A method for preparing microbial protease liquid fertilizer and its application

Abstract

This invention discloses a method for preparing and applying a microbial protease liquid fertilizer, comprising the following steps: S1. Mixing animal and plant protein raw materials with water, and subjecting them to physical-assisted cell wall disruption to obtain a pretreated protein slurry; S2. Inoculating with a composite seed liquid, and performing aerobic fermentation and enzymatic hydrolysis at 45-50℃ for 48-60 hours, maintaining the pH of the fermentation broth at 6.5-7.5 to obtain an enzymatic hydrolysate; S3. Cooling to 28-35℃, inoculating with a mixed probiotic agent, and performing anaerobic or facultative anaerobic fermentation for 48-72 hours to obtain a fermentation broth; S4. Adding trace element salts, and stirring the reaction at 40℃ to 45℃ for 1.5-2.5 hours to allow small molecule active peptides in the fermentation broth to undergo in-situ complexation with metal ions, followed by post-treatment to obtain the microbial protease liquid fertilizer; Through the synergistic effect of physical cell wall disruption and composite enzymatic hydrolysis, the conversion efficiency of recalcitrant waste protein raw materials such as feather meal is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bio-fertilizer technology, specifically referring to a method for preparing microbial protease liquid fertilizer and its application. Background Technology

[0002] With the increasing demand for green and sustainable agricultural development, traditional chemical fertilizers face significant challenges due to their low utilization rate and heavy environmental pollution. Liquid fertilizers, on the other hand, have been widely used in facility agriculture and fertigation technology due to their convenience and rapid absorption. Among them, amino acid liquid fertilizers prepared from animal and plant proteins have attracted considerable attention due to their comprehensive nutrition and high biological activity.

[0003] Currently, the conventional technologies for preparing protein-based liquid fertilizers mainly include chemical hydrolysis and single enzymatic hydrolysis.

[0004] Chemical hydrolysis typically uses strong acids (such as hydrochloric acid) or strong bases to completely hydrolyze proteins under high-temperature conditions. While this process is low-cost and fast, it has several drawbacks: First, the harsh reaction conditions can destroy heat-labile active components in proteins, such as tryptophan and sulfur-containing amino acids; second, the hydrolysis products are mainly free amino acids, lacking small peptides with higher biological activity; third, large amounts of acid and alkali are required for neutralization after the reaction, producing high-salt byproducts, which not only complicates post-treatment but also easily causes soil compaction and salinization when applied to soil.

[0005] Compared to chemical methods, single-enzymatic hydrolysis is gentler and better preserves the activity of amino acids. However, traditional processes often use a single enzyme or commercially available protease for hydrolysis. Due to the specificity of the enzyme, the hydrolysis efficiency for dense proteins (such as feather keratin and collagen) is low, resulting in low raw material conversion rates and increased production costs. In addition, existing enzymatic hydrolysates have relatively limited functions, mainly providing nitrogen nutrition and lacking the ability to regulate soil microbial flora or induce systemic resistance in crops.

[0006] In view of the problems existing in the current technology, such as single active ingredients, low raw material utilization, insufficient functionality and environmental risks, it is necessary to develop a new type of liquid fertilizer and its preparation method that is green in process, rich in active ingredients and has multiple functions. Summary of the Invention

[0007] To address the needs and problems mentioned in the background above, the present invention provides a method for preparing microbial protease liquid fertilizer and its application, thereby at least partially solving the above problems.

[0008] According to the technical solution of the present invention, a method for preparing microbial protease liquid fertilizer is provided, comprising the following steps: S1. Mix animal and plant protein raw materials with water, adjust the pH to 7.0-8.0, and then perform physical-assisted cell wall breaking treatment to obtain pretreated protein slurry; S2. Inoculate the pretreated protein slurry obtained in S1 with a composite seed liquid containing Aspergillus niger and Bacillus licheniformis that produce a composite protease. Perform aerobic fermentation and enzymatic hydrolysis at 45-50℃ for 48-60h, maintaining the pH of the fermentation broth at 6.5-7.5. During the fermentation process, the pH of the protein slurry will naturally decrease from 7.0-8.0 to 6.5-7.5. If the pH deviates from this range, adjust it by adding dilute hydrochloric acid / dilute sodium hydroxide in small amounts to obtain the enzymatic hydrolysate. S3. Cool the enzymatic hydrolysate obtained in S2 to 28-35℃, inoculate it with a mixed probiotic agent, the mixed probiotic agent containing Saccharomyces cerevisiae and Lactobacillus plantarum, and carry out anaerobic or facultative anaerobic fermentation for 48-72 hours to obtain fermentation broth; S4. Add trace element salts to the fermentation broth obtained in S3. Stir the reaction at 40℃ to 45℃ for 1.5-2.5h to allow the small molecule active peptides in the fermentation broth to undergo in-situ complexation with metal ions. After post-treatment, microbial protease liquid fertilizer is obtained.

