Method for producing vitamin B12 through aerobic fermentation of pseudomonas denitrificans

Abstract

The invention relates to the technical field of biological fermentation, in particular to a method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans, which comprises the following steps: breeding Pseudomonas denitrificans strains, and inoculating to an optimized slant culture medium for activation culture; preparing a seed culture medium, and inoculating the activated strain into the seed culture medium for culturing according to the inoculum size; preparing a fermentation culture medium, and modifying the fermentation culture medium; inoculating the seed solution into a fermentation culture medium according to the inoculum size, and carrying out aerobic fermentation culture; sodium citrate and betaine are added in the fermentation process, the dissolved oxygen concentration is controlled, and the pH value is monitored; after fermentation is finished, vitamin B12 is extracted and purified. According to the method for producing the vitamin B12 through aerobic fermentation of the pseudomonas denitrificans, the yield and quality of the vitamin B12 are remarkably improved by obtaining strains with high synthesis capacity, optimizing components and proportions of a slant culture medium, a seed culture medium and a fermentation culture medium and adopting an adsorbent preparation technology.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation, and particularly relates to a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans. Background Art

[0002] Vitamin B12 is a general term for a class of cobalamin compounds containing cobalt, and was first discovered as the "anti-pernicious anemia factor". Vitamin B12 is an important bioactive substance and also a growth factor for many microorganisms and animals, and is widely used in medicine, food and animal husbandry. Due to the extremely complex molecular structure of vitamin B12, its total chemical synthesis requires more than 70 reaction steps and is costly, so currently vitamin B12 is almost entirely produced by microbial fermentation.

[0003] In nature, there are two different pathways for the biosynthesis of vitamin B12, namely the aerobic pathway and the anaerobic pathway. The anaerobic pathway mainly occurs in some archaea and bacteria, such as certain Propionibacterium and Clostridium, etc. These microorganisms synthesize vitamin B12 through different enzyme systems and reaction paths under anaerobic or hypoxic conditions. The aerobic pathway mainly occurs in certain specific bacteria, such as Pseudomonas denitrificans. These bacteria can synthesize vitamin B12 from simple organic precursors through a series of enzyme-catalyzed reactions under aerobic conditions.

[0004] However, the aerobic pathway has strict requirements for the oxygen concentration. Too high will generate reactive oxygen species that damage microbial cells, too low will inhibit the activity of related enzymes, and controlling the oxygen concentration requires complex equipment and delicate operations, and the fermentation equipment has high requirements; the aerobic environment is prone to the growth of miscellaneous bacteria, and the cost of pollution detection and prevention and control is high; the enzyme-catalyzed reactions involved in the aerobic pathway are complex, the requirement for multi-enzyme cooperation is high, and the stability of intermediate products is poor, and side reactions are likely to occur, affecting the synthesis efficiency. Summary of the Invention

[0005] The present application provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans to solve the problems of slow growth, difficult transformation, complex pathway, high cost, etc. of vitamin B12 in related technologies.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans, and the method comprises the following steps: selecting a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, and inoculating the selected strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 5%-10% (v / v), and culturing with shaking at 28-30°C and 180-220 r / min for 24-36 hours; preparing a fermentation medium and performing modification treatment on the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 10%-15% (v / v), and performing aerobic fermentation culture at 30-32°C and 180-220 r / min for 120-168 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 4-6 mg / L, and monitoring the pH value in real time to maintain it at 6.5-7.5; after the fermentation is completed, extracting and purifying vitamin B12.

[0007] Further, the components of the modified fermentation medium are proportioned by weight, including: glucose: 10-20 parts; corn steep liquor: 20-50 parts; acetylthreonine: 0.05-0.1 part; adenosine acid: 0.01-0.03 part; zirconia: 1-5 parts; phytotrin: 0.5-2 parts; 5-methyl-6-benzimidazolyl-nucleotide: 0.01-0.03 part.

[0008] Further, the phytotrin is made from astragalus extract by specific biotechnology. Among them, advanced extraction technology is used to extract plant active ingredients, and after mixing, the optimized strain by gene editing is inoculated. It is fermented by the Triple-MAX triple glycolysis process of microbial engineering, and the temperature and other conditions are controlled in three stages, so that the strain decomposes nutrients, generates intermediate products and constructs the basic structure of phytotrin. The "Multi-Point" enzymatic oligopeptide process of enzyme engineering is used to add enzyme preparations to modify oligopeptides and polysaccharides under specific mild conditions to improve the activity and stability. The "Bio-Synthesis biosynthesis" process of synthetic biology is used to promote microorganisms to convert the previous products into phytotrin containing 38 medium-quality elements, 32 high-quality elements and 28 colloidal elements in a suitable environment.

