Method for preparing fertilizer by extracting protein from valine fermentation liquor

Abstract

The invention discloses a method for preparing a fertilizer by extracting protein from valine fermentation broth, and relates to the technical field of fertilizer preparation, the method comprises the steps of valine fermentation broth pretreatment, protein extraction and protein purification, the valine fermentation broth pretreatment adopts a magnetic field pretreatment device, the method has the advantages that the subsequent extraction process is more efficient through magnetic field pretreatment and aggregation of protein molecules, impurities can be further removed in the subsequent steps due to the change of physical properties of the impurities, and in the magnetic field pretreatment process, the impurities can be effectively removed in the subsequent steps. The fermentation liquor continuously and circularly flows in the magnetic field, so that the magnetic field can play a sufficient role on each part of the fermentation liquor, the aggregation state of protein of the valine fermentation liquor pretreated by the magnetic field is more stable, and the distribution of impurities is more beneficial to subsequent separation operation.

Description

Technical Field

[0001] The invention relates to the technical field of fertilizer preparation, in particular to a method for preparing fertilizer by extracting protein from valine 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's requirements for fertilizers are getting higher and higher. They not only require them to have rich nutrients, but also expect their sources to be greener and more sustainable. As an important amino acid, valine is widely used in the fields of medicine, food and feed. At present, the production of valine usually adopts the fermentation method. During the fermentation process, a large amount of valine fermentation liquid will be produced. In addition to valine, these fermentation liquids also contain a certain amount of nutrients such as protein. However, the existing treatment methods often only focus on the extraction of valine, and fail to make full use of resources such as protein in the fermentation liquid. The remaining fermentation liquid containing protein and other substances is usually discharged as waste liquid, which not only causes a great waste of resources, but also causes certain pollution to the environment. With the development of agriculture, the demand for high-quality fertilizers is growing. Protein, as an important nutrient, can be used to prepare high-efficiency fertilizers. If protein can be extracted from valine fermentation broth and prepared into fertilizer, it can not only achieve comprehensive utilization of resources and reduce environmental pollution, but also provide high-quality fertilizer products for agricultural production. At present, although there are some methods for extracting protein from fermentation broth, there are problems such as low extraction efficiency, low product purity, and complex operation in the protein extraction of valine fermentation broth. At the same time, the existing fertilizer preparation methods also have certain limitations in the combination of nutrients and the improvement of fertilizer efficiency. For this reason, we propose a method for extracting protein from valine fermentation broth to prepare fertilizer. Summary of the invention

[0003] The object of the present invention is to provide a method for preparing fertilizer by extracting protein from valine fermentation broth.

[0004] In order to solve the problems raised in the above background technology, the present invention provides the following technical solutions: a method for extracting protein from valine fermentation broth to prepare fertilizer, comprising pretreatment of valine fermentation broth, protein extraction and protein purification, wherein the valine fermentation broth pretreatment uses a magnetic field pretreatment device to treat the fermentation broth using a magnetic field of specific intensity and frequency, and the specific operation steps of extracting protein from valine fermentation broth to prepare fertilizer are as follows: Step 1: preliminarily filtering the valine fermentation broth, and transferring it to a magnetic field pretreatment device after filtering to aggregate the protein in the valine fermentation broth and change the physical properties of the impurities therein; Step 2: introducing a biosorbent, adding it to the pretreated valine fermentation broth, stirring and mixing at room temperature, and using the biosorbent to adsorb protein. After the adsorption reaction is completed, transferring it to a corrosion-resistant container, and using a special separation device to separate the protein from the fermentation broth; Step 3: protein purification, placing the separated biosorbent adsorbed with the protein in a specific desorption solution, desorbing the protein from the biosorbent, and ultrafiltration to obtain a high-purity protein solution; Step 4: Transfer the purified protein solution into a stirring container, add an organic carbon source, a phosphorus source, a potassium source and trace elements, wherein the trace elements include iron, zinc, copper and manganese, which are added in the form of sulfates and chelates, stir and mix, add biologically active substances, and obtain a mixed fertilizer material, which is then made into a finished protein fertilizer through solidification technology; Step 5: Conduct a comprehensive quality inspection on the prepared protein fertilizer. Once qualified, it will be packaged in an environmentally friendly manner and stored in a dry and dark place.

[0005] As a further scheme of the present invention: in the step 1, the valine fermentation broth is preliminarily filtered using a multi-stage filtering device, wherein the first-stage filtration uses a filter with a pore size of 100 um to remove larger particles of impurities, the second-stage filtration uses a filter with a pore size of 50 um to remove smaller particles of impurities, and the third-stage filtration uses a filter with a pore size of 20 um to remove tiny impurities, thereby obtaining a crude valine fermentation broth.

[0006] As a further solution of the present invention: in the step 1, the crude valine fermentation broth is transferred to a magnetic field pretreatment device, which is composed of a group of high-strength permanent magnets, and its magnetic field strength is set to 1.5 Tesla, and the magnetic field frequency is adjusted to 50 Hz, so that the protein in the crude valine fermentation broth is aggregated, and the magnetic field pretreatment time is set to 45 min-60 min to obtain a semi-finished valine fermentation broth.