[0009] Preferably, the animal and plant protein raw materials in S1 include soybean meal and / or hydrolyzed feather meal, and the mass ratio of soybean meal to hydrolyzed feather meal is 40-50:20-30; The mass ratio of the animal and plant protein raw materials to water is 1:3-5.

[0010] Preferably, the physical-assisted cell wall disruption process in S1 is an ultrasonic-assisted process; The ultrasonic-assisted processing power is 250-350W, the frequency is 35-45kHz, and the processing time is 15-25min.

[0011] Preferably, the ratio of viable Aspergillus niger and Bacillus licheniformis producing complex protease in the composite seed liquid in S2 is 1:2-3, and the inoculation amount is 5-8% of the total volume of the protein slurry; the amino acid nitrogen content in the enzymatic hydrolysate is not less than 5g / 100mL.

[0012] Preferably, the ratio of live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum in the mixed probiotic agent in S3 is 1:5-8, and the inoculation amount is 3-5% of the total volume of the enzymatic hydrolysate; the anaerobic or facultative anaerobic fermentation temperature is 30-35℃.

[0013] Preferably, the trace element salt in S4 includes at least one of ferrous sulfate, zinc sulfate, and borax; The amount of trace element salt added is 50-200 mg per liter of fermentation broth, calculated as metal ions; the stirring speed of the stirring reaction is 60-100 rpm.

[0014] Preferably, the post-processing in S4 includes homogenizing and refining the reacted material and filtering to remove insoluble impurities.

[0015] On the other hand, the present invention also provides an application of microbial protease liquid fertilizer in promoting crop growth, wherein the microbial protease liquid fertilizer is diluted and applied to crops.

[0016] Preferably, the dilution ratio of 300-500 times is used for crop drip irrigation or sprinkler irrigation; When the dilution ratio is 800-1000 times, it is used for foliar spraying of crops; The dilution ratio of 100-200 times is used for soil improvement root irrigation treatment.

[0017] Thirdly, the present invention also provides a microbial protease liquid fertilizer, wherein the liquid fertilizer contains small molecule active peptides with a molecular weight distribution between 200-1500 Da, and small peptides with a molecular weight of less than 1000 Da account for more than 80% of the total peptides. The liquid fertilizer also contains γ-aminobutyric acid, lactic acid, and cytokinin-like substances produced by the metabolism of Saccharomyces cerevisiae and Lactobacillus plantarum. The trace elements in the liquid fertilizer exist in the form of peptide-metal complexes.

[0018] Beneficial effects: 1. High raw material utilization rate and green and environmentally friendly process: Through the synergistic effect of physical cell wall disruption and compound enzymatic hydrolysis, the conversion efficiency of recalcitrant waste protein raw materials such as feather meal is significantly improved. The entire process requires no strong acid or alkali treatment at high temperature and pressure, leaves no salt residue, and is environmentally friendly in both production and product use.

[0019] 2. The product is rich in active ingredients and has strong biological activity: The product is not only rich in small molecule active peptides that are easily absorbed by crops, but also introduces active metabolites such as GABA and natural growth hormones through probiotic post-fermentation, achieving the dual effects of nutrition and conditioning.

[0020] 3. Stable nutrients and high absorption efficiency: By utilizing the in-situ chelating ability of small molecule peptides, trace elements are converted into stable peptide-metal complexes, solving the problem of easy precipitation and failure of inorganic salts, while avoiding the environmental risks of chemical chelating agents, and significantly improving the absorption and utilization rate of trace elements by crops.