[0009] Further, the components of the slant medium are proportioned by weight, including: sucrose: 30-35 parts; peptone: 8-12 parts; corn steep liquor: 8-12 parts; (NH4)2SO4: 0.2-0.3 part; (NH4)2HPO4: 1.2-1.8 parts; MnSO4・H2O: 0.08-0.12 part; ZnSO4・7H2O: 0.08-0.12 part; agar: 18-22 parts.

[0010] Further, the components of the seed culture medium are proportioned by weight and include: sucrose: 32-38 parts; peptone: 18-22 parts; KH2PO4: 4-6 parts; (NH4)2SO4: 1.5-2.5 parts; (NH4)2HPO4: 0.6-1.0 part; MgSO4: 1.2-1.8 parts; ZnSO4・7H2O: 0.01-0.03 part; MnSO4・H2O: 0.15-0.25 part.

[0011] Further, the mass concentration of the sodium citrate is 0.3-0.4%, and the concentration of the betaine is 25-40 mmol / L.

[0012] Further, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0013] Further, the preparation process of the adsorbent includes: adding an MOFs precursor and a resin into ethanol according to a mass ratio of 1:2-1:5, stirring evenly to form a suspension; slowly dropping a selenium source solution into the suspension under vigorous stirring, and keeping the reaction system evenly mixed; after the reaction ends, washing the product by centrifugation, and drying the washed product at 60-80 °C for 12 hours to obtain the adsorbent.

[0014] Further, the MOFs precursor is 2-methylimidazole, and the selenium source is sodium selenite.

[0015] Further, the eluent is a PBS buffer solution.

[0016] Further, the purification includes ion exchange resin and gel filtration.

[0017] The beneficial effects obtained by the present invention are as follows: 1. The present invention first selects a Pseudomonas denitrificans strain with high synthesis ability and optimizes its traits through genetic engineering or mutagenesis breeding. Then, the fermentation medium is modified and optimized to provide precise nutrients to meet the needs of the bacteria. During the fermentation process, an optimized medium and precisely proportioned nutrient components are used to improve the vitality and metabolic efficiency of the bacteria. At the same time, specific components are added to promote enzymatic reactions and reduce costs; 2. Through the modified fermentation medium, the present invention uses glucose as the main carbon source to quickly provide energy and carbon skeletons for the bacteria, promoting the rapid arrival of its logarithmic growth phase. At the same time, corn steep liquor powder is used as the nitrogen source, supporting the synthesis of biological macromolecules such as bacterial proteins and nucleic acids, as well as the generation of enzymes related to vitamin B12 synthesis. The two work together to ensure the vigorous metabolism and rapid proliferation of the bacteria in the early stage. In addition, adenosine and acetylthreonine play a key role in metabolic regulation. They regulate relevant metabolic pathways according to the energy state of the bacteria, maintaining the metabolic balance in different fermentation stages. 5-Methyl-6-benzimidazolyl-nucleotide, as a key precursor for vitamin B12 synthesis, acts synergistically with other nutrients provided by glucose, corn steep liquor powder, etc., jointly constructing the complex molecular structure of vitamin B12. Finally, the addition of zirconia effectively adsorbs the harmful substances generated during the fermentation process, maintaining the physical and chemical properties of the fermentation broth stable and providing good environmental conditions for the synthesis of vitamin B12. The synergistic effect of these components jointly promotes the efficient synthesis and yield optimization of vitamin B12; 3. The adsorbent composed of MOFs precursor and resin in the present invention has a high specific surface area and a rich pore structure. During the synthesis process of MOFs materials, regular crystal structures are formed, and there are uniformly sized and interconnected pores inside. The sizes of these pores are usually in the nanometer range, which can provide sufficient adsorption sites for vitamin B12 molecules. The presence of resin not only helps to maintain the overall structural stability of the adsorbent but may also further increase the diversity and connectivity of the pores. When the vitamin B12 solution contacts the adsorbent, vitamin B12 molecules can enter these pores through diffusion and interact with the active sites on the inner wall of the pores, thus achieving efficient adsorption; 4. The present invention introduces a selenium source, and the doping of sodium selenite changes the electronic structure of the adsorbent. The coordination groups around the cobalt ion in the vitamin B12 molecule may interact with the electron cloud changed due to selenium doping on the adsorbent surface. This interaction may be a weak chemical bonding or electron transfer process, making the vitamin B12 molecule more easily adsorbed on the adsorbent surface, thereby improving the adsorption capacity and adsorption rate of the adsorbent. At the same time, during the vitamin B12 extraction process, there may be some oxidizing substances in the solution, which may damage the structure and performance of the adsorbent. Selenium atoms can consume the oxidizing substances in the solution through their own redox reactions, protecting the structural integrity of the adsorbent; 5. The extraction and purification process after fermentation in the present invention is a key link to improve the purity of vitamin B12. By combining microwave-assisted extraction technology with boiling method and ultrasonic disruption method, it can more effectively break the bacterial cells and release vitamin B12, while reducing the dissolution of impurities. During the purification process, by using the method of combining ion exchange resin and gel filtration, it can effectively remove the impurities in the extract, significantly improving the purity of vitamin B12; 6. The production method of the present invention has been optimized and verified through a large number of experiments, and has good stability and repeatability. The equipment and technology used are all common in industrial production, easy to operate and maintain, meeting the requirements of large-scale industrial production, and can stably produce high-quality vitamin B12. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the following description of the embodiments in conjunction with the drawings, wherein: Figure 1 is the scanning electron micrograph of the adsorbent during the preparation provided in Example 1 of the present invention; Figure 2 is the scanning electron micrograph of the adsorbent during the preparation provided in Example 2 of the present invention; Figure 3 is the scanning electron micrograph of the adsorbent during the preparation provided in Example 3 of the present invention; Figure 4 is the scanning electron micrograph of the adsorbent during the preparation provided in Example 4 of the present invention; Figure 5 is the scanning electron micrograph of the adsorbent during the preparation provided in Example 5 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The technical solutions of the present invention are illustrated by specific specific examples below. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these clearly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope in which the present invention can be implemented.