[0007] As a further solution of the present invention: in the step 2, a biosorbent is prepared, the biosorbent is composited by genetically modified microbial cells and nanomaterials, a gene fragment containing a specific protein domain coding sequence is introduced into the genome of Bacillus by molecular biological means, so that it is stably expressed on the surface of the cell, the structural gene-engineered Bacillus cells are composited with the nanomaterials, the nanomaterials are selected from carbon nanotubes, the Bacillus cells and the carbon nanotubes are stirred and mixed at a mass ratio of 1.1-1.4:0.5-0.7 at 25° C.-27° C. to prepare the biosorbent, which is added to the semi-finished valine fermentation broth at 25° C. and added in an amount of 10 g / L for mixing, and stirred at a stirring rate of 150 r / min-200 r / min for 2 hours.

[0008] As a further solution of the present invention: in the step 2, an electric field separation device is used to separate the protein from the valine fermentation broth. An electric field with a strength of 50 V / cm is applied to the valine fermentation broth. The biosorbent moves toward a specific electrode under the action of the electric field, thereby separating the biosorbent adsorbed with protein.

[0009] As a further solution of the present invention: in the step 3, the desorption solution is composed of a buffer salt, a surfactant and a reducing agent, wherein the buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration range of 0.1 mol / L-0.2 mol / L, the surfactant is a non-ionic surfactant Tween-20 with a concentration of 0.5%-1%, and the reducing agent is dithiothreitol with a concentration of 5 mmol / L-10 mmol / L. The biosorbent adsorbed with protein is placed in the desorption solution, the amount of the desorption solution is 10 mL / g corresponding to the biosorbent, the desorption temperature is controlled between 30° C. and 35° C., and the desorption process lasts for 2 h to 3 h.

[0010] As a further solution of the present invention: in the step three, after the desorption is completed, the protein-containing solution is subjected to ultrafiltration treatment, and the ultrafiltration adopts multi-stage ultrafiltration, and is filtered in sequence with ultrafiltration membranes with molecular weight cutoffs of 10kDa, 5kDa and 3kDa respectively, and the filtration environment pressure range is 0.2MPa-0.5MPa.

[0011] As a further solution of the present invention: in the step 4, the added amounts of the organic carbon source, phosphorus source, potassium source and trace elements account for 10%-15%, 5%-8%, 8%-12% and 1%-2% of the protein solution respectively; the purified protein solution is added into a stirring container, stirred at a stirring rate of 100r / min-120r / min for 30min, and after adding the bioactive substance, solidification treatment is carried out to prepare a finished protein fertilizer.

[0012] As a further scheme of the present invention: in the step 5, the quality of the finished protein fertilizer is tested, including protein content, nutrient element ratio, activity of biologically active substances and other relevant indicators, wherein other relevant indicators include moisture content, pH value and particle size, the moisture content is set to 10%-15% of the total mass of the finished protein fertilizer, the pH value is set between 6.5-8.5, and the particle size is controlled between 1mm-5mm. After the finished protein fertilizer is tested and qualified, it is packaged and stored between 10℃-30℃, and the relative humidity is controlled at 70%.

[0013] By adopting the above technical solution, compared with the prior art, the beneficial effects of the present invention are: 1. The present invention uses magnetic field pretreatment to aggregate protein molecules, making the subsequent extraction process more efficient, and the change in the physical properties of impurities is conducive to further removing impurities in subsequent steps. During the magnetic field pretreatment process, the fermentation liquid continuously circulates in the magnetic field to ensure that the magnetic field can fully play a role in each part of the fermentation liquid. The aggregation state of the protein in the valine fermentation liquid after magnetic field pretreatment is more stable, and the distribution of impurities is more conducive to subsequent separation operations. By performing solidification technology operations on the purified protein solution, the fertilizer raw material prepared can be solidified into granules or blocks to obtain a finished protein fertilizer. This finished product has good stability, is easy to store, transport and use, and ensures the quality of the finished protein fertilizer. 2. The present invention prepares the biosorbent and adopts a method of combining genetically engineered Bacillus cells with nanomaterials. The biosorbent not only has a high selective adsorption capacity, but also the addition of nanomaterials enhances the stability and adsorption performance of the adsorbent, making the protein extraction process more efficient and accurate, reducing the waste of resources, and improving the protein recovery rate. By adding a desorption solution and multi-stage filtration operation of the ultrafiltration process, the mildness and efficiency of protein desorption from the biosorbent are ensured, and residual impurities and small molecules are removed to obtain a high-purity protein solution. The high-purity protein makes the protein content in the prepared fertilizer higher and more nutritious, which can better meet the needs of crops for nutrients such as protein and promote the growth and development of crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure is a schematic diagram of a specific process of extracting protein to prepare 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 present invention discloses a method for extracting protein from valine fermentation liquid to prepare fertilizer, comprising pretreatment of valine fermentation liquid, protein extraction and protein purification. The valine fermentation liquid pretreatment uses a magnetic field pretreatment device to treat the fermentation liquid using a magnetic field of specific intensity and frequency. The specific operation steps of extracting protein from valine fermentation liquid to prepare fertilizer are as follows: Step 1: preliminarily filtering the valine fermentation broth, and transferring it to a magnetic field pretreatment device after filtering to aggregate the protein in the valine fermentation broth and change the physical properties of the impurities therein; Step 2: introducing a biosorbent, adding it to the pretreated valine fermentation broth, stirring and mixing at room temperature, and using the biosorbent to adsorb protein. After the adsorption reaction is completed, transferring it to a corrosion-resistant container, and using a special separation device to separate the protein from the fermentation broth; Step 3: protein purification, placing the separated biosorbent adsorbed with the protein in a specific desorption solution, desorbing the protein from the biosorbent, and ultrafiltration to obtain a high-purity protein solution; Step 4: Transfer the purified protein solution into a stirring container, add an organic carbon source, a phosphorus source, a potassium source and trace elements, wherein the trace elements include iron, zinc, copper and manganese, which are added in the form of sulfates and chelates, stir and mix, add biologically active substances, and obtain a mixed fertilizer material, which is then made into a finished protein fertilizer through solidification technology; Step 5: Conduct a comprehensive quality inspection on the prepared protein fertilizer. Once qualified, it will be packaged in an environmentally friendly manner and stored in a dry and dark place.