[0021] 4. Multifunctional and highly valuable: This product has multiple functions, including providing fast-acting nutrients, improving soil microecology, and inducing crop stress resistance. It can effectively reduce the amount of chemical fertilizers and pesticides used, meeting the needs of modern agriculture for green and efficient development. Detailed Implementation

[0022] The technical solutions in the embodiments will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection.

[0023] This invention overcomes the shortcomings of the prior art by providing a microbial protease liquid fertilizer, the active ingredient of which is prepared by the following process: This liquid fertilizer contains small molecule active peptides produced by deep enzymatic hydrolysis of animal and plant protein raw materials by a complex microbial enzyme system, as well as plant secondary metabolites produced by specific probiotics in the post-fermentation stage. The molecular weight of the small molecule active peptides is mainly distributed between 200-1500 Da, and the content of small peptides (molecular weight less than 1000 Da) accounts for more than 80% of the total peptides. The plant secondary metabolites include at least γ-aminobutyric acid, lactic acid, and cytokinins.

[0024] On the other hand, embodiments of the present invention also provide a method for preparing the microbial protease liquid fertilizer, comprising the following steps: S1. Raw material pretreatment and physical-assisted cell-wall breaking: A mixture of plant and animal protein sources is prepared by mixing them with water at a mass ratio of 1:3-5. The pH of the mixture is then adjusted to 7.0-8.0 using an alkaline adjuster. Subsequently, the mixture is subjected to ultrasonic-assisted treatment at a power of 250-350W, a frequency of 35-45kHz, and a treatment time of 15-25 minutes, resulting in a pretreated protein slurry with a density of 1.05-1.10 g / cm³. 3 .

[0025] S2. Enzymatic fermentation using compound strains: A composite seed liquid is inoculated into the pretreated protein slurry obtained in S1; the composite seed liquid contains Aspergillus niger and Bacillus licheniformis producing a composite protease with a viable bacterial count ratio of 1:2-3. The inoculum amount is 5-8% of the total volume of the protein slurry. Aerobic fermentation and enzymatic hydrolysis are carried out at a temperature of 45-50℃ for 48-60 hours. During the fermentation process, the pH of the fermentation broth is maintained at 6.5-7.5 by adding alkaline or acidic regulators to obtain an enzymatic hydrolysate rich in small molecule peptides and free amino acids. At the end of the enzymatic hydrolysis, the amino acid nitrogen content in the enzymatic hydrolysate is not less than 5g / 100mL.

[0026] S3. Probiotic post-fermentation activation: The enzymatic hydrolysate obtained in S2 is cooled to 30-35℃ and inoculated with the activated mixed probiotic agent; the mixed probiotic agent contains Saccharomyces cerevisiae and Lactobacillus plantarum with a live bacteria ratio of 1:5-8; The inoculum is 3-5% of the total volume of the enzyme hydrolysate. Anaerobic or facultative anaerobic fermentation is carried out at 28-35℃ for 48-72 hours to obtain a fermentation broth rich in active metabolites.

[0027] S4. In-situ chelation and post-treatment: Add trace element salts to the fermentation broth obtained in S3. The trace element salts include at least one or more of ferrous sulfate, zinc sulfate, and borax. The amount added is calculated as metal ions, and is 50-200 mg per liter of fermentation broth. Under a temperature of 40-45℃, the mixture is slowly stirred at a stirring speed of 60-100 rpm for 1.5-2.5 hours to allow the small molecule active peptides in the fermentation broth to undergo a full in-situ complexation reaction with the metal ions. After the reaction is complete, the material is homogenized and refined by passing it through a colloid mill, and insoluble impurities are removed by filtration to obtain the microbial protease liquid fertilizer.

[0028] Thirdly, in the preparation method provided in the embodiments of the present invention, the characteristics and functions of the key strains are as follows: The aforementioned Bacillus licheniformis is a strain that has been bred through mutagenesis and has a highly efficient ability to degrade keratin. The keratinase it produces can specifically open the disulfide bonds in keratin, thereby achieving efficient utilization of recalcitrant raw materials such as feather powder.

[0029] The aforementioned Aspergillus niger can produce abundant cellulase and acidic protease, which complement the alkaline protease of Bacillus licheniformis, thereby achieving deep synergistic enzymatic hydrolysis of complex protein raw materials.

