Method for preparing bio-fertilizer by combining compound probiotics and leucine fermentation liquor

Abstract

The invention discloses a method for preparing a biological fertilizer by combining compound probiotics and leucine fermentation liquor, and relates to the technical field of fertilizer preparation, the method comprises the following steps: carrying out magnetic reinforcement treatment on the probiotics, carrying out ultrasonic-assisted extraction on the leucine fermentation liquor, and activating and propagating the compound probiotics, and after the treatment on the compound probiotics and the leucine fermentation liquor is completed, carrying out ultrasonic-assisted extraction on the leucine fermentation liquor to obtain the biological fertilizer. The preparation method has the advantages that the bacterial liquid is placed in a magnetic field for pretreatment, so that the activity and the stability of the probiotics can be enhanced, the probiotics can play a better role in the subsequent fermentation and application process, the metabolic activity of the probiotics subjected to magnetic strengthening treatment is remarkably improved, and the metabolic activity of the probiotics subjected to magnetic strengthening treatment is remarkably improved. According to the invention, the leucine fermentation broth is used as a raw material, organic matters in the soil can be more effectively decomposed, more nutrients are released to be absorbed by crops, ultrasonic-assisted extraction is carried out on the leucine fermentation broth, and in the treatment process, ultrasonic treatment can destroy bacterial cells, so that leucine is more fully released, and the extraction rate and purity of leucine are improved.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, and in particular to a method for preparing biological fertilizer by combining composite probiotics with leucine fermentation liquid. Background Art

[0002] In modern agricultural production, efficient and environmentally friendly fertilizers are crucial to improving crop yields and quality. With the continuous advancement of science and technology, people have higher and higher requirements for fertilizers. They not only require them to have rich nutrients, but also expect them to have greener and more sustainable sources. Compound probiotics can improve soil structure. They decompose organic matter in the soil by secreting various enzymes and organic acids, increase soil permeability and water retention, and promote the formation of soil aggregate structure. Secondly, compound probiotics can improve soil fertility. They can fix nitrogen, dissolve phosphorus and potassium, and convert nutrients in the soil that are difficult for crops to absorb into available forms, providing sufficient nutrition for crop growth. In addition, compound probiotics can also enhance the stress resistance of crops. They can produce antibacterial substances, inhibit the growth of pathogens in the soil, and reduce the occurrence of crop diseases. At the same time, they can also induce crops to produce systemic resistance and improve the adaptability of crops to drought, salinity, low temperature and other adversities. Leucine is an important amino acid and plays an important role in the growth and development of crops. Leucine can promote the growth of crop roots, increase the absorption area of ​​the roots, and improve the absorption capacity of crops for water and nutrients. At the same time, leucine can also enhance the stress resistance of crops and improve their adaptability to adversity. At present, leucine is mainly produced by fermentation. Leucine fermentation liquid contains rich leucine and other beneficial ingredients, such as vitamins, amino acids, etc. However, the application of leucine fermentation liquid in the agricultural field is still in its infancy. On the one hand, the production cost of leucine fermentation liquid is high, which limits its large-scale application in the agricultural field. On the other hand, the use method and dosage of leucine fermentation liquid need further research and optimization. At present, leucine fermentation liquid is mainly used by foliar spraying and soil irrigation, but different use methods and dosages have different effects on the growth and development of crops, and need to be selected according to specific circumstances. Compound probiotics can improve the structure of soil microbial communities, enhance the soil's water and fertilizer retention capacity, and improve the stress resistance of crops. Leucine in leucine fermentation liquid can promote crop root growth and improve the crop's ability to absorb nutrients. Through innovative preparation methods, compound probiotics and leucine fermentation liquid are efficiently combined, which can give full play to the advantages of both and prepare biological fertilizers with higher performance to meet the needs of sustainable development of modern agriculture. To this end, we propose a method for preparing biological fertilizers by combining compound probiotics with leucine fermentation liquid. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing a biofertilizer by combining composite probiotics with leucine fermentation liquid.

[0004] In order to solve the problems raised in the above background technology, the present invention provides the following technical solutions: a method for preparing a biofertilizer by combining a composite probiotic with a leucine fermentation broth, comprising magnetic enhancement treatment of probiotics, ultrasonic-assisted extraction of leucine fermentation broth, and activation and expansion of the composite probiotics. After the composite probiotics and the leucine fermentation broth are treated, they are mixed and fermented to prepare the biofertilizer. The specific operation steps of preparing the biofertilizer by combining the composite probiotic with the leucine fermentation broth are as follows: Step 1: selecting a composite probiotic material and a specific genetically engineered strain for leucine fermentation, and preparing a leucine fermentation liquid after leucine fermentation by the specific genetically engineered strain; Step 2: Stir and mix the composite probiotics with sterile saline to fully disperse the probiotics in the saline, place the mixture in a magnetic field after dispersion, and perform ultrasound-assisted extraction on the prepared leucine fermentation liquid; Step 3, placing the composite probiotic bacterial solution after magnetic enhancement treatment in a constant temperature oscillating incubator, culturing and activating it, and then adding it to the expansion culture medium for expansion culture. During this period, the number of live bacteria in the bacterial solution should be regularly tested to ensure that the number of live bacteria after expansion culture is controlled at more than 5 billion / mL, so as to obtain the composite probiotic bacterial solution after expansion culture; Step 4: performing multiple ultrafiltration treatments on the leucine fermentation broth after ultrasound-assisted extraction, and after the ultrafiltration is completed, performing ion exchange chromatography on the leucine fermentation broth, eluting the solution after the chromatography, collecting the leucine eluate, and concentrating the leucine eluate under vacuum to obtain a high-concentration leucine solution; Step 5, stirring and mixing the composite probiotic bacterial liquid after expansion and the high concentration leucine solution for fermentation, spray drying the solution after fermentation, adding humic acid, amino acids and trace elements, stirring and mixing, and extruding and granulating the solution to obtain a finished biofertilizer; Step 6: Conduct quality inspection on the finished bio-fertilizer products, bag the qualified ones, and store them in a dry and ventilated place.

[0005] As a further scheme of the present invention: in the step one, the composite probiotics include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus gelatinosa, Bacillus acidophilus and Bifidobacterium, and the proportions of the above six probiotic materials are 20%, 20%, 15%, 15%, 15% and 15% in order, respectively, and the total number of live bacteria of the six probiotic materials is greater than 2 billion / g.

[0006] As a further solution of the present invention: in the step 1, a specific genetically engineered strain is selected for leucine fermentation, and the formula of the fermentation medium is 50 g / L glucose, 10 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 5 g / L yeast powder and 5 g / L leucine precursor.

[0007] As a further scheme of the present invention: in the step 2, the composite probiotics are mixed according to a proportion and then added to sterile physiological saline, the number of viable bacteria of the composite probiotics is controlled to reach 200 million / mL, the mixed bacterial solution is placed in a magnetic field with a magnetic field strength of 1000 Gauss for 2h-3h, and the leucine fermentation solution after fermentation is subjected to ultrasonic-assisted extraction, and ultrasonic treatment with a frequency of 40kHz and a power of 500W is used for 30min to obtain a composite probiotic bacterial solution after magnetic enhancement.