[0017] In one embodiment of the present invention: in step 1, the valine fermentation broth is preliminarily filtered using a multi-stage filtration device, wherein the first-stage filtration uses a filter with a pore size of 100 um to remove larger particles of impurities, the second-stage filtration uses a filter with a pore size of 50 um to remove smaller particles of impurities, and the third-stage filtration uses a filter with a pore size of 20 um to remove tiny impurities, thereby obtaining a crude valine fermentation broth.

[0018] In one embodiment of the present invention: in step 1, the crude valine fermentation broth is transferred to a magnetic field pretreatment device, which is composed of a group of high-strength permanent magnets, and its magnetic field strength is set to 1.5 Tesla, and the magnetic field frequency is adjusted to 50 Hz. The protein in the crude valine fermentation broth is aggregated, and the magnetic field pretreatment time is set to 45min-60min to obtain a semi-finished valine fermentation broth.

[0019] In one embodiment of the present invention, in step 2, a biosorbent is prepared, the biosorbent is made of a composite of genetically modified microbial cells and nanomaterials, a gene fragment containing a specific protein domain coding sequence is introduced into the genome of Bacillus by molecular biological means, so that it is stably expressed on the surface of the cell, the structural gene-engineered Bacillus cells are composited with the nanomaterials, the nanomaterials are selected from carbon nanotubes, the Bacillus cells and the carbon nanotubes are stirred and mixed at a mass ratio of 1.1-1.4:0.5-0.7 at 25° C.-27° C. to prepare the biosorbent, which is added to the semi-finished valine fermentation broth at 25° C. and added in an amount of 10 g / L for mixing, and stirred at a stirring rate of 150 r / min-200 r / min for 2 hours.

[0020] In one embodiment of the present invention, in step 2, an electric field separation device is used to separate the protein from the valine fermentation broth. An electric field with a strength of 50 V / cm is applied to the valine fermentation broth. The biosorbent moves toward a specific electrode under the action of the electric field, thereby separating the biosorbent adsorbed with protein.

[0021] In one embodiment of the present invention: in step 3, the desorption solution is composed of a buffer salt, a surfactant and a reducing agent, wherein the buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration range of 0.1 mol / L-0.2 mol / L, the surfactant is a non-ionic surfactant Tween-20 with a concentration of 0.5%-1%, and the reducing agent is dithiothreitol with a concentration of 5 mmol / L-10 mmol / L. The biosorbent adsorbed with protein is placed in the desorption solution, the amount of the desorption solution is 10 mL / g for the biosorbent, the desorption temperature is controlled between 30° C. and 35° C., and the desorption process lasts for 2 h to 3 h.

[0022] In one embodiment of the present invention: in step three, after desorption is completed, the protein-containing solution is subjected to ultrafiltration treatment, and the ultrafiltration adopts multi-stage ultrafiltration, and is filtered in sequence with ultrafiltration membranes with molecular weight cutoffs of 10kDa, 5kDa and 3kDa respectively, and the filtration environment pressure range is 0.2MPa-0.5MPa.

[0023] In one embodiment of the present invention: in step 4, the added amounts of organic carbon source, phosphorus source, potassium source and trace elements account for 10%-15%, 5%-8%, 8%-12% and 1%-2% of the protein solution respectively, the purified protein solution is added into a stirring container, stirred at a stirring rate of 100r / min-120r / min for 30min, and after adding the bioactive substance, solidification treatment is carried out to prepare a finished protein fertilizer.

