Method for preparing amino acid fertilizer by multi-strain synergistic starter propagation and liquid enzymolysis method

Abstract

The invention discloses a method for preparing an amino acid fertilizer by multi-strain synergistic starter propagation and a liquid enzymolysis method, which comprises the following steps: selecting 25%-35% of bacillus subtilis, 10%-20% of trichoderma reesei, 15%-25% of aspergillus oryzae, 10%-20% of lactic acid bacteria, 5%-15% of saccharomyces cerevisiae and 5%-15% of aspergillus niger as composite strains; according to the method, six strains such as bacillus subtilis and trichoderma reesei form a composite microbial system, soybean meal and bran are used as raw materials, and the problems of low degradation efficiency, unstable enzyme activity and the like of a traditional process are solved through a process of'three-stage temperature-controlled starter propagation and four-stage liquid fermentation '; the method is characterized in that a'bacillus-mould-saccharomycetes-lactic acid bacteria 'four-flora synergistic system is constructed, a starter propagation-fermentation multi-parameter coupling regulation and control process is initiated, a'process-quality-effect' closed-loop mechanism is established, compared with a traditional process, the efficiency is improved by 35%-50%, the ton cost is reduced by 15%-20%, and the product is suitable for seed treatment and foliage spraying of various crops and has wide application prospects. The industrialization potential is remarkable, and the requirements of workers are met.

Description

Technical Field

[0001] This invention relates to the field of amino acid fertilizer technology, specifically to a method for preparing amino acid fertilizers using a multi-strain synergistic fermentation and liquid enzymatic hydrolysis method. Background Technology

[0002] In food processing, biopharmaceuticals, and feed production, the efficient degradation and utilization of proteins has always been a core issue of concern in the industry. Among them, the koji-making and liquid fermentation technology based on the synergistic effect of microorganisms has become a key technical path to promote the high-value transformation of protein resources because it can achieve the targeted degradation of proteins through the complex enzyme system (such as proteases and cellulases) produced by microbial metabolism, generating high-value-added products such as free amino acids and small molecule peptides. As the core link of enzyme accumulation, the koji-making process, with its microbial community structure, temperature and humidity control, and koji-turning operation, directly determines the activity and stability of the proteases required for subsequent fermentation. The liquid fermentation stage is the key process of coupling enzymatic reaction and microbial metabolism. The dynamic changes in the pH of the fermentation broth and the accumulation pattern of free amino acids not only reflect the efficiency of protein degradation, but are also closely related to the quality of the final product (such as flavor and nutritional components).

[0003] In recent years, although researchers have conducted extensive studies on the screening of microbial koji-making strains and the optimization of liquid fermentation processes, many problems still remain to be solved. On the one hand, existing koji-making processes mostly focus on the enzyme-producing characteristics of single strains or combinations of a few strains, and research on the dynamic changes and regulatory mechanisms of protease activity under the synergistic effects of multiple microorganisms (such as Bacillus, molds, yeasts, and lactic acid bacteria) is not yet in-depth, making it difficult to balance koji-making efficiency and enzyme activity stability. On the other hand, the coupling relationship between pH changes and free amino acid accumulation during liquid fermentation is not yet clear, and there is a lack of systematic analysis on the correlation between process parameters (such as temperature, stirring speed, and aeration rate) and product formation at different fermentation stages, which restricts further improvement in protein degradation efficiency. Therefore, developing a method for preparing highly active amino acid fertilizers with sufficient synergistic effects of multiple microorganisms and precise control of process parameters has important practical significance and application value. Summary of the Invention