[0008] As a further scheme of the present invention: in the step three, the composite probiotic bacterial liquid after magnetic strengthening treatment is placed in a constant temperature shaking incubator, and cultured at 35°C and 150 / min for 18h-24h. After the composite probiotics are fully activated, they are expanded. The formula of the expansion culture medium is 10g / L peptone, 5g / L beef extract, 5g / L sodium chloride and 10g / L glucose. The activated bacterial liquid is inoculated into the expansion culture medium at an inoculation rate of 5%, and cultured at a temperature of 35°C and an oscillation rate of 150r / min for 40h-48h, and the number of live bacteria after expansion is controlled to be above 5 billion / mL.

[0009] As a further scheme of the present invention: in the step 4, the leucine fermentation broth after ultrasound-assisted extraction is introduced into the ultrafiltration equipment through the feed pipe, the pressure pump is turned on, and the leucine fermentation broth is passed through the ultrafiltration membrane under the action of pressure, and the ultrafiltration membrane with a molecular weight cutoff of 3kDa-5kDa is selected to filter it, and the flow rate is set to 10L / min-12L / min. After the filtration is completed, ion exchange chromatography is performed on it, and the ultrafiltered leucine fermentation broth passes through the ion exchange resin column at a flow rate of 5L / min-8L / min. After the ion exchange resin is filled in the ion exchange resin pile, the buffer solution is slowly injected into the ion exchange resin pile, and the flow rate is controlled to be 1L / min-3L / min. The price buffer solution continues to flow through the ion exchange resin column until the pH value and ion concentration of the effluent are substantially consistent with the pH value and ion concentration of the buffer solution, thereby obtaining a chromatographic solution.

[0010] As a further scheme of the present invention: in the step 4, the solution after the chromatography is completed is eluted, and according to the type of ion exchange resin and the adsorption characteristics of leucine, for a strongly acidic cation exchange resin, a 0.5 mol / L-1.0 mol / L sodium chloride solution is used to elute it, and the elution flow rate is controlled at 3 L / min-5 L / min, and the leucine eluate is collected and placed in a vacuum concentration device, the vacuum system is turned on, and the pressure in the device is gradually reduced. At the same time, the leucine eluate is heated, the vacuum degree is set to 0.08 MPa-0.10 MPa, and the temperature is controlled at 40°C-50°C. According to the volume of the leucine eluate and the processing capacity of the equipment, the leucine eluate is concentrated for 2h-4h until a high-concentration leucine solution is obtained.

[0011] As a further solution of the present invention: in the step 5, the composite probiotic bacterial liquid after expansion is mixed with the high concentration leucine solution in a ratio of 1-2:1, the stirring rate is controlled at 50r / min-80r / min, and after mixing, it is transferred to a fermentation tank for fermentation, the temperature is controlled between 30°C and 35°C, the fermentation time is 36h-48h, and after fermentation, the fermentation liquid is transported to a spray drying equipment, the inlet temperature is set to 150°C-180°C, the outlet temperature is set to 70°C-90°C, and the spray rate is 5L / mi n-10L / min, the fermentation liquid is quickly dried into powder by spray drying, the dried powder is mixed with humic acid, amino acids and trace elements, the added amount of humic acid is 10%-20% of the total weight, the added amount of amino acids is 5%-10% of the total weight, and the added amount of trace elements is 1%-5%. After mixing, use a stirring device to stir at a stirring rate of 30r / min-50r / min for 30-60min. After stirring, the evenly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer.

[0012] As a further solution of the present invention: in the step six, the quality inspection includes appearance inspection, moisture determination and microbial activity detection, the particle size of the granular bio-fertilizer is set to 2mm-5mm, the moisture content is 5%-10%, the microbial culture temperature is 30℃-35℃, the culture time is 24h-48h, and after the quality inspection is qualified, the storage temperature of the bagged fertilizer is controlled between 5℃-30℃.

[0013] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are: 1. The present invention places the bacterial liquid into a magnetic field for pretreatment, so that the activity and stability of the probiotics can be enhanced, and the probiotics can play a better role in the subsequent fermentation and application process. The metabolic activity of the probiotics treated with magnetic enhancement is significantly improved, and the organic matter in the soil can be more effectively decomposed, and more nutrients can be released for crop absorption. The leucine fermentation liquid is subjected to ultrasonic-assisted extraction. During the treatment process, the ultrasonic treatment can destroy the bacterial cells, so that leucine is released more fully, and the extraction rate and purity of leucine are improved. At the same time, the ultrasonic treatment can promote the release of vitamins and amino acids in the fermentation liquid. The leucine fermentation liquid is subjected to ultrasonic-assisted extraction and multi-step treatment to obtain a high-concentration leucine solution, which is mixed and fermented with the composite probiotic bacterial liquid after expansion, so that the content of leucine and other beneficial components in the biological fertilizer is higher, which can better promote the growth of crop roots and increase the root absorption area, thereby improving the crop's ability to absorb water and nutrients; 2. The present invention improves the extraction rate and purity of leucine by subjecting the leucine fermentation broth to ultrafiltration, ion exchange chromatography and vacuum concentration, reduces the waste of raw materials, and thus reduces the production cost, which helps to promote the large-scale application of leucine fermentation broth in the agricultural field. The combination of leucine fermentation broth and composite probiotics provides a rich source of nutrition for soil microorganisms, attracts more types of microorganisms to colonize and reproduce in the soil, and the increase in the diversity of such microorganisms helps to improve the anti-interference ability of the soil. Even in the face of environmental changes or external pressures, the soil ecosystem can maintain a relatively stable function. The added nutrients such as humic acid, amino acids and trace elements enrich the nutritional components of the biofertilizer, and can meet the needs of crops for various nutrients at different growth stages, which helps to improve the yield and quality of crops, enhance the stress resistance of crops, and achieve sustainable development of agriculture. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a schematic diagram of a specific process for preparing biological fertilizer in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] The specific embodiments of the present invention are further described below in conjunction with the accompanying drawings. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0016] The method for preparing a biofertilizer by combining a composite probiotic with a leucine fermentation broth of the present invention comprises magnetic enhancement treatment of the probiotic, ultrasonic-assisted extraction of the leucine fermentation broth, and activation and expansion of the composite probiotic. After the composite probiotic and the leucine fermentation broth are treated, they are mixed and fermented. The specific operation steps of preparing a biofertilizer by combining the composite probiotic with the leucine fermentation broth are as follows: Step 1: selecting a composite probiotic material and a specific genetically engineered strain for leucine fermentation, and preparing a leucine fermentation liquid after leucine fermentation by the specific genetically engineered strain; Step 2: Stir and mix the composite probiotics with sterile saline to fully disperse the probiotics in the saline, place the mixture in a magnetic field after dispersion, and perform ultrasound-assisted extraction on the prepared leucine fermentation liquid; Step 3, placing the composite probiotic bacterial solution after magnetic enhancement treatment in a constant temperature oscillating incubator, culturing and activating it, and then adding it to the expansion culture medium for expansion culture. During this period, the number of live bacteria in the bacterial solution should be regularly tested to ensure that the number of live bacteria after expansion culture is controlled at more than 5 billion / mL, so as to obtain the composite probiotic bacterial solution after expansion culture; Step 4: performing multiple ultrafiltration treatments on the leucine fermentation broth after ultrasound-assisted extraction, and after the ultrafiltration is completed, performing ion exchange chromatography on the leucine fermentation broth, eluting the solution after the chromatography, collecting the leucine eluate, and concentrating the leucine eluate under vacuum to obtain a high-concentration leucine solution; Step 5, stirring and mixing the composite probiotic bacterial liquid after expansion and the high concentration leucine solution for fermentation, spray drying the solution after fermentation, adding humic acid, amino acids and trace elements, stirring and mixing, and extruding and granulating the solution to obtain a finished biofertilizer; Step 6: Conduct quality inspection on the finished bio-fertilizer products, bag the qualified ones, and store them in a dry and ventilated place.