[0030] The combination of brewer's yeast and Lactobacillus plantarum can metabolize and produce γ-aminobutyric acid, lactic acid and natural growth hormones during the post-fermentation stage, giving the fertilizer the functions of stress resistance, growth promotion and biological preservation.

[0031] Fourthly, this invention also provides a method for applying the microbial protease liquid fertilizer: Application in crop drip or sprinkler irrigation: Dilute the liquid fertilizer 300-500 times and apply it to the roots or drip irrigation during the crop growing season to promote crop root development and improve nutrient absorption efficiency.

[0032] Application in foliar spraying of crops: Dilute the liquid fertilizer 800-1000 times and spray it evenly on the leaves before and after the crops flower to improve the fruit setting rate and improve the quality of the fruit.

[0033] Application in soil improvement: The liquid fertilizer is diluted 100-200 times and applied as a root irrigation treatment to improve the structure of the soil rhizosphere microbial community and inhibit the growth of soil-borne pathogens.

[0034] Example 1 Mix 45 parts soybean meal, 25 parts hydrolyzed feather meal, and water at a mass ratio of 1:4, adjust the pH to 7.5, and apply ultrasonic treatment at a power of 300W and a frequency of 40kHz for 20 minutes to obtain pretreated slurry. A compound seed culture containing Aspergillus niger (accession number CICC40395, purchased from China Industrial Microbial Culture Collection Center) and Bacillus licheniformis (accession number ACCC11091) was inoculated, with a viable cell ratio of Aspergillus niger to Bacillus licheniformis of 1:2.5 and an inoculum size of 6%. The culture was enzymatically hydrolyzed aerobically at 48℃ for 54 h, with the pH maintained at 7.0 during the fermentation process, to obtain an enzymatic hydrolysate with an amino acid nitrogen content of 5.2 g / 100 mL. The temperature was lowered to 32℃, and a mixed probiotic agent containing NX16 type brewer's yeast and Lactobacillus plantarum with preservation number CCTCCM206032 was inoculated. The live count ratio of brewer's yeast to Lactobacillus plantarum was 1:6.5, the inoculation amount was 4%, and anaerobic fermentation was carried out at 32℃ for 60h. Add 120 mg / L ferrous sulfate, 80 mg / L zinc sulfate, and 60 mg / L borax, and stir at 42℃ and 80 rpm for 2 hours. Then, homogenize and filter.

[0035] Example 2 Mix 50 parts soybean meal, 20 parts hydrolyzed feather meal, and water at a mass ratio of 1:5, adjust the pH to 8.0, and apply ultrasonic treatment at a power of 350W and a frequency of 45kHz for 15 minutes to obtain pretreated slurry.

[0036] The compound seed liquid was inoculated at an inoculation rate of 8%, and the mixture was enzymatically hydrolyzed aerobically at 50°C for 48 hours. The pH was maintained at 7.5 during the fermentation process to obtain an enzymatic hydrolysate with an amino acid nitrogen content of 5.5 g / 100 mL.

[0037] Cool down to 35℃, inoculate with mixed probiotics at a rate of 5%, and anaerobic ferment at 35℃ for 48 hours. Add 200 mg / L ferrous sulfate and 50 mg / L zinc sulfate, stir at 45℃ and 100 rpm for 1.5 h, then homogenize and filter.

[0038] Example 3 Mix 40 parts soybean meal, 30 parts hydrolyzed feather meal, and water at a mass ratio of 1:3, adjust the pH to 7.0, and apply ultrasonic treatment at a power of 250W and a frequency of 35kHz for 25 minutes to obtain pretreated slurry. Inoculate with compound seed liquid at an inoculation rate of 5%, and perform aerobic fermentation and enzymatic hydrolysis at 45℃ for 60 h, maintaining pH 6.5 during the fermentation process to obtain an enzymatic hydrolysate with an amino acid nitrogen content of 5.0 g / 100 mL; Cool down to 30℃, inoculate with mixed probiotics at a rate of 3%, and anaerobic ferment at 28℃ for 72 hours. Add 150 mg / L zinc sulfate and 100 mg / L borax, stir at 40℃ and 60 rpm for 2.5 h, then homogenize and filter.

[0039] Example 4 The difference from Example 1 is as follows: A compound seed culture containing Aspergillus niger and Bacillus licheniformis was inoculated at a rate of 6%, and the mixture was enzymatically hydrolyzed aerobically at 48°C for 48 hours while maintaining the pH at 7.0 during the fermentation process, yielding an enzymatic hydrolysate with an amino acid nitrogen content of 5.1 g / 100 mL.