[0020] To better understand the above technical solution, the exemplary embodiments of the present invention will be described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention can be more thoroughly understood and the scope of the present invention can be fully conveyed to those skilled in the art.

[0021] Example 1 The present invention provides a method for aerobic fermentation of Pseudomonas denitrificans to produce vitamin B12, and the method comprises the following steps: breeding a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, and inoculating the selected strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 5% (v / v), and performing shaking culture at 28°C and 180 r / min for 24 hours; preparing a fermentation medium and performing modification treatment on the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 10% (v / v), and performing aerobic fermentation culture at 30°C and 180 r / min for 120 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 4 mg / L, and monitoring the pH value in real time to maintain it at 7.2; after the fermentation is completed, extracting and purifying vitamin B12.

[0022] Among them, the breeding method is to optimize its traits by genetic engineering or mutagenesis breeding.

[0023] It can be understood that in the embodiments of the present application, by breeding a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability and optimizing its fermentation conditions, including activation culture, seed medium preparation, fermentation medium modification, aerobic fermentation culture, and subsequent vitamin B12 extraction and purification, the high-efficiency, low-cost, and high-quality production of vitamin B12 is achieved.

[0024] In the embodiments of the present application, the components of the modified fermentation medium are proportioned by weight, and include: glucose: 10 parts; corn steep liquor: 20 parts; acetylthreonine: 0.05 part; adenosine monophosphate: 0.01 part; zirconium oxide: 1 part; phytohormone: 0.5 part; 5-methyl-6-(benzimidazol-1-yl)benzimidazole riboside: 0.01 part.

[0025] In the embodiments of the present application, the components of the slant medium are proportioned by weight, and include: sucrose: 30 parts; peptone: 8 parts; corn steep liquor: 8 parts; (NH4)2SO4: 0.2 part; (NH4)2HPO4: 1.2 parts; MnSO4·H2O: 0.08 part; ZnSO4·7H2O: 0.08 part; agar: 18 parts.

[0026] In the embodiments of the present application, the components of the seed medium are proportioned by weight and include: sucrose: 32 parts; peptone: 18 parts; KH2PO4: 4 parts; (NH4)2SO4: 1.5 parts; (NH4)2HPO4: 0.6 part; MgSO4: 1.2 parts; ZnSO4・7H2O: 0.01 part; MnSO4・H2O: 0.15 part.