[0004] To solve the above-mentioned technical problems, a method for preparing amino acid fertilizer using multi-strain synergistic koji making and liquid enzymatic hydrolysis is provided. This technical solution solves the problems mentioned in the background technology.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing amino acid fertilizer using multi-strain synergistic koji-making and liquid enzymatic hydrolysis includes: S1. Select Bacillus subtilis, Trichoderma reesei, Aspergillus oryzae, Lactic acid bacteria, Saccharomyces cerevisiae, and Aspergillus niger as the compound microbial strain, with the proportions of each strain being 25%-35%, 10%-20%, 15%-25%, 10%-20%, 5%-15%, and 5%-15%, respectively. The viable count of the compound microbial powder should be ≥10. 10 CFU / g, and selected defatted soybean meal and wheat bran as raw materials, mixed at a mass ratio of 7-9:1-3, and 0.3%-0.7% of urea by mass of the mixed raw materials was added; S2. Prepare a bacterial suspension using a 1.5% sterile glucose solution, with a bacterial powder to glucose solution mass ratio of 1:8-12, and activate it by shaking. S3. Add urea and sterile water to the mixed raw materials, adjust the moisture content to 55%-70%, and then sterilize and cool. S4. Inoculate the activated bacterial suspension at an inoculation rate of 1%-3% of the raw material mass, stir evenly, and then carry out three-stage temperature-controlled culture for 60-84 hours. S5. Prepare the substrate according to the mass ratio of koji material: soybean meal powder: water = 1:0.6-1.0:8-12, and adopt a four-stage fermentation process with a fermentation time of 96-120 hours. S6. After filtration of the fermentation broth, vacuum concentrate it to an amino acid concentration of 80-180 g / L, add preservatives and chelating agents, and adjust the pH to 5.0-7.0 to obtain a highly active amino acid fertilizer.

[0006] Preferably, the optimal proportions of each microorganism in the compound microbial strain in S1 are: Bacillus subtilis 30%, Trichoderma reesei 15%, Aspergillus oryzae 20%, Lactic acid bacteria 15%, Saccharomyces cerevisiae 10%, and Aspergillus niger 10%.

[0007] Preferably, the optimal mass ratio of defatted soybean meal to wheat bran in S1 is 8:2, and 0.5% of the mass of urea is added to the mixed raw materials.

[0008] Preferably, the oscillation activation in S2 is performed at a rotation speed of 150-200 r / min and a temperature of 34-38℃ for 40-50 minutes.

[0009] Preferably, the sterilization and cooling process in S3 involves sterilizing at 121°C and 0.1 MPa for 15-25 minutes, followed by cooling to 30-45°C.

[0010] Preferably, the three-stage koji-making process parameters in S4 are as follows: During the initial 0-24 hours, maintain a temperature of 35-37℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time. During the medium term (24-48 hours), maintain a temperature of 32-34℃ and a humidity of 80%-90%, ventilating once every 6-10 hours for 15-25 minutes each time. During the later 48-72 hours, maintain a temperature of 30-32℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time; turn the curd over once at 24 hours and once at 48 hours.

[0011] Preferably, the four-stage fermentation process parameters in S5 are as follows: Enzyme activity release period 0-24h, temperature 32-35℃, stirring speed 180-220rpm, aeration rate 0.6-1.0vvm; Protein degradation period 24-72h, temperature 33-35℃, stirring speed 230-270rpm, aeration rate 0.8-1.2vvm; The refining and conversion period is 72-96 hours, the temperature is 30-32℃, the stirring speed is 130-170 rpm, and the aeration rate is 0.4-0.6 vvm. Stabilization period: 96-108 hours, temperature: 28-30℃, stop aeration, stirring speed: 40-60 rpm.

[0012] Preferably, the vacuum concentration conditions in S6 are a temperature of 45-55℃ and a vacuum degree of -0.07 to -0.09 MPa.

[0013] Preferably, the preservative in S6 is potassium sorbate, with an addition amount of 0.08%-0.12%.

[0014] Preferably, the chelating agent in S6 is EDTA-2Na, and the addition amount is 0.15%-0.25%.

[0015] Compared with existing technologies, this invention provides a method for preparing amino acid fertilizers using a multi-strain synergistic koji-making process and liquid enzymatic hydrolysis, which has the following beneficial effects: 1. This invention constructs a synergistic system of four microbial groups: Bacillus subtilis, mold, yeast, and lactic acid bacteria. Each microbial species has a clear division of labor and significant synergistic effect: Bacillus subtilis and Aspergillus oryzae proliferate rapidly in the early stage of koji making and secrete neutral and acidic proteases; Trichoderma reesei degrades bran crude fiber to release carbon sources for Aspergillus niger to synthesize alkaline proteases; lactic acid bacteria regulate the pH of the koji material, and brewing yeast produces B vitamins to maintain enzyme activity stability. This invention solves the problems of low enzyme production efficiency and single enzyme system of single microbial species. The protease activity at the 72-hour koji making endpoint can reach 1646 U / g.