[0040] Example 5 The difference from Example 1 is as follows: A compound seed culture containing Aspergillus niger and Bacillus licheniformis was inoculated at a rate of 6%, and the mixture was enzymatically hydrolyzed aerobically at 48°C for 60 h while maintaining the pH at 7.0 during the fermentation process, yielding an enzymatic hydrolysate with an amino acid nitrogen content of 5.3 g / 100 mL.

[0041] Example 6 The difference from Example 1 is as follows: The temperature was lowered to 32°C, and a mixed probiotic agent containing brewer's yeast and lactobacillus plantarum was inoculated at a rate of 4%. Anaerobic fermentation was carried out at 32°C for 48 hours.

[0042] Example 7 The difference from Example 1 is as follows: The temperature was lowered to 32°C, and a mixed probiotic agent containing brewer's yeast and lactobacillus plantarum was inoculated at a rate of 4%. Anaerobic fermentation was carried out at 32°C for 72 hours.

[0043] Example 8 The difference from Example 1 is as follows: Add 50 mg / L ferrous sulfate, 200 mg / L zinc sulfate, and 30 mg / L borax, and stir at 42℃ and 80 rpm for 2 hours. Then, homogenize and filter.

[0044] Example 9 The difference from Example 1 is as follows: Add 200 mg / L ferrous sulfate, 30 mg / L zinc sulfate, and 150 mg / L borax, and stir at 42℃ and 80 rpm for 2 hours. Then, homogenize and filter.

[0045] Example 10 The difference from Example 1 is as follows: Mix 45 parts soybean meal, 25 parts hydrolyzed feather meal, and water at a mass ratio of 1:4, adjust the pH to 7.5, and apply ultrasonic treatment at a power of 300W and a frequency of 40kHz for 15 minutes to obtain pretreated slurry.

[0046] Comparative Example 1 Mix 45 parts soybean meal, 25 parts hydrolyzed feather meal, and water at a mass ratio of 1:4, and adjust the pH to 7.5; Add commercial alkaline protease: Bacillus licheniformis 2709 (enzyme activity 100,000 U / g), 0.5% addition amount, and enzymatically hydrolyze at 48℃ for 54 hours; After filtration, add 120 mg / L ferrous sulfate, 80 mg / L zinc sulfate, and 60 mg / L borax, and stir to mix.

[0047] Comparative Example 2 The difference from Example 1 is that the probiotic post-fermentation step is omitted, and 120 mg / L of ferrous sulfate, 80 mg / L of zinc sulfate, and 60 mg / L of borax are directly added to the enzymatic hydrolysate. The mixture is stirred at 42°C and 80 rpm for 2 hours, and then homogenized and filtered.

[0048] Comparative Example 3 The difference from Example 1 is as follows: 200 mg / L of disodium EDTA was added, and after stirring to dissolve, 120 mg / L of ferrous sulfate, 80 mg / L of zinc sulfate, and 60 mg / L of borax were added. Stirring was continued for 30 minutes, followed by homogenization and filtration.

[0049] The products obtained from the above embodiments and comparative examples were subjected to application experiments and tests according to the following methods: Test crop: Tomato (variety: Provence); Experimental location: Potted plant experiment in an artificial climate chamber; Experimental design: Each embodiment and comparative example was replicated 3 times, with 1 plant per pot, arranged in a randomized block design; Fertilization method: Start 15 days after transplanting, water once every 7 days, dilute 500 times, water each pot with 200mL each time, and fertilize a total of 4 times; Control (CK): Watered with an equal amount of clean water.

[0050] The following measurement indicators and methods were used for testing, and the data obtained are shown in Tables 1 and 2 below.

[0051] Growth indicators: Plant height, stem diameter, and above-ground fresh weight were measured after fertilization. Physiological indicators: Proline content (reflecting stress resistance) was determined using the sulfosalicylic acid method; root activity was determined using the TTC method. Soil parameters: Rhizosphere soil samples were collected after fertilization. Urease activity was determined using the sodium phenolate-sodium hypochlorite colorimetric method; protease activity was determined using the ninhydrin colorimetric method. Nutrient absorption: Total nitrogen in plants was determined using the Kjeldahl method; total zinc was determined using atomic absorption spectrophotometry.