[0024] In one embodiment of the present invention: in step five, the finished protein fertilizer is subjected to quality inspection, including protein content, nutrient element ratio, activity of biologically active substances and other relevant indicators, wherein other relevant indicators include moisture content, pH value and particle size, the moisture content is set to 10%-15% of the total mass of the finished protein fertilizer, the pH value is set between 6.5-8.5, the particle size is controlled between 1mm-5mm, and after the finished protein fertilizer is tested and qualified, it is packaged and stored between 10℃-30℃, and the relative humidity is controlled at 70%.

[0025] In one embodiment of the present invention: in the set magnetic field environment, the protein molecules in the crude valine fermentation broth begin to aggregate to a certain extent due to the effect of the magnetic field force. At the same time, some impurities in the crude valine fermentation broth, including small molecular organic matter, inorganic salts, etc., change their physical properties. Among them, paramagnetic impurities tend to aggregate in a specific area under the action of the magnetic field, and diamagnetic impurities are pushed to the area with weaker magnetic field. During the magnetic field pretreatment process, the crude valine fermentation broth.

[0026] In one embodiment of the present invention, when separating the protein from the valine fermentation broth, a magnetic field separation device can be used. First, a magnetic field with an intensity of 0.8 Tesla is applied to the valine fermentation broth. Since the biosorbent contains nanomaterials and has a certain degree of magnetism, under the action of the magnetic field, the biosorbent adsorbed with the protein will be quickly attracted and aggregated, thereby separating the protein from the fermentation broth.

[0027] 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.

[0028] In one embodiment of the present invention, the added bioactive substances include enzymes and beneficial microorganisms that promote plant growth, the enzymes include nitrogenase and phosphatase, and the beneficial microorganisms include Bacillus subtilis and Bacillus licheniformis, wherein the added amount of the enzyme substances is 100-200 units per kilogram of protein solution, and the added amount of the beneficial microorganisms is 1×10 6 -1×10 7colony forming units.

[0029] In one embodiment of the present invention: the curing technology is a cross-linking reaction of a polymer material, polyvinyl alcohol is selected as the raw material, and the fertilizer raw material obtained by mixing the purified protein solution with an organic carbon source, a phosphorus source, a potassium source and trace elements is transferred into a specific reaction container, and then the prepared polyvinyl alcohol is added, the reaction conditions are controlled, the cross-linking reaction is initiated, and appropriate stirring is performed during the reaction to ensure that the reaction proceeds evenly.

[0030] Embodiment 1 The valine fermentation liquid was subjected to multi-stage filtration. The first-stage filtration used a filter with a pore size of 100um to remove larger impurities. The second-stage filtration used a filter with a pore size of 50um to remove smaller impurities. During the filtration, attention was paid to the clarity of the filtrate. The third-stage filtration used a filter with a pore size of 20um to completely remove tiny impurities to obtain a crude valine fermentation liquid. The crude valine fermentation liquid was then transferred to a magnetic field pretreatment device. The magnetic field strength was set to 1.5 Tesla and the magnetic field frequency was set to 50Hz. The fermentation liquid was pretreated in the magnetic field for 45 minutes. During this process, the aggregation of proteins and the changes in the physical properties of impurities were closely observed. Impurities with paramagnetism tended to be more concentrated in a specific area under the action of the magnetic field, while impurities with antimagnetism would be pushed to an area with a weaker magnetic field. The biosorbent is prepared by selecting Bacillus, introducing a gene fragment containing a specific protein domain coding sequence into its genome by molecular biological means, so that it is stably expressed on the surface of the bacterium, and then stirring and mixing the modified Bacillus and carbon nanotubes at a mass ratio of 1.1:0.5 at 25° C. to prepare the biosorbent, adding the biosorbent to the pretreated fermentation liquid at an addition amount of 10 g / L, stirring at a stirring rate of 150 r / min at 25° C. for 2 hours to allow the biosorbent to fully adsorb the protein in the fermentation liquid, and after the adsorption is completed, using an electric field separation device to separate the protein from the fermentation liquid, and applying an electric field with a strength of 50 V / cm to the fermentation liquid; A desorption solution is prepared, which is composed of a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system (concentration of 0.1 mol / L), a non-ionic surfactant Tween-20 (concentration of 0.5%) and dithiothreitol (concentration of 5 mmol / L). The biosorbent adsorbed with the protein is placed in the desorption solution, and the amount of the desorption solution is 10 mL of the desorption solution per gram of the biosorbent. The desorption is carried out at 30°C for 2 hours. After the desorption is completed, an ultrafiltration treatment is carried out. First, an ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used for preliminary filtration at a pressure of 0.2 MPa to remove larger molecular impurities, and then an ultrafiltration membrane with a molecular weight cutoff of 5 kDa is used for filtration to obtain a relatively high-purity protein solution. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3 kDa is used for fine filtration at a pressure of 0.2 MPa to prepare a high-purity protein solution. The amount of organic carbon source, phosphorus source, potassium source and trace elements added was determined to be 10%, 5%, 8% and 1% of the protein solution, respectively. The purified protein solution was added to a stirring container and stirred at a stirring rate of 100 r / min for 30 min. Then, bioactive substances were added, including 100 units of nitrogenase per kilogram of protein solution, 100 units of phosphatase per kilogram of protein solution, and 1×10 Bacillus subtilis per kilogram of protein solution. 6 The colony forming units of Bacillus licheniformis were 1×10 per kg of protein solution. 6 The fertilizer raw materials are transferred into a specific reaction container, polyvinyl alcohol is added, the reaction temperature is controlled at 40°C, the pH value is 7, a cross-linking reaction is initiated, and appropriate stirring is performed during the reaction to ensure that the reaction proceeds evenly. The reaction time is 1 hour to prepare a finished protein fertilizer.