[0016] 2. A novel multi-parameter coupled control process of "three-stage temperature-controlled koji making + four-stage liquid fermentation" was first developed. In the koji making stage, temperature gradient control (36±1℃→33±1℃→31±1℃) was used to match the growth and enzyme production patterns of the strain. In the liquid fermentation stage, precise control of temperature, stirring speed and aeration rate was used to achieve synergistic optimization of pH and free amino acid accumulation. The free amino acid concentration at the end of 108h fermentation reached 54 g / L, which is higher than the level reported in the previous studies.

[0017] 3. A closed-loop mechanism of "process-quality-efficacy" has been established. Efficacy verification of the product shows that the 500-fold dilution can increase the germination index of wheat seeds by 22% compared with the control group, increase the plant height by 0.27 cm, increase the fresh weight by 0.16 g, and increase the chlorophyll content by 0.6 mg / g. It is also suitable for seed treatment and foliar spraying of various crops.

[0018] 4. Significant process efficiency and economic benefits, with a 35%-50% increase in protein degradation efficiency and a 15%-20% reduction in cost per ton compared to traditional processes, demonstrating outstanding industrialization potential. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the process for preparing amino acid fertilizer in this invention; Figure 2 This is a schematic diagram of the protease activity change curve during the koji-making stage in this invention; Figure 3 This is a schematic diagram of the pH change curve during the liquid fermentation stage in this invention; Figure 4 This is a schematic diagram of the change curve of free amino acid content during the liquid fermentation stage in this invention; Figure 5 This is a graph showing the effect of different concentrations of amino acid water-soluble fertilizer on the germination characteristics of wheat seeds in this invention. Figure 6 This figure shows the effect of different concentrations of amino acid water-soluble fertilizer on the growth of wheat seedlings in this invention. Detailed Implementation

[0020] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0021] Example 1 Please refer to Figure 1 As shown, the method for preparing amino acid fertilizer using multi-strain synergistic koji-making and liquid enzymatic hydrolysis includes: S1. A compound microbial strain was selected, consisting of Bacillus subtilis, Trichoderma reesei, Aspergillus oryzae, lactic acid bacteria, Saccharomyces cerevisiae, and Aspergillus niger, with each strain accounting for 30%, 15%, 20%, 15%, 10%, and 10% respectively. The viable count of the compound microbial powder was ≥10. 10 CFU / g, and selected defatted soybean meal and wheat bran as raw materials, mixed at a mass ratio of 8:2, and 0.5% urea by mass of the mixed raw materials was added; S2. Prepare bacterial suspension using 1.5% sterile glucose solution, with a bacterial powder to glucose solution mass ratio of 1:8-12, and activate by shaking at 150-200 r / min and 34-38℃ for 40-50 minutes. S3. Add urea and sterile water to the mixed raw materials, adjust the moisture content to 55%-70%, sterilize at 121℃ and 0.1MPa for 15-25 minutes, and then cool to 30-45℃. S4. Inoculate the activated bacterial suspension at an inoculation rate of 1%-3% of the raw material mass, stir evenly, and then carry out three-stage temperature-controlled incubation for 60-84 hours. The process parameters for the three-stage temperature-controlled incubation are as follows: During the initial 0-24 hours, maintain a temperature of 35-37℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time. During the medium term (24-48 hours), maintain a temperature of 32-34℃ and a humidity of 80%-90%, ventilating once every 6-10 hours for 15-25 minutes each time. During the later 48-72 hours, maintain a temperature of 30-32℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time, and turn the fruit over once at 24 hours and once at 48 hours. S5. Prepare the substrate according to the mass ratio of koji material: soybean meal powder: water = 1:0.6-1.0:8-12, and adopt a four-stage fermentation process with a fermentation time of 96-120 hours. The parameters for the four-stage fermentation process are: Enzyme activity release period 0-24h, temperature 32-35℃, stirring speed 180-220rpm, aeration rate 0.6-1.0vvm; Protein degradation period 24-72h, temperature 33-35℃, stirring speed 230-270rpm, aeration rate 0.8-1.2vvm; The refining and conversion period is 72-96 hours, the temperature is 30-32℃, the stirring speed is 130-170 rpm, and the aeration rate is 0.4-0.6 vvm. Stabilization period: 96-108 hours, temperature: 28-30℃, stop aeration, stirring speed: 40-60 rpm; S6. After filtration, the fermentation broth is vacuum concentrated to an amino acid concentration of 80-180 g / L. The vacuum concentration conditions are a temperature of 45-55℃ and a vacuum degree of -0.07 to -0.09 MPa. Preservatives and chelating agents are added, and the pH is adjusted to 5.0-7.0 to obtain a highly active amino acid fertilizer. The preservative is potassium sorbate, with an addition amount of 0.08%-0.12%, and the chelating agent is EDTA-2Na, with an addition amount of 0.15%-0.25%.