[0027] In the embodiments of the present application, the mass concentration of sodium citrate is 0.3%, and the concentration of betaine is 25 mmol / L.

[0028] In the embodiments of the present application, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0029] In the embodiments of the present application, as Figure 1 shown, the preparation process of the adsorbent includes: adding the MOFs precursor and the resin into ethanol according to a mass ratio of 1:2, and stirring evenly to form a suspension; slowly dropping the selenium source solution into the suspension under vigorous stirring to keep the reaction system evenly mixed; after the reaction ends, centrifuging and washing the product, and drying the washed product at 60 °C for 12 hours to obtain the adsorbent.

[0030] In the embodiments of the present application, the MOFs precursor is 2-methylimidazole, and the selenium source is sodium selenite.

[0031] In the embodiments of the present application, the eluent is PBS buffer.

[0032] In the embodiments of the present application, purification includes ion exchange resin and gel filtration.

[0033] Example 2 The present invention provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans, and the method includes the following steps: selecting a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, inoculating the selected strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 6% (v / v), and performing shake culture at 29 °C and 190 r / min for 27 hours; preparing a fermentation medium and performing a modification treatment on the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 11% (v / v), and performing aerobic fermentation culture at 31 °C and 190 r / min for 132 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 4.5 mg / L, and monitoring the pH value in real time to maintain it at 7.2; after the fermentation ends, extracting and purifying vitamin B12.

[0034] Among them, the breeding method is genetic engineering or mutagenesis breeding to optimize its traits.

[0035] It can be understood that in the embodiments of the present application, by breeding Pseudomonas denitrificans strains with high vitamin B12 synthesis ability and optimizing its fermentation conditions, including activation culture, seed medium preparation, fermentation medium modification, aerobic fermentation culture, and subsequent vitamin B12 extraction and purification, the high-efficiency, low-cost, and high-quality production of vitamin B12 is achieved.

[0036] In the embodiments of the present application, the components of the modified fermentation medium are proportioned by weight, including: glucose: 12; corn steep liquor: 28 parts; acetylthreonine: 0.06 part; adenosine monophosphate: 0.02 part; zirconium oxide: 2 parts; phytohormones: 0.8 part; 5-methyl-6-(benzimidazol-1-yl)methylaminopurine riboside: 0.02 part.

[0037] In the embodiments of the present application, the components of the slant medium are proportioned by weight, including: sucrose: 31 parts; peptone: 9 parts; corn steep liquor: 9 parts; (NH4)2SO4: 0.22 part; (NH4)2HPO4: 1.3 parts; MnSO4・H2O: 0.09 part; ZnSO4・7H2O: 0.09 part; agar: 19 parts.

[0038] In the embodiments of the present application, the components of the seed medium are proportioned by weight, including: sucrose: 33 parts; peptone: 19 parts; KH2PO4: 4.5 parts; (NH4)2SO4: 1.8 parts; (NH4)2HPO4: 0.7 part; MgSO4: 1.4 parts; ZnSO4・7H2O: 0.02 part; MnSO4・H2O: 0.17 part.

[0039] In the embodiments of the present application, the mass concentration of sodium citrate is 0.3%, and the concentration of betaine is 30 mmol / L.

[0040] In the embodiments of the present application, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0041] In the embodiments of the present application, as Figure 2 shown, the preparation process of the adsorbent includes: adding MOFs precursor and resin to ethanol at a mass ratio of 1:3, stirring evenly to form a suspension; slowly dropping the selenium source solution into the suspension under vigorous stirring to keep the reaction system evenly mixed; after the reaction, centrifuging and washing the product, and drying the washed product at 70 °C for 12 hours to obtain the adsorbent.

[0042] In the embodiments of the present application, the MOF precursor is 2-methylimidazole and the selenium source is sodium selenite.

[0043] In the embodiments of the present application, the eluent is PBS buffer.

[0044] In the embodiments of the present application, purification includes ion exchange resin and gel filtration.