[0017] In one embodiment of the present invention: in step one, the composite probiotics include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus gelatinosa, Bacillus acidophilus and Bifidobacterium, and the proportions of the above six probiotic materials are 20%, 20%, 15%, 15%, 15% and 15% in order, respectively, and the total number of live bacteria of the six probiotic materials is greater than 2 billion / g.

[0018] In one embodiment of the present invention: in step 1, a specific genetically engineered strain is selected for leucine fermentation, and the formula of the fermentation medium is 50 g / L glucose, 10 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 5 g / L yeast powder and 5 g / L leucine precursor.

[0019] In one embodiment of the present invention: in step 2, the composite probiotics are mixed in proportion and then added with sterile physiological saline, the viable count of the composite probiotics is controlled to reach 200 million / mL, the mixed bacterial solution is placed in a magnetic field with a magnetic field strength of 1000 Gauss for 2h-3h, and the leucine fermentation solution after fermentation is subjected to ultrasonic-assisted extraction, and ultrasonic treatment with a frequency of 40kHz and a power of 500W is used for 30min to obtain a composite probiotic bacterial solution after magnetic enhancement.

[0020] In one embodiment of the present invention: in step three, the composite probiotic bacterial liquid after magnetic enhancement treatment is placed in a constant temperature shaking incubator, and cultured at 35°C and 150 / min for 18h-24h. After the composite probiotics are fully activated, they are expanded. The formula of the expansion culture medium is 10g / L peptone, 5g / L beef extract, 5g / L sodium chloride and 10g / L glucose. The activated bacterial liquid is inoculated into the expansion culture medium at a 5% inoculation rate, and cultured at a temperature of 35°C and an oscillation rate of 150r / min for 40h-48h, and the number of live bacteria after expansion is controlled to be above 5 billion / mL.

[0021] In one embodiment of the present invention: in step 4, the leucine fermentation broth after ultrasound-assisted extraction is introduced into the ultrafiltration device through the feed pipe, the pressure pump is turned on, and the leucine fermentation broth is passed through the ultrafiltration membrane under the action of pressure, and the ultrafiltration membrane with a molecular weight cutoff of 3kDa-5kDa is selected to filter it, and the flow rate is set to 10L / min-12L / min. After the filtration is completed, ion exchange chromatography is performed on it, and the ultrafiltered leucine fermentation broth is passed through the ion exchange resin column at a flow rate of 5L / min-8L / min. After the ion exchange resin is filled in the ion exchange resin pile, the buffer solution is slowly injected into the ion exchange resin pile, and the flow rate is controlled to be 1L / min-3L / min. The price buffer solution continues to flow through the ion exchange resin column until the pH value and ion concentration of the effluent are substantially consistent with the pH value and ion concentration of the buffer solution, and a chromatographic solution is obtained.

[0022] In one embodiment of the present invention: in step 4, the solution after the chromatography is eluted, and according to the type of ion exchange resin and the adsorption characteristics of leucine, for a strongly acidic cation exchange resin, a 0.5 mol / L-1.0 mol / L sodium chloride solution is used to elute it, and the elution flow rate is controlled at 3 L / min-5 L / min. The leucine eluate is collected and placed in a vacuum concentration device, the vacuum system is turned on, and the pressure in the device is gradually reduced. At the same time, the leucine eluate is heated, the vacuum degree is set to 0.08 MPa-0.10 MPa, and the temperature is controlled at 40°C-50°C. According to the volume of the leucine eluate and the processing capacity of the equipment, it is concentrated for 2h-4h until a high-concentration leucine solution is obtained.

[0023] In one embodiment of the present invention: in step 5, the composite probiotic bacterial solution after expansion is mixed with the high concentration leucine solution in a ratio of 1-2:1, the stirring rate is controlled at 50r / min-80r / min, and after mixing, it is transferred to a fermentation tank for fermentation, the temperature is controlled between 30°C-35°C, the fermentation time is 36h-48h, and after fermentation, the fermentation liquid is transported to a spray drying device, the inlet temperature is set to 150°C-180°C, the outlet temperature is set to 70°C-90°C, and the spray rate is 5L / mi n-10L / min, the fermentation liquid is quickly dried into powder by spray drying, the dried powder is mixed with humic acid, amino acids and trace elements, the added amount of humic acid is 10%-20% of the total weight, the added amount of amino acids is 5%-10% of the total weight, and the added amount of trace elements is 1%-5%. After mixing, use a stirring device to stir at a stirring rate of 30r / min-50r / min for 30-60min. After stirring, the evenly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer.

[0024] In one embodiment of the present invention: in step six, the quality inspection includes appearance inspection, moisture determination and microbial activity detection, the particle size of the granular bio-fertilizer is set to 2mm-5mm, the moisture content is 5%-10%, the microbial culture temperature is 30℃-35℃, the culture time is 24h-48h, and after the quality inspection is qualified, the storage temperature of the bagged fertilizer is controlled between 5℃-30℃.

[0025] In one embodiment of the present invention: in the process of preparing biofertilizer, nano silicon dioxide and nano calcium carbonate can be introduced, and the nano silicon dioxide and nano calcium carbonate are mixed in a ratio of 1:1, and the added amount is 5% of the total mass of the biofertilizer. The nano material has a large specific surface area and good adsorption performance, and can absorb moisture and nutrients in the soil, reduce nutrient loss, and improve fertilizer utilization. At the same time, the nano material can also improve the soil structure and increase the air permeability and water retention of the soil.