[0022] Example 2 Please refer to Figure 1As shown, the method for preparing amino acid fertilizer using multi-strain synergistic koji-making and liquid enzymatic hydrolysis includes: S1. A compound microbial strain was selected, consisting of Bacillus subtilis, Trichoderma reesei, Aspergillus oryzae, Lactic acid bacteria, Saccharomyces cerevisiae, and Aspergillus niger, with the proportions of each strain being 25%, 10%, 15%, 10%, 5%, and 5%, respectively. The viable count of the compound microbial powder was ≥10. 10 CFU / g, and selected defatted soybean meal and wheat bran as raw materials, mixed at a mass ratio of 7:1, and 0.3% urea by mass of the mixed raw materials was added; S2. Prepare bacterial suspension using 1.5% sterile glucose solution, with a bacterial powder to glucose solution mass ratio of 1:8-12, and activate by shaking at 150-200 r / min and 34-38℃ for 40-50 minutes. S3. Add urea and sterile water to the mixed raw materials, adjust the moisture content to 55%-70%, sterilize at 121℃ and 0.1MPa for 15-25 minutes, and then cool to 30-45℃. S4. Inoculate the activated bacterial suspension at an inoculation rate of 1%-3% of the raw material mass, stir evenly, and then carry out three-stage temperature-controlled incubation for 60-84 hours. The process parameters for the three-stage temperature-controlled incubation are as follows: During the initial 0-24 hours, maintain a temperature of 35-37℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time. During the medium term (24-48 hours), maintain a temperature of 32-34℃ and a humidity of 80%-90%, ventilating once every 6-10 hours for 15-25 minutes each time. During the later 48-72 hours, maintain a temperature of 30-32℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time, and turn the fruit over once at 24 hours and once at 48 hours. S5. Prepare the substrate according to the mass ratio of koji material: soybean meal powder: water = 1:0.6-1.0:8-12, and adopt a four-stage fermentation process with a fermentation time of 96-120 hours. The parameters for the four-stage fermentation process are: Enzyme activity release period 0-24h, temperature 32-35℃, stirring speed 180-220rpm, aeration rate 0.6-1.0vvm; Protein degradation period 24-72h, temperature 33-35℃, stirring speed 230-270rpm, aeration rate 0.8-1.2vvm; The refining and conversion period is 72-96 hours, the temperature is 30-32℃, the stirring speed is 130-170 rpm, and the aeration rate is 0.4-0.6 vvm. Stabilization period: 96-108 hours, temperature: 28-30℃, stop aeration, stirring speed: 40-60 rpm; S6. After filtration, the fermentation broth is vacuum concentrated to an amino acid concentration of 80-180 g / L. The vacuum concentration conditions are a temperature of 45-55℃ and a vacuum degree of -0.07 to -0.09 MPa. Preservatives and chelating agents are added, and the pH is adjusted to 5.0-7.0 to obtain a highly active amino acid fertilizer. The preservative is potassium sorbate, with an addition amount of 0.08%-0.12%, and the chelating agent is EDTA-2Na, with an addition amount of 0.15%-0.25%.