[0045] Example 3 The present invention provides a method for aerobic fermentation of Pseudomonas denitrificans to produce vitamin B12, which includes the following steps: breeding a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, inoculating the selected strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 8% (v / v), and culturing it by shaking at 30 °C and 200 r / min for 30 hours; preparing a fermentation medium and modifying the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 13% (v / v), and performing aerobic fermentation culture at 32 °C and 200 r / min for 145 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 6 mg / L, and monitoring the pH value in real time to maintain it at 7.2; after the fermentation is completed, extracting and purifying vitamin B12.

[0046] Among them, the breeding method is to optimize its traits by genetic engineering or mutagenesis breeding.

[0047] It can be understood that in the embodiments of the present application, by breeding a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability and optimizing its fermentation conditions, including activation culture, seed medium preparation, fermentation medium modification, aerobic fermentation culture, and subsequent vitamin B12 extraction and purification, the high-efficiency, low-cost, and high-quality production of vitamin B12 is achieved.

[0048] In the embodiments of the present application, the components of the modified fermentation medium are proportioned by weight, including: glucose: 15 parts; corn steep liquor: 33 parts; acetylthreonine: 0.08 part; adenosine acid: 0.03 part; zirconia: 3 parts; phytotriol: 1.2 parts; 5-methyl-6-benzimidazolyl-nucleotide: 0.03 part.

[0049] In the embodiments of the present application, the components of the slant medium are proportioned by weight, including: sucrose: 33 parts; peptone: 10 parts; corn steep liquor: 10 parts; (NH4)2SO4: 0.25 part; (NH4)2HPO4: 1.5 parts; MnSO4・H2O: 0.1 part; ZnSO4・7H2O: 0.1 part; agar: 20 parts.

[0050] In the embodiments of the present application, the components of the seed medium are proportioned by weight and include: sucrose: 35 parts; peptone: 20 parts; KH2PO4: 5 parts; (NH4)2SO4: 2 parts; (NH4)2HPO4: 0.8 part; MgSO4: 1.5 parts; ZnSO4・7H2O: 0.03 part; MnSO4・H2O: 0.2 part.

[0051] In the embodiments of the present application, the mass concentration of sodium citrate is 0.4%, and the concentration of betaine is 35 mmol / L.

[0052] In the embodiments of the present application, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0053] In the embodiments of the present application, as Figure 3 shown, the preparation process of the adsorbent includes: adding the MOFs precursor and the resin to ethanol at a mass ratio of 1:3.8, stirring evenly to form a suspension; slowly dripping the selenium source solution into the suspension under vigorous stirring, and keeping the reaction system evenly mixed; after the reaction, washing the product by centrifugation, and drying the washed product at 80 °C for 12 hours to obtain the adsorbent.

[0054] In the embodiments of the present application, the MOFs precursor is 2-methylimidazole, and the selenium source is sodium selenite.

[0055] In the embodiments of the present application, the eluent is PBS buffer solution.

[0056] In the embodiments of the present application, purification includes ion exchange resin and gel filtration.

[0057] Example 4 The present invention provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans, and the method includes the following steps: screening a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, inoculating the screened strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 9% (v / v), and culturing it by shaking at 30 °C and 210 r / min for 33 hours; preparing a fermentation medium and performing a modification treatment on the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 14% (v / v), and performing aerobic fermentation culture at 32 °C and 210 r / min for 150 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 5.5 mg / L, and monitoring the pH value in real time to keep it at 7.2; after the fermentation is completed, extracting vitamin B12 and purifying it.

[0058] Among them, the breeding method is genetic engineering or mutagenesis breeding to optimize its traits.

[0059] It can be understood that in the embodiments of the present application, by breeding Pseudomonas denitrificans strains with high vitamin B12 synthesis ability and optimizing its fermentation conditions, including activation culture, seed medium preparation, fermentation medium modification, aerobic fermentation culture, and subsequent vitamin B12 extraction and purification, the high-efficiency, low-cost, and high-quality production of vitamin B12 is achieved.

[0060] In the embodiments of the present application, the components of the modified fermentation medium are proportioned by weight, including: glucose: 18 parts; corn steep liquor: 42 parts; acetylthreonine: 0.09 part; adenosine monophosphate: 0.03 part; zirconium oxide: 4 parts; phytohormone mixture: 1.6 parts; 5-methyl-6-(benzimidazol-1-yl)methylaminopurine ribonucleotide: 0.03 part.