[0026] In one embodiment of the present invention, there are two types of ion exchange resin columns: strongly acidic cation exchange resins, such as Amberlite IR-120, which have strongly acidic sulfonic acid groups and can undergo ion exchange adsorption with positively charged leucine. If leucine is positively charged at a specific pH, this type of resin is suitable for selection; and strongly basic anion exchange resins, such as Amberlite IRA-400, which have strongly basic quaternary ammonium groups and can be selected when leucine is negatively charged under specific conditions, wherein the specific conditions include a specific pH range, ionic strength, and temperature.

[0027] In one embodiment of the present invention: the materials of the buffer solution are divided into two types. For a strongly acidic cation exchange resin, an acetic acid-sodium acetate buffer solution can be selected. The main materials include acetic acid and sodium acetate. The ratio of the two is adjusted according to the required pH value (usually 4.0-6.0). An appropriate amount of acetic acid and sodium acetate are mixed in a certain molar ratio, and then the volume is adjusted to the required volume with distilled water. For a strongly basic anion exchange resin, a Tris-HCl buffer solution can be selected. The main materials are trishydroxymethylaminomethane (Tris) and hydrochloric acid. Tris is dissolved in water, and then the pH value is adjusted with hydrochloric acid (usually 7.0-9.0). A certain amount of Tris is dissolved in water, and then hydrochloric acid is added dropwise. At the same time, the pH value of the solution is monitored with a pH meter until the required pH value is reached, and finally the volume is adjusted to the required volume with distilled water.

[0028] In one embodiment of the present invention: ultrafiltration adopts a multi-stage ultrafiltration system, and the molecular weight cutoff of the ultrafiltration membrane decreases successively. First, an ultrafiltration membrane with a molecular weight cutoff of 10kDa is used for preliminary filtration to remove impurities with larger molecules, and then an ultrafiltration membrane with a molecular weight cutoff of 5kDa is used for further filtration to obtain a relatively high-purity protein solution. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3kDa is used for fine filtration to ensure the high purity of the protein solution. The pressure range during the ultrafiltration process is 0.2MPa-0.5MPa. Through multi-stage ultrafiltration, residual impurities and small molecules can be effectively removed to obtain a high-purity protein solution, providing high-quality protein raw materials for subsequent preparation of fertilizers.

[0029] In one embodiment of the present invention: during the expansion culture process, a sterile environment is maintained. Before operation, the incubator, culture medium and inoculation tools should be strictly sterilized. High-pressure steam sterilization can be used to ensure the sterilization effect. During the inoculation process, it should be carried out on a sterile operating table to avoid contamination by miscellaneous bacteria. During the expansion culture process, the growth status of the probiotics should be observed regularly. The turbidity and color changes of the culture medium can be observed with the naked eye to preliminarily judge the growth of the probiotics. The morphology and quantity changes of the probiotics can also be observed under a microscope to detect abnormal conditions in time.

[0030] Embodiment 1 Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus gelatinus, Bacillus acidophilus and Bifidobacterium are selected and mixed in a ratio of 20%, 20%, 15%, 15%, 15%, 15% to ensure that the total viable count of the six probiotic materials is 2.1 billion / g, and a specific genetically engineered strain is selected for leucine fermentation. The fermentation medium formula is 51 g / L glucose, 10.5 g / L ammonium sulfate, 5.2 g / L potassium dihydrogen phosphate, 1.1 g / L magnesium sulfate, 5.2 g / L yeast powder and 5.2 g / L leucine precursor; The composite probiotics were mixed in proportion and added to sterile physiological saline to control the viable bacterial count of the composite probiotics to 210 million / mL. The mixed bacterial solution was placed in a magnetic field with a magnetic field strength of 1020 Gauss for 2.2 hours. The leucine fermentation solution after fermentation was subjected to ultrasonic assisted extraction, using ultrasonic treatment at a frequency of 41kHz and a power of 510W for 30 minutes. The composite probiotic bacterial liquid after magnetic enhancement treatment was placed in a constant temperature shaking incubator, and was shaken and cultured for 19 hours at 35°C and 150r / min. After the composite probiotics were fully activated, expansion culture was carried out. The formula of the expansion culture medium was 10.5g / L peptone, 5.2g / L beef extract, 5.2g / L sodium chloride and 10.5g / L glucose. The activated bacterial liquid was inoculated into the expansion culture medium at a 5% inoculation rate, and cultured for 42 hours at a temperature of 35.2°C and an shaking rate of 152r / min. The number of viable bacteria after expansion culture was controlled at 5.1 billion / mL; The leucine fermentation broth after ultrasound-assisted extraction was subjected to multi-stage ultrafiltration treatment. First, an ultrafiltration membrane with a molecular weight cutoff of 10kDa was used for preliminary filtration to remove larger molecular impurities at a pressure of 0.25MPa. Then, an ultrafiltration membrane with a molecular weight cutoff of 5kDa was used for further filtration at a pressure of 0.3MPa. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3kDa was used for fine filtration at a pressure of 0.35MPa. After the filtration was completed, ion exchange chromatography was performed. The ultrafiltered leucine fermentation broth was passed through an Amberlite-filled filtration system at a flow rate of 6L / min. IR-120 ion exchange resin column, after the ion exchange resin is loaded in the ion exchange resin column, acetic acid-sodium acetate buffer (acetic acid and sodium acetate are mixed in a molar ratio of 1:2.2, and the volume is fixed to the required volume with distilled water, and the pH value is 5.2) is slowly injected into the ion exchange resin column, and the flow rate is controlled at 1.5L / min until the pH value and ion concentration of the effluent are basically consistent with the pH value and ion concentration of the buffer solution, and the solution after the chromatography is eluted with 0.7mol / L sodium chloride solution, and the elution flow rate is controlled at 3.5L / min, and the leucine eluate is collected and placed in a vacuum concentration device, and the vacuum system is turned on to gradually reduce the pressure in the device, and the leucine eluate is heated at the same time, and the vacuum degree is set to 0.085MPa, and the temperature is controlled at 42°C, and concentrated for 2.5h to obtain a high-concentration leucine solution; The composite probiotic bacterial liquid after expansion is mixed with the high-concentration leucine solution in a ratio of 1.2:1, and the stirring rate is controlled at 60r / min. After mixing, it is transferred to a fermentation tank for fermentation, and the temperature is controlled at 31°C for 38h. After fermentation, the fermentation liquid is transported to a spray drying device, and the inlet temperature is set to 155°C, the outlet temperature is set to 75°C, and the spray rate is 6L / min. The dried powder is mixed with humic acid, amino acids and trace elements, and the addition amount of humic acid is 12% of the total weight, the addition amount of amino acids is 6% of the total weight, and the addition amount of trace elements is 2%. After mixing, use a stirring device to stir at a stirring rate of 35r / min for 40min. After stirring, the uniformly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer. At the same time, nano-silicon dioxide and nano-calcium carbonate are introduced, and the two are mixed in a ratio of 1:1, and the addition amount is 5% of the total mass of the biofertilizer; The finished biofertilizer products are quality inspected, including appearance inspection, moisture determination and microbial activity detection. The particle size of granular biofertilizer is 2.5mm-4mm, the moisture content is 6%, the microbial culture temperature is 31°C, and the culture time is 30h. After passing the quality inspection, the storage temperature of bagged fertilizer is controlled between 8°C and 28°C.