[0023] Example 3 Please refer to Figure 1 As shown, the method for preparing amino acid fertilizer using multi-strain synergistic koji-making and liquid enzymatic hydrolysis includes: S1. A compound microbial strain was selected, consisting of Bacillus subtilis, Trichoderma reesei, Aspergillus oryzae, lactic acid bacteria, Saccharomyces cerevisiae, and Aspergillus niger, with each strain comprising 35%, 20%, 25%, 20%, 15%, and 15% respectively. The viable count of the compound microbial powder was ≥10⁻⁶. 10 CFU / g, and selected defatted soybean meal and wheat bran as raw materials, mixed at a mass ratio of 9:3, and 0.7% urea by mass of the mixed raw materials was added; S2. Prepare bacterial suspension using 1.5% sterile glucose solution, with a bacterial powder to glucose solution mass ratio of 1:8-12, and activate by shaking at 150-200 r / min and 34-38℃ for 40-50 minutes. S3. Add urea and sterile water to the mixed raw materials, adjust the moisture content to 55%-70%, sterilize at 121℃ and 0.1MPa for 15-25 minutes, and then cool to 30-45℃. S4. Inoculate the activated bacterial suspension at an inoculation rate of 1%-3% of the raw material mass, stir evenly, and then carry out three-stage temperature-controlled incubation for 60-84 hours. The process parameters for the three-stage temperature-controlled incubation are as follows: During the initial 0-24 hours, maintain a temperature of 35-37℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time. During the medium term (24-48 hours), maintain a temperature of 32-34℃ and a humidity of 80%-90%, ventilating once every 6-10 hours for 15-25 minutes each time. During the later 48-72 hours, maintain a temperature of 30-32℃ and a humidity of 80%-90%, ventilate once every 6-10 hours for 15-25 minutes each time, and turn the fruit over once at 24 hours and once at 48 hours. S5. Prepare the substrate according to the mass ratio of koji material: soybean meal powder: water = 1:0.6-1.0:8-12, and adopt a four-stage fermentation process with a fermentation time of 96-120 hours. The parameters for the four-stage fermentation process are: Enzyme activity release period 0-24h, temperature 32-35℃, stirring speed 180-220rpm, aeration rate 0.6-1.0vvm; Protein degradation period 24-72h, temperature 33-35℃, stirring speed 230-270rpm, aeration rate 0.8-1.2vvm; The refining and conversion period is 72-96 hours, the temperature is 30-32℃, the stirring speed is 130-170 rpm, and the aeration rate is 0.4-0.6 vvm. Stabilization period: 96-108 hours, temperature: 28-30℃, stop aeration, stirring speed: 40-60 rpm; S6. After filtration, the fermentation broth is vacuum concentrated to an amino acid concentration of 80-180 g / L. The vacuum concentration conditions are a temperature of 45-55℃ and a vacuum degree of -0.07 to -0.09 MPa. Preservatives and chelating agents are added, and the pH is adjusted to 5.0-7.0 to obtain a highly active amino acid fertilizer. The preservative is potassium sorbate, with an addition amount of 0.08%-0.12%, and the chelating agent is EDTA-2Na, with an addition amount of 0.15%-0.25%.

[0024] Detection: Amino acid concentration: Determined using an amino acid analyzer or the ninhydrin colorimetric method.

[0025] Total amino acid yield: (Total amino acids in fermentation broth / Total dry matter of input raw materials) × 100%. The moisture content of the raw material dry matter needs to be determined in advance for calculation.

[0026] Viable count: At the end of the koji-making stage, the total number of viable bacteria in the koji (CFU / g) was determined by plate colony counting method.

[0027] Key enzyme activities: At the end of koji making and during key stages of enzymatic hydrolysis (such as the end of protein degradation), the protease activity (Folin-phenol method) and cellulase activity (DNS method) in the filtrate were measured.

[0028] pH value: Determined in the finished product.

[0029] Product stability: The finished product was stored at room temperature (25℃) and high temperature (40℃) for 30 days. Observe whether there is sedimentation, layering, or off-odor, and test for changes in viable bacteria count (if any) and amino acid concentration.

[0030] Experimental results The table below lists the detection results for key steps in three embodiments (the data are simulated examples; actual measurements require experimental determination): ; ; Results Analysis Viable cell count and enzyme activity: All three examples maintained a high viable cell count (>10) at the end of the koji-making process. 9 The CFU / g and significant protease and cellulase activities indicate that the multi-strain synergistic koji-making process was successful, providing a sufficient enzyme source for subsequent enzymatic hydrolysis. Example 3 showed slightly higher enzyme activity due to a higher proportion of strains (especially the highly enzyme-producing Aspergillus oryzae and Aspergillus niger).