[0061] In the embodiments of the present application, the components of the slant medium are proportioned by weight, including: sucrose: 34 parts; peptone: 11 parts; corn steep liquor: 11 parts; (NH4)2SO4: 0.28 part; (NH4)2HPO4: 1.7 parts; MnSO4・H2O: 0.11 part; ZnSO4・7H2O: 0.11 part; agar: 21 parts.

[0062] In the embodiments of the present application, the components of the seed medium are proportioned by weight, including: sucrose: 37 parts; peptone: 21 parts; KH2PO4: 5.5 parts; (NH4)2SO4: 2.3 parts; (NH4)2HPO4: 0.9 part; MgSO4: 1.7 part; ZnSO4・7H2O: 0.03 part; MnSO4・H2O: 0.23 part.

[0063] In the embodiments of the present application, the mass concentration of sodium citrate is 0.4%, and the concentration of betaine is 38 mmol / L.

[0064] In the embodiments of the present application, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0065] In the embodiments of the present application, as Figure 4 shown, the preparation process of the adsorbent includes: adding MOFs precursor and resin into ethanol according to a mass ratio of 1:4.5, stirring evenly to form a suspension; slowly dropping the selenium source solution into the suspension under vigorous stirring to keep the reaction system evenly mixed; after the reaction ends, centrifuging and washing the product, and drying the washed product at 80 °C for 12 hours to obtain the adsorbent.

[0066] In the embodiments of the present application, the MOF precursor is 2-methylimidazole, and the selenium source is sodium selenite.

[0067] In the embodiments of the present application, the eluent is PBS buffer.

[0068] In the embodiments of the present application, purification includes ion exchange resin and gel filtration.

[0069] Example 5 The present invention provides a method for aerobic fermentation of Pseudomonas denitrificans to produce vitamin B12. The method includes the following steps: selecting a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability, inoculating the selected strain onto an optimized slant medium for activation culture; preparing a seed medium, inoculating the activated strain into the seed medium at an inoculation amount of 10% (v / v), and performing shake culture at 30 °C and 220 r / min for 36 hours; preparing a fermentation medium and performing modification treatment on the fermentation medium; inoculating the seed liquid into the fermentation medium at an inoculation amount of 15% (v / v), and performing aerobic fermentation culture at 32 °C and 220 r / min for 168 hours; adding sodium citrate and betaine during the fermentation process, controlling the dissolved oxygen concentration to be 6 mg / L, and monitoring the pH value in real time to maintain it at 7.2; after the fermentation is completed, extracting and purifying vitamin B12.

[0070] Among them, the selection method is to optimize its traits by genetic engineering or mutagenesis breeding.

[0071] It can be understood that in the embodiments of the present application, by selecting a Pseudomonas denitrificans strain with high vitamin B12 synthesis ability and optimizing its fermentation conditions, including activation culture, seed medium preparation, fermentation medium modification, aerobic fermentation culture, and subsequent vitamin B12 extraction and purification, the high-efficiency, low-cost, and high-quality production of vitamin B12 is achieved.

[0072] In the embodiments of the present application, the components of the modified fermentation medium are proportioned by weight, including: glucose: 20 parts; corn steep liquor: 50 parts; acetylthreonine: 0.1 part; adenosine monophosphate: 0.03 part; zirconia: 5 parts; phytotriol: 2 parts; 5-methyl-6-benzimidazolyl-nucleotide: 0.03 part.

[0073] In the embodiments of the present application, the components of the slant medium are proportioned by weight, including: sucrose: 35 parts; peptone: 12 parts; corn steep liquor: 12 parts; (NH4)2SO4: 0.3 part; (NH4)2HPO4: 1.8 parts; MnSO4・H2O: 0.12 part; ZnSO4・7H2O: 0.12 part; agar: 22 parts.

[0074] In the embodiment of the present application, the components of the seed culture medium are proportioned by weight and include: sucrose: 38 parts; peptone: 22 parts; KH2PO4: 6 parts; (NH4)2SO4: 2.5 parts; (NH4)2HPO4: 1.0 part; MgSO4: 1.8 parts; ZnSO4・7H2O: 0.03 part; MnSO4・H2O: 0.25 part.

[0075] In the embodiment of the present application, the mass concentration of sodium citrate is 0.4%, and the concentration of betaine is 40 mmol / L.

[0076] In the embodiment of the present application, after extracting vitamin B12, it further includes: adsorbing with an adsorbent; separating the nanoparticles adsorbed with vitamin B12; eluting vitamin B12 from the nanoparticles according to an eluent, and measuring the content of vitamin B12.