[0031] Embodiment 2 Select composite probiotic materials, mix them in proportion to ensure that the total number of live bacteria is 2.3 billion / g, select specific genetically engineered strains for leucine fermentation, and the fermentation medium formula is 53g / L glucose, 11g / L ammonium sulfate, 5.5g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 5.5g / L yeast powder and 5.5g / L leucine precursor; The composite probiotics were mixed in proportion and then added to sterile saline to control the viable count of the composite probiotics to 220 million / mL. The mixed bacterial solution was placed in a magnetic field with a magnetic field strength of 1050 Gauss for 2.5 hours. The leucine fermentation solution after fermentation was subjected to ultrasonic assisted extraction, using an ultrasonic treatment of 43kHz and 530W for 30 minutes. The composite probiotic bacterial liquid after magnetic enhancement treatment was placed in a constant temperature shaking incubator and cultured at 35.5°C and 155r / min for 21h. After the composite probiotics were fully activated, expansion culture was carried out. The formula of the expansion culture medium was 11g / L peptone, 5.5g / L beef extract, 5.5g / L sodium chloride and 11g / L glucose. The activated bacterial liquid was inoculated into the expansion culture medium at a 5% inoculation rate and cultured at a temperature of 35.5°C and an oscillation rate of 155r / min for 44h. The number of viable bacteria after expansion culture was controlled at 5.3 billion / mL. The leucine fermentation broth after ultrasound-assisted extraction was subjected to multi-stage ultrafiltration treatment. First, an ultrafiltration membrane with a molecular weight cutoff of 10kDa was used for preliminary filtration at a pressure of 0.3MPa, and then an ultrafiltration membrane with a molecular weight cutoff of 5kDa was used for further filtration at a pressure of 0.35MPa. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3kDa was used for fine filtration at a pressure of 0.4MPa. After the filtration was completed, ion exchange chromatography was performed. The ultrafiltered leucine fermentation broth was passed through an Amberlite-filled filtration system at a flow rate of 7L / min. IRA-400 ion exchange resin column, after the ion exchange resin is loaded in the ion exchange resin column, Tris-HCl buffer is slowly injected into the ion exchange resin column, and the flow rate is controlled to be 2L / min, until the pH value and ion concentration of the effluent are basically consistent with the pH value and ion concentration of the buffer solution, and the solution after the chromatography is eluted with 0.8mol / L sodium chloride solution, and the elution flow rate is controlled at 4L / min, and the leucine eluate is collected and placed in a vacuum concentration device, and the vacuum system is turned on to gradually reduce the pressure in the device, and the leucine eluate is heated at the same time, and the vacuum degree is set to 0.09MPa, and the temperature is controlled at 45°C, and concentrated for 3h to obtain a high-concentration leucine solution; The composite probiotic bacterial liquid after expansion is mixed with the high-concentration leucine solution in a ratio of 1.5:1, and the stirring rate is controlled at 70r / min. After mixing, it is transferred to a fermentation tank for fermentation, and the temperature is controlled at 33°C. The fermentation time is 42h. After fermentation, the fermentation liquid is transported to a spray drying device, and the inlet temperature is set to 165°C, the outlet temperature is set to 80°C, and the spray rate is 8L / min. The dried powder is mixed with humic acid, amino acids and trace elements. The amount of humic acid added is 15% of the total weight, the amount of amino acids added is 7% of the total weight, and the amount of trace elements added is 3%. After mixing, use a stirring device to stir at a stirring rate of 40r / min for 45min. After stirring, the uniformly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer. At the same time, nano-silicon dioxide and nano-calcium carbonate are introduced, and the two are mixed in a ratio of 1:1, and the amount added is 5% of the total mass of the biofertilizer; The finished biofertilizer products are quality inspected, including appearance inspection, moisture determination and microbial activity detection. The particle size of granular biofertilizer is 3mm-4.5mm, the moisture content is 7%, the microbial culture temperature is 33℃, and the culture time is 36h. After passing the quality inspection, the storage temperature of bagged fertilizer is controlled between 10℃-25℃.

[0032] Embodiment 3 Select composite probiotic materials, mix them in proportion to ensure that the total number of live bacteria is 2.5 billion / g, select specific genetically engineered strains for leucine fermentation, and the fermentation medium formula is 55g / L glucose, 11.5g / L ammonium sulfate, 5.8g / L potassium dihydrogen phosphate, 1.3g / L magnesium sulfate, 5.8g / L yeast powder and 5.8g / L leucine precursor; The composite probiotics were mixed in proportion and then added to sterile saline to control the viable count of the composite probiotics to 230 million / mL. The mixed bacterial solution was placed in a magnetic field with a magnetic field strength of 1080 Gauss for 2.8 hours. The leucine fermentation solution after fermentation was subjected to ultrasonic assisted extraction, using ultrasonic treatment at a frequency of 44kHz and a power of 540W for 30 minutes. The composite probiotic bacterial liquid after magnetic enhancement treatment was placed in a constant temperature shaking incubator and cultured at 36°C and 158 r / min for 22 hours. After the composite probiotics were fully activated, expansion culture was carried out. The formula of the expansion culture medium was 11.5 g / L peptone, 5.8 g / L beef extract, 5.8 g / L sodium chloride and 11.5 g / L glucose. The activated bacterial liquid was inoculated into the expansion culture medium at a 5% inoculation rate and cultured at a temperature of 36°C and an oscillation rate of 158 r / min for 46 hours. The number of viable bacteria after expansion culture was controlled at 5.5 billion / mL. The leucine fermentation broth after ultrasound-assisted extraction was subjected to multi-stage ultrafiltration treatment, first using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa for preliminary filtration at a pressure of 0.35 MPa, then using an ultrafiltration membrane with a molecular weight cutoff of 5 kDa for further filtration at a pressure of 0.4 MPa, and finally using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa for fine filtration at a pressure of 0.45 MPa. After the filtration was completed, ion exchange chromatography was performed, and the ultrafiltered leucine fermentation broth was passed through an ion exchange resin column filled with Amberlite IR-120 at a flow rate of 7.5 L / min. After the ion exchange resin was filled in the ion exchange resin column, acetic acid-sodium acetate buffer (acetic acid and sodium acetate were mixed at a flow rate of 7.5 L / min) was added. Mix according to the molar ratio of 1:2.5, dilute to the required volume with distilled water, pH value is 5.5), slowly inject into the ion exchange resin column, control the flow rate at 2.2L / min, until the pH value and ion concentration of the effluent are basically consistent with the pH value and ion concentration of the buffer solution, elute the solution after chromatography with 0.9mol / L sodium chloride solution, and control the elution flow rate at 4.5L / min. Collect the leucine eluate and place it in a vacuum concentration device, turn on the vacuum system, gradually reduce the pressure in the device, and heat the leucine eluate at the same time, set the vacuum degree to 0.095MPa, control the temperature at 48°C, and concentrate for 3.5h to obtain a high-concentration leucine solution; The composite probiotic bacterial liquid after expansion is mixed with the high-concentration leucine solution in a ratio of 1.8:1, and the stirring rate is controlled at 75r / min. After mixing, it is transferred to a fermentation tank for fermentation, and the temperature is controlled at 34°C. The fermentation time is 45h. After fermentation, the fermentation liquid is transported to a spray drying device, and the inlet temperature is set to 170°C, the outlet temperature is set to 85°C, and the spray rate is 9L / min. The dried powder is mixed with humic acid, amino acids and trace elements. The amount of humic acid added is 18% of the total weight, the amount of amino acids added is 8% of the total weight, and the amount of trace elements added is 4%. After mixing, use a stirring device to stir at a stirring rate of 45r / min for 50min. After stirring, the uniformly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer. At the same time, nano-silicon dioxide and nano-calcium carbonate are introduced, and the two are mixed in a ratio of 1:1, and the amount added is 5% of the total mass of the biofertilizer; The finished biofertilizer products are quality inspected, including appearance inspection, moisture determination and microbial activity detection. The particle size of granular biofertilizer is 3.5mm-5mm, the moisture content is 8%, the microbial culture temperature is 34℃, and the culture time is 40h. After passing the quality inspection, the storage temperature of bagged fertilizer is controlled between 12℃-28℃.