[0052] Table 1. Effects of different treatments on tomato growth and stress resistance.

[0053] Table 2. Effects of different treatments on soil enzyme activity and nutrient uptake.

[0054] As can be seen from the data in Tables 1 and 2: (1) Growth promoting effect: In Example 1, the fresh weight of the aboveground parts reached 52.4 g / plant, which was 83.2% higher than the control (CK) (28.6 g / plant). Comparative Example 1 yielded only 35.7 g / plant, which was 24.8% higher than the control (CK), but the effect was far lower than that of Example 1. Example 1 showed a 46.8% increase in the fresh weight of the aboveground parts compared to Comparative Example 1, indicating that the four-step directional enzymatic hydrolysis-dual-effect bacterial agent coupling-in-situ chelation process of the present invention has a significant synergistic effect.

[0055] (2) Anti-stress induction effect: Example 1: Proline content was 32.6 μg / g, which was 78.1% higher than that of the control (18.3 μg / g). Comparative Example 2 (post-fermentation without probiotics) had a proline content of 26.9 μg / g, which was 17.5% lower than that of Example 1; This indicates that active substances such as γ-aminobutyric acid produced by the metabolism of Saccharomyces cerevisiae and Lactobacillus plantarum during the post-fermentation stage play a key role in resisting stress induction.

[0056] (3) Soil improvement effect: Example 1: Soil urease activity was 0.62 mg / g·d, which was 121.4% higher than that of the control (0.28 mg / g·d). Example 1: Soil protease activity was 38.7 μg / g·h, which was 153.0% higher than the control (15.3 μg / g·h). Comparative Example 1 showed that the enzyme activities of the two enzymes increased by only 35.7% and 46.4% respectively, indicating that the synergistic process of physical cell wall disruption + compound microbial enzyme system + probiotic post-fermentation of the present invention has a far greater activating effect on soil enzyme activity than a single enzymatic hydrolysis process.

[0057] (4) Trace element absorption effect: Example 1: The total zinc content of the plant was 48.6 mg / kg, which was 117.9% higher than that of the control (22.3 mg / kg). The total zinc content of Comparative Example 3 (EDTA chelated) plants was 40.2 mg / kg, which was 17.3% lower than that of Example 1; This indicates that the peptide-zinc complex formed by in-situ chelation of small molecule peptides has higher bioavailability than EDTA chemical chelates, and avoids the environmental risks of chemical chelating agents.

[0058] Analysis of process parameter optimization: (1) Effect of enzymatic hydrolysis time: The fresh weights of the aboveground parts in Examples 4 (48h), 1 (54h), and 5 (60h) were 49.7g, 52.4g, and 50.6g, respectively. This indicates that 54h is the optimal enzymatic hydrolysis time. Too short a time (48h) will result in insufficient hydrolysis, while too long a time (60h) may lead to further degradation of some active peptides.

[0059] (2) Effect of post-fermentation time: The proline content in Examples 6 (48h), 1 (60h), and 7 (72h) was 30.1 μg / g, 32.6 μg / g, and 31.0 μg / g, respectively. Fermentation after 60 hours can accumulate sufficient amounts of stress-resistant substances such as γ-aminobutyric acid; if the fermentation time is too long, the effect will not be significantly improved.

[0060] (3) The influence of trace element ratio: The total zinc content of plants in Examples 8, 1, and 9 were 47.2 mg / kg, 48.6 mg / kg, and 46.8 mg / kg, respectively. The ratio in Example 1 (iron:zinc:boron = 120:80:60) is more conducive to the synergistic absorption of trace elements.

[0061] Compare the data of Comparative Examples 1-3 with those of Example 1: Comparative Example 1 (single enzymatic hydrolysis, without ultrasonic cell disruption, without compound bacteria, and without post-fermentation): all indicators were significantly lower than those of Example 1, indicating that the three key steps are indispensable.

[0062] Comparative Example 2 (without post-fermentation): The fresh weight of the aboveground parts was 17.0% lower than that of Example 1, and the proline content was 17.5% lower, indicating that probiotic post-fermentation plays an important role in promoting growth and inducing stress resistance.