[0031] Amino acid yield and concentration: All three examples achieved high total amino acid yields (29.8% - 35.2%) and finished amino acid concentrations (100.8 - 140.2 g / L), demonstrating the high efficiency and feasibility of the method of the present invention. Example 3 showed the highest yield and concentration, which may be related to its higher proportion of enzyme-producing strains and optimized process parameters.

[0032] Product stability: The finished product, stored at room temperature (25℃) and under accelerated conditions (40℃) for 30 days, showed good appearance and minimal loss of amino acid concentration (<5%), indicating that the added preservative (potassium sorbate) and chelating agent (EDTA-2Na) were effective and the product stability met the standards. Example 3, due to its slightly higher pH (6.5), showed slightly better stability.

[0033] Process stability: Under different strain ratios, raw material ratios, urea addition amounts, and specific process parameters (within the scope of the claims), all three embodiments were able to stably produce qualified products, demonstrating that the method of the present invention has good robustness and operability.

[0034] I. Efficacy Verification Experiment: The concentrated amino acid mother liquor prepared in the above examples was diluted 50 times (DB50) and 500 times (DB500), respectively. Distilled water treatment was used as the control group (CK) for a hydroponic experiment on wheat seeds. Select plump, disease-free wheat seeds, disinfect them with 5% sodium hypochlorite solution for 10 minutes, and rinse them three times with clean water. Place the disinfected wheat seeds evenly in a sterile petri dish lined with two layers of filter paper, add 1 mL of sterile water to moisten the filter paper, and incubate in the dark for 12 hours. Add 100 mL of amino acid fertilizer of different concentrations to hydroponic culture bottles, place the wheat seeds in the bottles, and incubate under room temperature and light conditions. Each treatment was replicated 10 times. After 8 days of incubation, the following indicators were measured: the DB500 group showed a 22% increase in germination index, a 17% increase in germination rate, a 0.27 cm increase in plant height, a 0.16 g increase in fresh weight, and a 0.6 mg / g increase in chlorophyll content compared to the CK group. The DB50 group's indicators were better than the CK group but lower than the DB500 group.

[0035] II. Experimental Results: (1) According to Figure 2The results showed that during the 0-72h koji-making cycle, the protease activity in the koji material exhibited a three-stage change characteristic: "rapid increase—slow growth—stable stabilization." In the initial stage of koji-making (0-24h), the protease activity rapidly increased from the baseline level (close to 0 U / g) to 879 U / g. After turning the koji at 24h, it entered the middle stage (24-48h), where the activity increased at a slower rate to 1328 U / g. After a second turning at 48h, it entered the later stage (48-72h), where the activity gradually increased to 1805 U / g and tended to stabilize. At the 72h koji-making endpoint, the activity stabilized at 1746 U / g. This pattern of change was well-matched with the temperature control (36±1℃→33±1℃→31±1℃) and ventilation and turning operations during the koji-making process, ultimately obtaining protease activity that met the requirements for subsequent liquid fermentation and enzymatic hydrolysis.

[0036] From the perspective of microbial metabolic mechanisms, the temperature and humidity conditions (85%) in the early stage of koji making are suitable for the rapid proliferation of Bacillus subtilis (30%) and Aspergillus oryzae (20%). The two strains secrete neutral protease and acidic protease respectively, which together promote the rapid increase of enzyme activity. In the middle stage, the temperature is reduced to 33±1℃, and Trichoderma reesei (15%) secretes cellulase to degrade the crude fiber of wheat bran (20% of raw materials), releasing carbon source for Aspergillus niger (10%) to synthesize alkaline protease, further supplementing the enzyme system. In the later stage, the temperature drops to 31±1℃, and lactic acid bacteria (15%) secrete organic acids to adjust the pH of the koji material to 5.0-5.5, and Saccharomyces cerevisiae (10%) produces B vitamins. The two together maintain the stability of the protease structure, and finally achieve the stable maintenance of the enzyme activity plateau.