[0031] Embodiment 2 The valine fermentation liquid was subjected to multi-stage filtration, with the aperture of the first-stage filter screen being 102um, the aperture of the second-stage filter screen being 52um, and the aperture of the third-stage filter screen being 21um, to ensure that impurities were fully removed, and the crude valine fermentation liquid was obtained, which was transferred to a magnetic field pretreatment device, with the magnetic field intensity set to 1.5 Tesla, the magnetic field frequency adjusted to 48Hz, and the pretreatment time being 48min; When preparing the biosorbent, the bacillus cells and the carbon nanotubes were stirred and mixed at a mass ratio of 1.15:0.55 at 25° C., the biosorbent was added to the fermentation broth at an addition amount of 11 g / L, and stirred at a stirring rate of 155 r / min at 24° C. for 2 hours. After the adsorption was completed, an electric field separation device was used to apply an electric field with an intensity of 48 V / cm in the fermentation broth to separate the protein and the fermentation broth; The desorption solution is composed of a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system (concentration of 0.11 mol / L), Tween-20 (concentration of 0.6%) and dithiothreitol (concentration of 6 mmol / L). The amount of desorption solution is 10.5 mL per gram of biosorbent. Desorption is carried out at 30°C for 2 hours. After desorption, ultrafiltration is performed. First, an ultrafiltration membrane with a molecular weight cutoff of 10.5 kDa is used for preliminary filtration at a pressure of 0.22 MPa, and then an ultrafiltration membrane with a molecular weight cutoff of 5.2 kDa is used for filtration. Finally, an ultrafiltration membrane with a molecular weight cutoff of 3.1 kDa is used for fine filtration at a pressure of 0.22 MPa. The amount of organic carbon source, phosphorus source, potassium source and trace elements added accounted for 11%, 5.5%, 8.5% and 1.2% of the protein solution, respectively. The purified protein solution was added to a stirring container and stirred at a stirring rate of 100 r / min for 30 min. Bioactive substances were added, with 110 units of nitrogenase per kilogram of protein solution, 110 units of phosphatase per kilogram of protein solution, and 1.2×10 Bacillus subtilis per kilogram of protein solution. 6 Colony forming units of Bacillus licheniformis are 1.2×10 per kilogram of protein solution. 6 The product was solidified by polyvinyl alcohol at 40℃, pH value of 7.2 and reaction time of 1h.

[0032] Embodiment 3 The valine fermentation liquid was filtered through multiple stages, the aperture of the first-stage filter was 105um, the aperture of the second-stage filter was 53um, and the aperture of the third-stage filter was 22um. The crude valine fermentation liquid was transferred to a magnetic field pretreatment device, the magnetic field intensity was 1.48 Tesla, the magnetic field frequency was 49Hz, and the pretreatment time was 50min. To prepare a biosorbent, bacillus cells and carbon nanotubes were stirred and mixed at a mass ratio of 1.25:0.6 at 24.5° C., the biosorbent was added to the fermentation broth in an amount of 12 g / L, and stirred at a stirring rate of 160 r / min at 24.5° C. for 2.2 h. An electric field with a strength of 49 V / cm was applied to the fermentation broth using an electric field separation device; The desorption solution consisted of a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system (concentration of 0.13 mol / L), Tween-20 (concentration of 0.7%) and dithiothreitol (concentration of 7 mmol / L). The amount of desorption solution was 11 mL per gram of biosorbent. The desorption was carried out at 32 °C for 2.2 h. Ultrafiltration treatment used ultrafiltration membranes with molecular weight cutoffs of 11 kDa, 5.5 kDa and 3.2 kDa, respectively, at a pressure of 0.25 MPa. The amount of organic carbon source, phosphorus source, potassium source and trace elements added accounted for 12%, 6%, 9% and 1.5% of the protein solution respectively. The purified protein solution was added to a stirring container and stirred at a stirring rate of 105r / min for 32min. Bioactive substances were added, 130 units of nitrogenase per kilogram of protein solution, 130 units of phosphatase per kilogram of protein solution, and 1.5×10 Bacillus subtilis per kilogram of protein solution. 6 Colony forming units of Bacillus licheniformis are 1.5×10 per kilogram of protein solution. 6 The colony forming units were solidified, and polyvinyl alcohol was selected as the polymer material in the treatment process. The reaction temperature was 43°C, the pH value was 7.5, and the reaction time was 1.3h.

