no rancid odor. After being thawed in running water to a core temperature of 0 to 2 degrees Celsius, they are immediately put into the crushing process. The interval from thawing to the start of enzymatic hydrolysis does not exceed 90 minutes to reduce the excessive generation of bitter peptides caused by endogenous protease autolysis.

8. The method for preparing water-soluble organic fertilizer for soil improvement using a complex of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: Before chopping, the fruit and vegetable residues are soaked in a sodium hypochlorite solution with a mass concentration of 0.5% for three minutes for disinfection, and then rinsed twice with deionized water to remove pesticide residues and bacteria on the surface. The water used for pre-cooking is softened water treated with activated carbon and reverse osmosis with a conductivity of no more than 50 microsiemens per centimeter. After pre-cooking, the cooling rate is no less than 3 degrees Celsius per minute to prevent the Maillard reaction from causing browning of the fruit and vegetable extract and loss of active polyphenols.

9. The method for preparing water-soluble organic fertilizer for soil improvement using a compound of deep-sea fish protein and fruit and vegetable fermentation products according to claim 1, characterized in that: The physicochemical indicators of the prepared deep-sea fish protein and fruit and vegetable fermentation compound biological bacteria soil amendment water-soluble organic fertilizer are as follows: pH 6.0 to 7.5 (1:250 dilution for water solubility), total water-soluble organic matter not less than 120 grams per liter, organic nitrogen in the form of amino acids and small peptides accounting for not less than 78% of the total nitrogen, calcium and magnesium content not greater than 0.3 grams per liter to avoid forming insoluble precipitates with organic acid radicals, the survival rate of live bacteria in the finished product is not less than 70% of the initial value within 6 months under the storage conditions of 4 to 10 degrees Celsius in the dark, and there is no flocculation, no visible suspended matter, and no odor within 24 hours after dilution 500 times for water solubility.

10. The use of the deep-sea fish protein and fruit and vegetable fermentation compound biological soil-improving water-soluble organic fertilizer prepared according to any one of claims 1-9 in acidified soils, secondary salinized soils, and compacted and degraded soils produced by continuous cropping in facilities, characterized in that: The water-soluble organic fertilizer is diluted with 3 to 8 kilograms of water per mu (unit of land area) and applied by drip irrigation or fertigation at a ratio of 300 to 500 times. It is applied once every 20 to 30 days and continuously for no less than three times. This can reduce the destruction rate of soil aggregate structure in the topsoil by more than 40%, increase soil microbial biomass carbon by more than 50%, reduce soil salinity by more than 30%, and reduce the number of rhizosphere pathogens such as Fusarium oxysporum and Pythium by more than one order of magnitude.

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