[0033] Embodiment 4: Select composite probiotic materials, mix them in proportion to ensure that the total number of live bacteria is 2.4 billion / g, select specific genetically engineered strains for leucine fermentation, and the fermentation medium formula is 54g / L glucose, 11.2g / L ammonium sulfate, 5.6g / L potassium dihydrogen phosphate, 1.2g / L magnesium sulfate, 5.6g / L yeast powder and 5.6g / L leucine precursor; The composite probiotics were mixed in proportion and then added to sterile saline to control the viable count of the composite probiotics to 225 million / mL. The mixed bacterial solution was placed in a magnetic field with a magnetic field strength of 1060 Gauss for 2.6 hours. The leucine fermentation solution after fermentation was subjected to ultrasonic assisted extraction, using an ultrasonic treatment of 42kHz and 520W for 30 minutes. The composite probiotic bacterial liquid after magnetic enhancement treatment was placed in a constant temperature shaking incubator and cultured at 35.8°C and 156r / min for 20h. After the composite probiotics were fully activated, expansion culture was carried out. The formula of the expansion culture medium was 11g / L peptone, 5.4g / L beef extract, 5.4g / L sodium chloride and 11g / L glucose. The activated bacterial liquid was inoculated into the expansion culture medium at a 5% inoculation rate and cultured at a temperature of 35.8°C and an oscillation rate of 156r / min for 44h. The number of viable bacteria after expansion culture was controlled at 5.2 billion / mL. The leucine fermentation broth after ultrasound-assisted extraction was subjected to multi-stage ultrafiltration treatment. First, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa was used for preliminary filtration at a pressure of 0.32 MPa, and then an ultrafiltration membrane with a molecular weight cutoff of 5 kDa was used for further filtration at a pressure of 0.38 MPa. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3 kDa was used for fine filtration at a pressure of 0.42 MPa. After the filtration was completed, ion exchange chromatography was performed. The ultrafiltered leucine fermentation broth was passed through an ion exchange resin column filled with Amberlite IRA-400 at a flow rate of 7 L / min. After the ion exchange resin was filled in the ion exchange resin column, , slowly inject Tris-HCl buffer into the ion exchange resin column, control the flow rate to 1.8L / min, until the pH value and ion concentration of the effluent are basically consistent with the pH value and ion concentration of the buffer, elute the solution after chromatography with 0.85mol / L sodium chloride solution, control the elution flow rate to 4.2L / min, collect the leucine eluate and place it in a vacuum concentration device, turn on the vacuum system, gradually reduce the pressure in the device, heat the leucine eluate at the same time, set the vacuum degree to 0.092MPa, control the temperature at 46°C, and concentrate for 3.2h to obtain a high-concentration leucine solution; The composite probiotic bacterial liquid after expansion is mixed with the high-concentration leucine solution in a ratio of 1.6:1, and the stirring rate is controlled at 65r / min. After mixing, it is transferred to a fermentation tank for fermentation, and the temperature is controlled at 32°C. The fermentation time is 40h. After fermentation, the fermentation liquid is transported to a spray drying device, and the inlet temperature is set to 160°C, the outlet temperature is set to 78°C, and the spray rate is 7L / min. The dried powder is mixed with humic acid, amino acids and trace elements. The amount of humic acid added is 14% of the total weight, the amount of amino acids added is 7% of the total weight, and the amount of trace elements added is 3%. After mixing, use a stirring device to stir at a stirring rate of 40r / min for 45min. After stirring, the uniformly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer. At the same time, nano-silicon dioxide and nano-calcium carbonate are introduced, and the two are mixed in a ratio of 1:1, and the addition amount is 5% of the total mass of the biofertilizer; The finished biofertilizer products are quality inspected, including appearance inspection, moisture determination and microbial activity detection. The particle size of granular biofertilizer is 3mm-4.5mm, the moisture content is 7%, the microbial culture temperature is 32℃, and the culture time is 36h. After passing the quality inspection, the storage temperature of bagged fertilizer is controlled between 10℃-26℃.