[0033] Embodiment 4: The valine fermentation liquid was subjected to multi-stage filtration, the aperture of the first-stage filter screen was 108um, the aperture of the second-stage filter screen was 54um, and the aperture of the third-stage filter screen was 23um. The crude valine fermentation liquid was transferred to a magnetic field pretreatment device, the magnetic field intensity was 1.5 Tesla, the magnetic field frequency was 50Hz, and the pretreatment time was 50min; To prepare a biosorbent, bacillus cells and carbon nanotubes were stirred and mixed at a mass ratio of 1.3:0.65 at 25.5° C., the biosorbent was added to the fermentation broth in an amount of 13 g / L, and stirred at a stirring rate of 170 r / min at 25° C. for 2.3 h. An electric field with a strength of 51 V / cm was applied to the fermentation broth using an electric field separation device; The desorption solution consisted of a sodium dihydrogen phosphate-sodium dihydrogen phosphate buffer system (concentration of 0.15 mol / L), Tween-20 (concentration of 0.8%) and dithiothreitol (concentration of 8 mmol / L). The amount of desorption solution was 11.5 mL per gram of biosorbent. The desorption was carried out at 33 °C for 2 h. Ultrafiltration treatment used ultrafiltration membranes with molecular weight cutoffs of 11.5 kDa, 5.8 kDa and 3.3 kDa, respectively, at a pressure of 0.28 MPa. The amount of organic carbon source, phosphorus source, potassium source and trace elements added accounted for 13.5%, 6.5%, 9.5% and 1.8% of the protein solution, respectively. The purified protein solution was added to a stirring container and stirred at a stirring rate of 108r / min for 33min. Bioactive substances were added, 150 units of nitrogenase per kilogram of protein solution, 150 units of phosphatase per kilogram of protein solution, and 1.8×10 Bacillus subtilis per kilogram of protein solution. 6 Colony forming units of Bacillus licheniformis are 1.8×10 per kilogram of protein solution. 6The colony forming units were solidified, polyvinyl alcohol was selected as the polymer material, the reaction temperature was 45°C, the pH value was 7.8, and the reaction time was 1.5h.

[0034] Embodiment 5 The valine fermentation liquid was subjected to multi-stage filtration, the aperture of the first-stage filter screen was 110um, the aperture of the second-stage filter screen was 55um, and the aperture of the third-stage filter screen was 24um. The crude valine fermentation liquid was transferred to a magnetic field pretreatment device, the magnetic field intensity was 1.5 Tesla, the magnetic field frequency was 50Hz, and the pretreatment time was 55min. To prepare a biosorbent, bacillus cells and carbon nanotubes were stirred and mixed at a mass ratio of 1.35:0.7 at 26° C., the biosorbent was added to the fermentation broth in an amount of 14 g / L, and stirred at a stirring rate of 180 r / min at 26° C. for 2.4 h. An electric field with a strength of 52 V / cm was applied to the fermentation broth using an electric field separation device; The desorption solution consisted of a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system (concentration of 0.18 mol / L), Tween-20 (concentration of 0.9%) and dithiothreitol (concentration of 9 mmol / L). The amount of desorption solution was 12 mL per gram of biosorbent. The desorption was carried out at 34 °C for 2.4 h. Ultrafiltration treatment used ultrafiltration membranes with molecular weight cutoffs of 12 kDa, 6 kDa and 3.4 kDa, respectively, at a pressure of 0.32 MPa. The amount of organic carbon source, phosphorus source, potassium source and trace elements added accounted for 14.5%, 7%, 10% and 1.9% of the protein solution, respectively. The purified protein solution was added to a stirring container and stirred at a stirring rate of 110 r / min for 34 min. Bioactive substances were added, 170 units of nitrogenase per kilogram of protein solution, 170 units of phosphatase per kilogram of protein solution, and 1.9×10 Bacillus subtilis per kilogram of protein solution. 6 Colony forming units of Bacillus licheniformis are 1.9×10 per kilogram of protein solution. 6 The colony forming units were solidified, polyvinyl alcohol was selected as the polymer material, the reaction temperature was 47°C, the pH value was 8, and the reaction time was 1.7h.

[0035] The quality of the protein fertilizer products prepared in the above five groups of embodiments was tested, and the testing method and specific results are as follows: Protein content detection: The operation steps are as follows: using the Kjeldahl method, taking a certain amount of protein fertilizer finished product sample, and calculating the nitrogen content in the sample through digestion, distillation, titration and other steps, and then multiplying it by the conversion coefficient to obtain the protein content.

[0036] Nutrient element ratio detection: The operation steps are as follows: after the fertilizer sample is processed by chemical analysis method and spectrophotometry, the content of organic carbon source is determined by measuring the absorbance at a specific wavelength.

[0037] Activity detection of biologically active substances: The operation steps are to determine the activity of the enzyme by measuring the rate of the enzyme reaction, mix the fertilizer sample with a specific substrate, react for a period of time under suitable conditions, and measure the data during the reaction.

[0038] Moisture content detection: The operation steps are as follows: the fertilizer sample is dried to a constant weight at a certain temperature using a drying method, and the mass difference before and after drying is calculated to determine the moisture content.