[0077] In the embodiment of the present application, as Figure 5 shown, the preparation process of the adsorbent includes: adding MOFs precursor and resin to ethanol at a mass ratio of 1:5, stirring evenly to form a suspension; slowly dropping the selenium source solution into the suspension under vigorous stirring, and keeping the reaction system evenly mixed; after the reaction ends, centrifuging and washing the product, and drying the washed product at 80 °C for 12 hours to obtain the adsorbent.

[0078] In the embodiment of the present application, the MOFs precursor is 2-methylimidazole, and the selenium source is sodium selenite.

[0079] In the embodiment of the present application, the eluent is PBS buffer solution.

[0080] In the embodiment of the present application, purification includes ion exchange resin and gel filtration.

[0081] Comparative Example 1 This comparative example provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans. The difference from Example 1 is only that the fermentation medium does not contain acetylthreonine and zirconia, and the reduced amounts of acetylthreonine and zirconia are apportioned to glucose, and the other components, component contents, and preparation methods are the same as those in Example 1.

[0082] Comparative Example 2 This comparative example provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans. The difference from Example 1 is only that after extracting vitamin B12, no adsorbent is prepared for adsorption, and direct purification treatment is carried out, and the other components, component contents, and preparation methods are the same as those in Example 1.

[0083] Comparative Example 3 This comparative example provides a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans. The difference from Example 1 is only that the fermentation medium does not contain acetylthreonine and zirconia. The reduced amounts of acetylthreonine and zirconia are evenly distributed to glucose. After extracting vitamin B12, no adsorbent is prepared for adsorption, and direct purification treatment is carried out. The other components, component contents, and preparation methods are the same as those in Example 1.

[0084] Performance Test For the methods for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans prepared in Examples 1 - 5 and Comparative Examples 1 - 3 respectively, the content of vitamin B12 was accurately determined by a high-performance liquid chromatograph. The content of vitamin B12 in the samples was calculated using specific chromatographic conditions and standard curves, and the purity was evaluated. The results are shown in Table 1 below.

[0085] Table 1 Detection Results

[0086] As shown in the detection results in Table 1, the yields of vitamin B12 in Examples 1 - 5 are all significantly higher than those in Comparative Examples 1 - 3. This indicates that the synergistic effect of each component in the modified fermentation medium and the use of the adsorbent in the present invention play an important role in increasing the yield of vitamin B12. In Comparative Example 1, acetylthreonine and zirconia were not added, resulting in a significant decrease in yield, indicating that these two components play a key promoting role in the growth of the bacteria and the synthesis of vitamin B12 during the fermentation process.

[0087] In terms of the purity of vitamin B12, Examples 1 - 5 also show relatively high purity, all above 90%, while the purity of Comparative Examples 1 - 3 is relatively low. This is because in the extraction and purification process after fermentation in the present invention, the combination of microwave-assisted extraction technology with boiling method and ultrasonic disruption method can more effectively break the bacteria and release vitamin B12, while reducing the dissolution of impurities. Then, combined with the purification method of ion exchange resin and gel filtration, it can effectively remove the impurities in the extract, thus significantly improving the purity of vitamin B12. In Comparative Example 2, no adsorbent was used for adsorption and direct purification treatment was carried out, resulting in a decrease in purity, indicating that the adsorbent plays an important role in removing impurities and improving purity.

[0088] In terms of production cost, although the production costs of Examples 1 - 5 are slightly higher than those of Comparative Examples 1 - 3, considering their relatively high yields and purities, the method of the present invention still has obvious advantages in economic benefits. Although Comparative Example 3 has the lowest production cost, its yield and purity are not ideal and cannot meet the market demand for high-quality vitamin B12.

[0089] In summary, the method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans of the present invention realizes the efficient and high-quality production of vitamin B12 through measures such as optimizing the fermentation medium, using adsorbents, and improving the extraction and purification processes, and has good application prospects and economic benefits.