[0034] Embodiment 5 Select composite probiotic materials, mix them in proportion to ensure that the total number of live bacteria is 2.6 billion / g, select specific genetically engineered strains for leucine fermentation, and the fermentation medium formula is 56g / L glucose, 12g / L ammonium sulfate, 5.9g / L potassium dihydrogen phosphate, 1.4g / L magnesium sulfate, 5.9g / L yeast powder and 5.9g / L leucine precursor; The composite probiotics were mixed in proportion and then added to sterile saline to control the viable count of the composite probiotics to 240 million / mL. The mixed bacterial solution was placed in a magnetic field with a magnetic field strength of 1100 Gauss for 3 hours. The leucine fermentation solution after fermentation was subjected to ultrasonic assisted extraction, using an ultrasonic treatment of 45kHz and 550W for 30 minutes. The composite probiotic bacterial liquid after magnetic enhancement treatment was placed in a constant temperature shaking incubator, and cultured at 36.5°C and 160r / min for 23h. After the composite probiotics were fully activated, expansion culture was carried out. The formula of the expansion culture medium was 12g / L peptone, 6g / L beef extract, 6g / L sodium chloride and 12g / L glucose. The activated bacterial liquid was inoculated into the expansion culture medium at a 5% inoculation rate, and cultured at a temperature of 36.5°C and an oscillation rate of 160r / min for 48h. The number of viable bacteria after expansion culture was controlled at 5.8 billion / mL. The leucine fermentation broth after ultrasound-assisted extraction was subjected to multi-stage ultrafiltration treatment. First, an ultrafiltration membrane with a molecular weight cutoff of 10kDa was used for preliminary filtration at a pressure of 0.4MPa, and then an ultrafiltration membrane with a molecular weight cutoff of 5kDa was used for further filtration at a pressure of 0.45MPa. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3kDa was used for fine filtration at a pressure of 0.5MPa. After the filtration was completed, ion exchange chromatography was performed. The ultrafiltered leucine fermentation broth was passed through an Amberlite-filled filtration system at a flow rate of 8L / min. IR-120 ion exchange resin column, after the ion exchange resin is loaded in the ion exchange resin column, acetic acid-sodium acetate buffer (acetic acid and sodium acetate are mixed in a molar ratio of 1:2.8, and the volume is fixed to the required volume with distilled water, and the pH value is 5.8) is slowly injected into the ion exchange resin column, and the flow rate is controlled at 2.5L / min until the pH value and ion concentration of the effluent are basically consistent with the pH value and ion concentration of the buffer solution, and the solution after the chromatography is eluted with a 1mol / L sodium chloride solution, and the elution flow rate is controlled at 5L / min, and the leucine eluate is collected and placed in a vacuum concentration device, and the vacuum system is turned on to gradually reduce the pressure in the device, and the leucine eluate is heated at the same time, and the vacuum degree is set to 0.1MPa, and the temperature is controlled at 50°C, and concentrated for 4h to obtain a high-concentration leucine solution; The composite probiotic bacterial liquid after expansion is mixed with the high-concentration leucine solution in a ratio of 2:1, and the stirring rate is controlled at 80r / min. After mixing, it is transferred to a fermentation tank for fermentation, and the temperature is controlled at 35°C. The fermentation time is 48h. After fermentation, the fermentation liquid is transported to a spray drying device, and the inlet temperature is set to 180°C, the outlet temperature is set to 90°C, and the spray rate is 10L / min. The dried powder is mixed with humic acid, amino acids and trace elements. The amount of humic acid added is 20% of the total weight, the amount of amino acids added is 9% of the total weight, and the amount of trace elements added is 4%. After mixing, use a stirring device to stir at a stirring rate of 50r / min for 60min. After stirring, the uniformly mixed material is transported to an extrusion granulation device to prepare a finished biofertilizer. At the same time, nano-silicon dioxide and nano-calcium carbonate are introduced, and the two are mixed in a ratio of 1:1, and the amount added is 5% of the total mass of the biofertilizer; The finished biofertilizer products are quality inspected, including appearance inspection, moisture determination and microbial activity detection. The particle size of granular biofertilizer is 4mm-5mm, the moisture content is 9%, the microbial culture temperature is 35℃, and the culture time is 48h. After passing the quality inspection, the storage temperature of bagged fertilizer is controlled between 15℃-30℃.

[0035] Appearance inspection, Steps: Randomly select a certain number of biofertilizer particles and observe their color, shape and uniformity with the naked eye under good light.

[0036] Moisture determination, Steps: Using the drying method, a certain amount of biofertilizer sample is weighed, placed in an oven, and dried to constant weight at a specific temperature. The moisture content is determined by calculating the mass difference before and after drying.

[0037] Microbial activity detection, Steps: Use the plate count method to dilute the biofertilizer sample, spread it on a specific culture medium, culture it at 30℃-35℃ for 24h-48h, and then count the number of colonies to determine the number of live probiotics.

[0038] Based on the above five groups of embodiments, the quality of the finished biofertilizer is summarized, and the specific data are as follows: Embodiment 1

[0039] Appearance inspection: The particles are brown, regular in shape, well-proportioned, and have no obvious agglomeration. Moisture content: Determined by drying method, the moisture content is 5.8%, which meets the standard; Microbial activity detection: After culturing on a specific culture medium, the number of colonies was counted, and the number of live bacteria was 5.3 billion / mL, which met the requirement for the number of live bacteria after expansion culture; The particle size of the granular biofertilizer is 2.5mm-4mm, the moisture content is 6%, the microbial culture temperature is 31°C, and the culture time is 30h.

[0040] Embodiment 2 Appearance inspection: The particles are dark brown in color, regular in shape, and have good uniformity; Moisture determination: The moisture content is 6.5%, which is within the standard range; Microbial activity test: The number of live bacteria is 5.5 billion / mL, and the microbial activity is good; The particle size of the granular biofertilizer is 3.0mm-4.5mm, the moisture content is 7%, the microbial culture temperature is 33°C, and the culture time is 36h.

[0041] Embodiment 3 Appearance inspection: The particles are black, regular in shape, uniform and free of lumps; Moisture determination: The moisture content was measured to be 7.2%; Microbial activity detection: the number of live bacteria reached 5.7 billion / mL; The particle size of the granular biofertilizer is 3.5mm-5.0mm, the moisture content is 8%, the microbial culture temperature is 34°C, and the culture time is 40h.

[0042] Embodiment 4: Appearance inspection: The particles are brown, regular in shape, and highly uniform; Moisture determination: moisture content is 6.8%; Microbial activity test: The number of viable bacteria is 5.4 billion / mL; The particle size of the granular biofertilizer is 3.0mm-4.5mm, the moisture content is 7%, the microbial culture temperature is 32°C, and the culture time is 36h.

[0043] Embodiment 5 Appearance inspection: black particles, regular shape, high uniformity; Moisture determination: The moisture content is 8.5%, which meets the standard; Microbial activity test: The number of viable bacteria was 5.9 billion / mL; The particle size of the granular biofertilizer is 4.0mm-5.0mm, the moisture content is 9%, the microbial culture temperature is 35°C, and the culture time is 48h.

[0044] In summary, the feasibility and effectiveness of extracting protein from valine fermentation broth to prepare fertilizer in the five embodiments of the present invention have important practical significance and application value.

[0045] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the scope of protection of the present invention.

[0046] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0047] The above contents are merely examples and explanations of the present invention. Various modifications or additions to the specific embodiments described or replacements in similar ways by technicians in the technical field shall fall within the protection scope of the present invention as long as they do not deviate from the invention or exceed the scope defined by the claims.