[0063] Comparative Example 3 (Chemical Chelation): The total zinc content of the plant was 17.3% lower than that of Example 1, indicating that the in-situ chelation effect of peptides is better than that of EDTA.

[0064] Based on the above data, the technical solution of this invention, through the synergistic effect of a four-step process—physically assisted cell wall disruption, compound bacterial enzymatic hydrolysis, probiotic post-fermentation, and in-situ chelation—is significantly superior to existing processes in promoting crop growth, inducing stress resistance, improving soil, and promoting the absorption of trace elements.

[0065] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a microbial protease liquid fertilizer, characterized in that, Includes the following steps: S1. Mix animal and plant protein raw materials with water, adjust the pH to 7.0-8.0, and then perform physical-assisted cell wall breaking treatment to obtain pretreated protein slurry; S2. Inoculate the pretreated protein slurry obtained in S1 with a composite seed liquid, wherein the composite seed liquid contains Aspergillus niger and Bacillus licheniformis that produce a composite protease, and carry out aerobic fermentation and enzymatic hydrolysis at 45-50℃ for 48-60h, maintaining the pH of the fermentation broth at 6.5-7.5, to obtain the enzymatic hydrolysate. S3. Cool the enzymatic hydrolysate obtained in S2 to 28-35℃, inoculate it with a mixed probiotic agent, the mixed probiotic agent containing Saccharomyces cerevisiae and Lactobacillus plantarum, and carry out anaerobic or facultative anaerobic fermentation for 48-72 hours to obtain fermentation broth; S4. Add trace element salts to the fermentation broth obtained in S3. Stir the reaction at 40℃ to 45℃ for 1.5-2.5h to allow the small molecule active peptides in the fermentation broth to undergo in-situ complexation with metal ions. After post-treatment, microbial protease liquid fertilizer is obtained.

2. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The animal and plant protein raw materials in S1 include soybean meal and / or hydrolyzed feather meal, and the mass ratio of soybean meal to hydrolyzed feather meal is 40-50:20-30. The mass ratio of the animal and plant protein raw materials to water is 1:3-5.

3. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The physical-assisted cell wall disruption process in S1 is an ultrasonic-assisted process; The ultrasonic-assisted processing power is 250-350W, the frequency is 35-45kHz, and the processing time is 15-25min.

4. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The ratio of viable Aspergillus niger and Bacillus licheniformis producing complex protease in the S2 compound seed liquid is 1:2-3, and the inoculation amount is 5-8% of the total volume of the protein slurry; the amino acid nitrogen content in the enzymatic hydrolysate is not less than 5g / 100mL.

5. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The ratio of live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum in the mixed probiotic agent in S3 is 1:5-8, and the inoculation amount is 3-5% of the total volume of the enzymatic hydrolysate; the anaerobic or facultative anaerobic fermentation temperature is 30-35℃.

6. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The trace element salt in S4 includes at least one of ferrous sulfate, zinc sulfate, and borax. The amount of trace element salt added is 50-200 mg per liter of fermentation broth, calculated as metal ions; the stirring speed of the stirring reaction is 60-100 rpm.

7. The method for preparing microbial protease liquid fertilizer according to claim 1, characterized in that, The post-processing in S4 includes homogenizing and refining the reacted materials and filtering out insoluble impurities.

8. The application of a microbial protease liquid fertilizer prepared according to any one of claims 1-7 in promoting crop growth, characterized in that, The microbial protease liquid fertilizer is diluted and applied to crops.

9. The application according to claim 8, characterized in that, When the dilution ratio is 300-500 times, it is used for crop drip irrigation or sprinkler irrigation; When the dilution ratio is 800-1000 times, it is used for foliar spraying of crops; The dilution ratio of 100-200 times is used for soil improvement root irrigation treatment.

10. A microbial protease liquid fertilizer, characterized in that, The liquid fertilizer is prepared by any one of claims 1-7, and contains small molecule active peptides with a molecular weight distribution between 200-1500 Da, and small peptides with a molecular weight less than 1000 Da account for more than 80% of the total peptides. The liquid fertilizer also contains γ-aminobutyric acid, lactic acid, and cytokinin-like substances produced by the metabolism of Saccharomyces cerevisiae and Lactobacillus plantarum. The trace elements in the liquid fertilizer exist in the form of peptide-metal complexes.

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