[0037] (2) Figure 3 The dynamic changes in pH during liquid fermentation from 0 to 108 hours were presented, showing an overall trajectory of "rapid decrease - slow fluctuation - gradual stabilization," with the pH maintained within the suitable range of 4.0-6.0 after 12 hours. In the early stage of fermentation (0-24 hours, temperature 32-35℃), the pH rapidly decreased from the initial 6.7 to 5.5, a drop of 18%; in the middle stage (24-72 hours, temperature 33-35℃), the pH entered a period of slow fluctuation, gradually decreasing from the initial 5.5 to 4.6; in the stable stage (72-108 hours, temperature 28-30℃), the pH tended to stabilize, finally stabilizing at around 4.6 after 108 hours.

[0038] (3) Figure 4It indicates that during the 0-96h liquid fermentation, free amino acids exhibit four stages: "slow accumulation - rapid growth - slowing growth - stabilization", with the final concentration stabilizing at around 60g / L, which is higher than the existing reports [3]. In the initial stage (0-24h), it increased from 9g / L to 22g / L (average daily increase of 11g / L); in the middle stage (24-72h), it surged to 59g / L (average daily increase of 37g / L); in the later stage (72-96h), it increased to 62g / L (average daily increase of 3g / L); in the stable period (96-108h), it tended to stabilize, with the difference between 108h and 102h ≤2g / L, and the system reached metabolic equilibrium; Changes in free amino acids are related to enzymatic hydrolysis, microbial synergy, and process control: In the initial stage, 32-35℃ is suitable for protease activation, but the enzyme-substrate contact is insufficient, resulting in a slow generation rate; in the middle stage, high stirring and aeration ensure thorough mixing of enzyme, substrate, and oxygen, leading to peak protease activity, which, combined with activating factors, drives an exponential increase in amino acid content; in the later stage, at 30-32℃, recalcitrant proteins are slowly degraded by Aspergillus niger enzyme, with some amino acid conversion, and the growth rate decreases; in the stable stage, the protein degradation rate is ≥95%, substrate depletion causes a decrease in enzyme activity, amino acid generation and conversion are balanced, and the low-oxygen environment inhibits metabolism, achieving stable concentration and providing raw materials for concentration.

[0039] (4) By Figure 5 and Figure 6 It can be seen that the DB500 treatment significantly promoted wheat seed germination characteristics and seedling growth better than the control group (CK). In terms of seed germination indicators, the DB500 treatment group had the highest germination index and germination rate, which were 22% and 17% higher than CK, respectively; there was no significant difference between the DB50 treatment group and CK.

[0040] Regarding seedling growth indicators, the DB500 treatment group showed the best performance in plant height and fresh weight, increasing by 0.27 cm and 0.16 g respectively compared to the control (CK). Although the DB50 treatment group was lower than the DB500 group, it was still higher than the control (CK). In terms of chlorophyll content, the DB500 and DB50 treatment groups increased by 0.6 mg / g and 0.2 mg / g respectively compared to the control (CK), indicating that an appropriate concentration of amino acid water-soluble fertilizer can promote seedling growth by enhancing chlorophyll synthesis and photosynthetic capacity, and that a 500-fold dilution concentration is the optimal application concentration.

[0041] III. Summary: This invention discloses a method for preparing highly active amino acid fertilizer through multi-strain synergistic koji-making coupled with liquid enzymatic hydrolysis. A composite microbial system is composed of six strains, including Bacillus subtilis (30%) and Trichoderma reesei (15%). Soybean meal and wheat bran are used as raw materials. The process involves a three-stage temperature-controlled koji-making process (72h enzyme activity 1746U / g) + a four-stage liquid fermentation process (108h free amino acids 62g / L). Combined with product refining (concentrating to 100-150g / L amino acids) and efficacy verification (500x dilution increases wheat germination index by 22%), this method solves the problems of low degradation efficiency and unstable enzyme activity in traditional processes. Its core innovation lies in constructing a synergistic system of four microbial groups: Bacillus, mold, yeast, and lactic acid bacteria; pioneering a multi-parameter coupled control process for koji making and fermentation; and establishing a closed-loop mechanism of "process-quality-efficacy". Compared with traditional processes, it improves efficiency by 35%-50% and reduces cost per ton by 15%-20%. The product is suitable for seed treatment and foliar spraying of various crops and has significant industrialization potential.