[0048] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a biofertilizer by combining a composite probiotic and a leucine fermentation broth, comprising magnetic enhancement treatment of the probiotic, ultrasound-assisted extraction of the leucine fermentation broth, and activation and expansion of the composite probiotic. After the composite probiotic and the leucine fermentation broth are treated, they are mixed and fermented, and the method is characterized in that: The specific operation steps of combining the composite probiotics with the leucine fermentation liquid to prepare the biofertilizer are as follows: Step 1: selecting a composite probiotic material and a specific genetically engineered strain for leucine fermentation, and preparing a leucine fermentation liquid after leucine fermentation by the specific genetically engineered strain; Step 2: Stirring and mixing the composite probiotics with sterile physiological saline, and placing the mixture in a magnetic field for treatment, and performing ultrasound-assisted extraction on the prepared leucine fermentation liquid; Step 3, placing the composite probiotic bacterial liquid after magnetic enhancement treatment in a constant temperature shaking incubator, culturing and activating it, and then adding the culture medium for expansion, to obtain the composite probiotic bacterial liquid after expansion; Step 4: performing multiple ultrafiltration treatments on the leucine fermentation broth after ultrasound-assisted extraction, and after the ultrafiltration is completed, performing ion exchange chromatography on the leucine fermentation broth, eluting the solution after the chromatography, collecting the leucine eluate, and concentrating the leucine eluate under vacuum to obtain a high-concentration leucine solution; Step 5, stirring and mixing the composite probiotic bacterial liquid after expansion and the high concentration leucine solution for fermentation, spray drying the solution after fermentation, adding humic acid, amino acids and trace elements, stirring and mixing, and extruding and granulating the solution to obtain a finished biofertilizer; Step 6: Conduct quality inspection on the finished bio-fertilizer products, bag the qualified ones, and store them in a dry and ventilated place.

2. The method for preparing biofertilizer by combining composite probiotics and leucine fermentation liquid according to claim 1, characterized in that: In the step 1, the composite probiotics include Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus gelatinosa, Bacillus acidophilus and Bifidobacterium, and the proportions of the above six probiotic materials are 20%, 20%, 15%, 15%, 15% and 15% respectively in order, and the total number of live bacteria of the six probiotic materials is greater than 2 billion / g.

3. The method for preparing biofertilizer by combining composite probiotics and leucine fermentation liquid according to claim 1, characterized in that: In the step 1, a specific genetically engineered strain is selected for leucine fermentation, and the formula of the fermentation medium is 50 g / L glucose, 10 g / L ammonium sulfate, 5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 5 g / L yeast powder and 5 g / L leucine precursor.

4. The method for preparing biofertilizer by combining composite probiotics and leucine fermentation liquid according to claim 1, characterized in that: In the step 2, the composite probiotics are mixed in proportion and then added with sterile physiological saline, the number of viable bacteria of the composite probiotics is controlled to reach 200 million / mL, the mixed bacterial solution is placed in a magnetic field with a magnetic field strength of 1000 Gauss for 2h-3h, and the leucine fermentation solution after fermentation is subjected to ultrasonic-assisted extraction, and ultrasonic treatment with a frequency of 40kHz and a power of 500W is used for 30min to obtain a composite probiotic bacterial solution after magnetic enhancement.

5. The method for preparing biofertilizer by combining composite probiotics with leucine fermentation liquid according to claim 1, characterized in that: In the step three, the composite probiotic bacterial liquid after magnetic enhancement treatment is placed in a constant temperature shaking incubator, and cultured at 35°C and 150 / min for 18h-24h. After the composite probiotics are fully activated, they are expanded. The formula of the expansion culture medium is 10g / L peptone, 5g / L beef extract, 5g / L sodium chloride and 10g / L glucose. The activated bacterial liquid is inoculated into the expansion culture medium at an inoculation rate of 5%, and cultured at a temperature of 35°C and an oscillation rate of 150r / min for 40h-48h, and the number of live bacteria after expansion is controlled to be above 5 billion / mL.

6. The method for preparing biofertilizer by combining composite probiotics with leucine fermentation liquid according to claim 1, characterized in that: In the step 4, the leucine fermentation broth after ultrasound-assisted extraction is introduced into the ultrafiltration equipment through the feed pipe, the pressure pump is turned on, the leucine fermentation broth is passed through the ultrafiltration membrane under the action of pressure, and the ultrafiltration membrane with a molecular weight cutoff of 3kDa-5kDa is selected to filter it, and the flow rate is set to 10L / min-12L / min. After the filtration is completed, ion exchange chromatography is performed on it, and the ultrafiltered leucine fermentation broth is passed through the ion exchange resin column at a flow rate of 5L / min-8L / min. After the ion exchange resin is filled in the ion exchange resin pile, the buffer solution is slowly injected into the ion exchange resin pile, and the flow rate is controlled to be 1L / min-3L / min. The price buffer solution continues to flow through the ion exchange resin column until the pH value and ion concentration of the effluent are substantially consistent with the pH value and ion concentration of the buffer solution, thereby obtaining a chromatographic solution.

7. The method for preparing biofertilizer by combining composite probiotics with leucine fermentation liquid according to claim 1, characterized in that: In the step 4, the solution after the chromatography is completed is eluted, and according to the type of ion exchange resin and the adsorption characteristics of leucine, for a strongly acidic cation exchange resin, a 0.5 mol / L-1.0 mol / L sodium chloride solution is used to elute it, and the elution flow rate is controlled at 3 L / min-5 L / min. The leucine eluate is collected and placed in a vacuum concentration device, the vacuum system is turned on, and the pressure in the device is gradually reduced. At the same time, the leucine eluate is heated, the vacuum degree is set to 0.08 MPa-0.10 MPa, and the temperature is controlled at 40° C.-50° C. According to the volume of the leucine eluate and the processing capacity of the equipment, it is concentrated for 2h-4h until a high-concentration leucine solution is obtained.

8. The method for preparing biofertilizer by combining composite probiotics with leucine fermentation liquid according to claim 1, characterized in that: In the step 5, the composite probiotic bacterial liquid after expansion is mixed with the high concentration leucine solution in a ratio of 1-2:1, the stirring rate is controlled at 50r / min-80r / min, and after mixing, it is transferred to a fermentation tank for fermentation, the temperature is controlled between 30°C-35°C, the fermentation time is 36h-48h, and after fermentation, the fermentation liquid is transported to a spray drying equipment, the inlet temperature is set to 150°C-180°C, the outlet temperature is set to 70°C-90°C, and the spray speed is 5L / min-10L / min, the fermentation liquid is quickly dried into powder by spray drying, the dried powder is mixed with humic acid, amino acids and trace elements, the added amount of humic acid is 10%-20% of the total weight, the added amount of amino acids is 5%-10% of the total weight, and the added amount of trace elements is 1%-5%. After mixing, a stirring device is used to stir at a stirring rate of 30r / min-50r / min for 30-60min. After stirring, the evenly mixed material is conveyed to an extrusion granulation device to prepare a finished biofertilizer.

9. The method for preparing biofertilizer by combining composite probiotics with leucine fermentation liquid according to claim 1, characterized in that: In step six, the quality inspection includes appearance inspection, moisture determination and microbial activity detection. The particle size of the granular bio-fertilizer is set to 2mm-5mm, the moisture content is 5%-10%, the microbial culture temperature is 30℃-35℃, and the culture time is 24h-48h. After the quality inspection is qualified, the storage temperature of the bagged fertilizer is controlled between 5℃-30℃.

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