[0039] pH value detection: Operation steps: Use a pH meter to dissolve the fertilizer sample in a certain amount of water, stir evenly, and insert the pH meter electrode for measurement.

[0040] Particle size detection: The operation steps are as follows: using the sieving method, the fertilizer sample is passed through standard sieves of different apertures, the mass of the fertilizer particles remaining on each sieve is measured, and the particle size distribution is determined.

[0041] The test results are as follows: Embodiment 1

[0042] Protein content: The protein content is 22% of the total weight of the fertilizer; Nutrient element ratio: organic carbon source content is 10% of the total fertilizer mass, phosphorus source content is 5% of the total fertilizer mass, potassium source content is 8% of the total fertilizer mass, and trace elements content is 1% of the total fertilizer mass; Activity of biologically active substances: nitrogenase activity is 15ug / h of nitrogen fixation per gram of fertilizer, phosphatase activity is 7ug / h of inorganic phosphorus produced by hydrolyzing phosphate ester per gram of fertilizer, and the sum of Bacillus subtilis and Bacillus licheniformis contains 2.0×10 6 colony-forming units; Moisture content: The moisture content is 11% of the total mass of the finished protein fertilizer; pH value: pH value is 7; Particle size: The particle size is mainly distributed between 1.5mm-2.0mm.

[0043] Embodiment 2 Protein content: The protein content is 23% of the total weight of the fertilizer; Nutrient element ratio: organic carbon source content is 11% of the total fertilizer mass, phosphorus source content is 5.5% of the total fertilizer mass, potassium source content is 8.5% of the total fertilizer mass, and trace elements content is 1.2% of the total fertilizer mass; Activity of biologically active substances: nitrogenase activity is 16ug / h of nitrogen fixation per gram of fertilizer, phosphatase activity is 8ug / h of inorganic phosphorus produced by hydrolyzing phosphate ester per gram of fertilizer, and the sum of Bacillus subtilis and Bacillus licheniformis contains 2.4×10 6 colony-forming units; Moisture content: The moisture content is 12% of the total mass of the finished protein fertilizer; pH: pH 7.2 Particle size: The particle size is mainly distributed between 1.6mm-2.2mm.

[0044] Embodiment 3 Protein content: The protein content is 24% of the total weight of the fertilizer; Nutrient element ratio: organic carbon source content is 12% of the total fertilizer mass, phosphorus source content is 6% of the total fertilizer mass, potassium source content is 9% of the total fertilizer mass, and trace elements content is 1.5% of the total fertilizer mass; The activity of biologically active substances: nitrogenase activity is 17ug / h of nitrogen fixation per gram of fertilizer, phosphatase activity is 9ug / h of inorganic phosphorus produced by hydrolyzing phosphate ester per gram of fertilizer, and the sum of Bacillus subtilis and Bacillus licheniformis contains 3.0×10 6 colony-forming units; Moisture content: The moisture content is 13% of the total mass of the finished protein fertilizer; pH value: pH value is 7.5; Particle size: The particle size is mainly distributed between 1.8mm-2.5mm.

[0045] Embodiment 4: Protein content: The protein content is 25% of the total weight of the fertilizer; Nutrient element ratio: organic carbon source content is 13.5% of the total fertilizer mass, phosphorus source content is 6.5% of the total fertilizer mass, potassium source content is 9.5% of the total fertilizer mass, and trace elements content is 1.8% of the total fertilizer mass; Activity of biologically active substances: nitrogenase activity is 18ug / h of nitrogen fixation per gram of fertilizer, phosphatase activity is 9.5ug / h of inorganic phosphorus produced by hydrolyzing phosphate ester per gram of fertilizer, and the sum of Bacillus subtilis and Bacillus licheniformis contains 3.6×10 6 colony-forming units; Moisture content: The moisture content is 14% of the total mass of the finished protein fertilizer; pH value: pH value is 7.8; Particle size: The particle size is mainly distributed between 2.0mm-2.8mm.

[0046] Embodiment 5 Protein content: The protein content is 26% of the total weight of the fertilizer; Nutrient element ratio: organic carbon source content is 14.5% of the total fertilizer mass, phosphorus source content is 7% of the total fertilizer mass, potassium source content is 10% of the total fertilizer mass, and trace elements content accounts for 1.9% of the total fertilizer mass; Activity of biologically active substances: nitrogenase activity is 19ug / h of nitrogen fixation per gram of fertilizer, phosphatase activity is 10ug / h of inorganic phosphorus produced by hydrolyzing phosphate ester per gram of fertilizer, and the sum of Bacillus subtilis and Bacillus licheniformis contains 3.8×10 6 colony-forming units; Moisture content: The moisture content is 15% of the total mass of the finished protein fertilizer; pH value: pH value is 8.0; Particle size: The particle size is mainly distributed between 2.2mm-3.0mm.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 extracting protein from valine fermentation broth to prepare fertilizer, comprising pretreatment of the valine fermentation broth, protein extraction and protein purification, wherein the valine fermentation broth is pretreated by a magnetic field pretreatment device, and the fermentation broth is treated by a magnetic field of a specific intensity and frequency, and the method is characterized in that: The specific operation steps of extracting protein from valine fermentation broth to prepare fertilizer are as follows: Step 1: preliminarily filtering the valine fermentation broth, and transferring it to a magnetic field pretreatment device after filtering to aggregate the protein in the valine fermentation broth and change the physical properties of the impurities therein; Step 2: introducing a biosorbent, adding the biosorbent to the pretreated valine fermentation broth, stirring and mixing at room temperature, adsorbing protein with the biosorbent, and separating the protein from the fermentation broth using a special separation device; Step 3: protein purification, placing the separated biosorbent adsorbed with the protein in a specific desorption solution, desorbing the protein from the biosorbent, and ultrafiltration to obtain a high-purity protein solution; Step 4: transfer the purified protein solution into a stirring container, add an organic carbon source, a phosphorus source, a potassium source and trace elements, stir and mix, add biologically active substances, obtain a mixed fertilizer material, and make it into a finished protein fertilizer through solidification technology; Step 5: Conduct a comprehensive quality inspection on the prepared protein fertilizer. Once qualified, it will be packaged in an environmentally friendly manner and stored in a dry and dark place.