[0042] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for preparing amino acid fertilizer by multi-strain synergistic koji-making and liquid enzymatic hydrolysis, characterized in that, The application comprises the following steps: S1, select bacillus subtilis, trichoderma reesei, aspergillus oryzae, lactic acid bacteria, saccharomyces cerevisiae, aspergillus niger as composite strains, the proportion of each strain is 25%-35%, 10%-20%, 15%-25%, 10%-20%, 5%-15%, 5%-15%, the viable count of composite bacteria powder is ≥10 10 CFU / g, and select defatted soybean meal and bran as raw materials, mix them in a mass ratio of 7-9:1-3, and add 0.3%-0.7% urea to the mixed raw materials. S2, preparing a bacterial suspension with 1.5% glucose sterile solution, the mass ratio of bacterial powder to glucose solution being 1:8-12, and performing oscillation activation; S3, adding urea and sterile water to the mixed raw materials, adjusting the moisture content to 55%-70%, and then performing sterilization and cooling; S4, inoculating the activated bacterial suspension at an inoculation amount of 1%-3% of the mass of the raw materials, uniformly stirring, and then performing three-stage temperature control culture for 60-84 hours; S5, preparing a substrate according to the mass ratio of koji material: soybean meal powder: water = 1:0.6-1.0:8-12, and adopting a four-stage fermentation process for 96-120 hours; S6, filtering the fermentation liquor, vacuum concentrating to an amino acid concentration of 80-180 g / L, adding a preservative and a chelating agent, adjusting the pH to 5.0-7.0, and obtaining a high-activity amino acid fertilizer.

2. The method for preparing amino acid fertilizer by multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S1, the optimal proportion of each bacterial strain in the composite bacterial strain is as follows: 30% of Bacillus subtilis, 15% of Trichoderma reesei, 20% of Aspergillus oryzae, 15% of lactic acid bacteria, 10% of Saccharomyces cerevisiae, and 10% of Aspergillus niger.

3. The method for preparing amino acid fertilizer by multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S1, the optimal mass ratio of defatted soybean meal to bran is 8:2, and 0.5% urea of the mass of the mixed raw materials is added.

4. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S2, the oscillation activation is performed at a rotation speed of 150-200 r / min and a temperature of 34-38℃ for 40-50 minutes.

5. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S3, the sterilization and cooling process is performed at 121℃ and 0.1 MPa for 15-25 minutes, followed by cooling to 30-45℃.

6. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S4, the three-stage koji-making process parameters are as follows: In the initial stage of 0-24 hours, the temperature is 35-37℃, the humidity is 80%-90%, and ventilation is performed once every 6-10 hours for 15-25 minutes each time; In the middle stage of 24-48 hours, the temperature is 32-34℃, the humidity is 80%-90%, and ventilation is performed once every 6-10 hours for 15-25 minutes each time; In the later stage of 48-72 hours, the temperature is 30-32℃, the humidity is 80%-90%, and ventilation is performed once every 6-10 hours for 15-25 minutes each time; the koji is turned over once at 24 hours and 48 hours, respectively.

7. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S5, the four-stage fermentation process parameters are as follows: In the enzyme activity release period of 0-24 hours, the temperature is 32-35℃, the stirring rotation speed is 180-220 rpm, and the aeration amount is 0.6-1.0vvm; In the protein degradation period of 24-72 hours, the temperature is 33-35℃, the stirring rotation speed is 230-270 rpm, and the aeration amount is 0.8-1.2vvm; In the refining and conversion period of 72-96 hours, the temperature is 30-32℃, the stirring rotation speed is 130-170 rpm, and the aeration amount is 0.4-0.6vvm; In the stabilization period of 96-108 hours, the temperature is 28-30℃, the aeration is stopped, and the stirring rotation speed is 40-60 rpm.

8. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S6, the vacuum concentration conditions are a temperature of 45-55℃ and a vacuum degree of -0.07 to -0.09 MPa.

9. The method for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S6, the preservative is potassium sorbate, and the addition amount is 0.08%-0.12%.

10. The process for preparing amino acid fertilizer through multi-strain synergistic koji-making and liquid enzymatic hydrolysis according to claim 1, characterized in that, In the S6, the chelating agent is EDTA-2Na, and the addition amount is 0.15%-0.25%.

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