2. A method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 1, the valine fermentation broth is preliminarily filtered using a multi-stage filtering device, wherein the first-stage filtration uses a filter with a pore size of 100 um to remove larger particles of impurities, the second-stage filtration uses a filter with a pore size of 50 um to remove smaller particles of impurities, and the third-stage filtration uses a filter with a pore size of 20 um to remove tiny impurities, thereby obtaining a crude valine fermentation broth.

3. A method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 1, the crude valine fermentation liquid is transferred into a magnetic field pretreatment device, which is composed of a group of high-strength permanent magnets. The magnetic field strength is set to 1.5 Tesla, and the magnetic field frequency is adjusted to 50 Hz. The protein in the crude valine fermentation liquid is aggregated, and the magnetic field pretreatment time is set to 45 min-60 min to obtain a semi-finished valine fermentation liquid.

4. A method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 2, a biosorbent is prepared. The biosorbent is made of a composite of genetically modified microbial cells and nanomaterials. A gene fragment containing a specific protein domain coding sequence is introduced into the genome of Bacillus by molecular biological means to stably express it on the cell surface. The structural gene-engineered Bacillus cells are composited with the nanomaterials. The nanomaterial is carbon nanotubes. The Bacillus cells and the carbon nanotubes are stirred and mixed at a mass ratio of 1.1-1.4:0.5-0.7 at 25° C.-27° C. to prepare the biosorbent. The biosorbent is added to the semi-finished valine fermentation broth at 25° C. and mixed in an amount of 10 g / L. The mixture is stirred at a stirring rate of 150 r / min-200 r / min for 2 hours.

5. The method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 2, an electric field separation device is used to separate the protein from the valine fermentation broth. An electric field with a strength of 50V / cm is applied to the valine fermentation broth. The biosorbent moves toward a specific electrode under the action of the electric field, thereby separating the biosorbent adsorbed with protein.

6. The method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 3, the desorption solution is composed of a buffer salt, a surfactant and a reducing agent, wherein the buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration range of 0.1 mol / L-0.2 mol / L, the surfactant is a non-ionic surfactant Tween-20 with a concentration of 0.5%-1%, and the reducing agent is dithiothreitol with a concentration of 5 mmol / L-10 mmol / L. The biosorbent adsorbed with protein is placed in the desorption solution, the amount of the desorption solution is 10 mL / g corresponding to the biosorbent, the desorption temperature is controlled between 30° C. and 35° C., and the desorption process lasts for 2 h to 3 h.

7. The method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step three, after desorption is completed, the protein-containing solution is subjected to ultrafiltration treatment, and the ultrafiltration adopts multi-stage ultrafiltration, and is filtered in sequence using ultrafiltration membranes with molecular weight cutoffs of 10kDa, 5kDa and 3kDa, respectively, and the filtration environment pressure range is 0.2MPa-0.5MPa.

8. The method for preparing fertilizer by extracting protein from valine fermentation broth according to claim 1, characterized in that: In the step 4, the added amounts of the organic carbon source, phosphorus source, potassium source and trace elements account for 10%-15%, 5%-8%, 8%-12% and 1%-2% of the protein solution respectively. The purified protein solution is added into a stirring container and stirred at a stirring rate of 100 r / min-120 r / min for 30 min. After adding the bioactive substance, the solution is solidified to prepare a finished protein fertilizer.

9. The method for extracting protein from valine fermentation broth to prepare fertilizer according to claim 1, characterized in that: In the step 5, the quality of the finished protein fertilizer is tested, including protein content, nutrient element ratio, activity of biologically active substances and other relevant indicators, wherein other relevant indicators include moisture content, pH value and particle size. The moisture content is set to 10%-15% of the total mass of the finished protein fertilizer, the pH value is set between 6.5-8.5, and the particle size is controlled between 1mm-5mm. After the finished protein fertilizer is tested and qualified, it is packaged and stored between 10°C-30°C, and the relative humidity is controlled at 70%.

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