[0090] The embodiment of the present application proposes a method for aerobic fermentation of vitamin B12 using Pseudomonas denitrificans. In this method, the Pseudomonas denitrificans strain is selected and optimized, and genetic engineering or mutagenesis breeding techniques are used to enhance its vitamin B12 synthesis ability. Subsequently, the fermentation medium is precisely modified and optimized, with glucose and corn steep liquor dry powder as the main raw materials, supplemented with key components such as adenylate and acetylthreonine, to synergistically promote the efficient synthesis of vitamin B12. At the same time, zirconia is added to maintain the stability of the fermentation broth. In the extraction and purification stage, a MOFs-resin composite adsorbent is used, and the adsorption performance is improved by doping with a selenium source. Combining microwave-assisted extraction, boiling method, ultrasonic disruption method, and ion exchange resin and gel filtration technology, vitamin B12 is effectively extracted and purified. The entire production method has been verified by experiments to have good stability and strong repeatability, and the equipment and technologies used are all suitable for large-scale industrial production, and can stably produce high-quality vitamin B12. Thus, the problems of slow growth, difficult transformation, complex pathways, and high costs of vitamin B12 in the related technologies are solved.

[0091] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans, characterized in that, The method includes the following steps: Select and breed Pseudomonas denitrificans strains with high vitamin B12 synthesis ability, and inoculate the selected strains onto an optimized slant medium for activation culture; Prepare a seed medium, inoculate the activated bacteria into the seed medium at an inoculation amount of 5%-10% (v / v), and shake culture at 28-30 °C and 180-220 r / min for 24-36 hours; Prepare a fermentation medium and modify the fermentation medium; Inoculate the seed liquid into the fermentation medium at an inoculation amount of 10%-15% (v / v), and carry out aerobic fermentation culture at 30-32 °C and 180-220 r / min for 120-168 hours; Add sodium citrate and betaine during the fermentation process, control the dissolved oxygen concentration to be 4-6 mg / L, and monitor the pH value in real time to maintain it at 6.5-7.5; After the fermentation is completed, extract and purify vitamin B12.

2. The method for producing vitamin B12 by aerobic fermentation using Pseudomonas denitrificans according to claim 1, wherein The components of the modified fermentation medium are in parts by weight and include: Glucose: 10-20 parts; Corn steep liquor: 20-50 parts; Acetylthreonine: 0.05-0.1 part; Adenylic acid: 0.01-0.03 part; Zirconia: 1-5 parts; Plant triterpenoids: 0.5-2 parts; 5-Methyl-6-benzimidazolyl-nucleotide: 0.01-0.03 part.

3. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans according to claim 1, characterized in that, The components of the slant medium are in parts by weight and include: Sucrose: 30-35 parts; Peptone: 8-12 parts; Corn steep liquor: 8-12 parts; (NH4)2SO4: 0.2-0.3 part; (NH4)2HPO4: 1.2-1.8 parts; MnSO4・H2O: 0.08-0.12 part; ZnSO4・7H2O: 0.08-0.12 part; Agar: 18-22 parts.

4. A method for producing vitamin B12 by aerobic fermentation using Pseudomonas denitrificans, according to claim 3, characterized in that, The components of the seed medium are in parts by weight and include: Sucrose: 32-38 parts; Peptone: 18-22 parts; KH2PO4: 4-6 parts; (NH4)2SO4: 1.5-2.5 parts; (NH4)2HPO4: 0.6-1.0 part; MgSO4: 1.2-1.8 parts; ZnSO4・7H2O: 0.01-0.03 part; MnSO4・H2O: 0.15-0.25 part.

5. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans according to claim 1, characterized in that, The mass concentration of the sodium citrate is 0.3-0.4%, and the concentration of the betaine is 25-40 mmol / L.

6. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans according to claim 1, characterized in that, After extracting vitamin B12, it further includes: Using an adsorbent for adsorption; Separating the nanoparticles adsorbed with vitamin B12; Eluting vitamin B12 from the nanoparticles according to an eluent and measuring the content of vitamin B12.

7. A method for producing vitamin B12 by aerobic fermentation using Pseudomonas denitrificans, according to claim 6, characterized in that, The preparation process of the adsorbent includes: Adding MOFs precursor and resin to ethanol at a mass ratio of 1:2-1:5, and stirring evenly to form a suspension; Slowly dripping the selenium source solution into the suspension under vigorous stirring to keep the reaction system evenly mixed; After the reaction is completed, wash the product by centrifugation, and dry the washed product at 60-80 °C for 12 hours to obtain the adsorbent.

8. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans according to claim 7, characterized in that, The MOF precursor is 2-methylimidazole, and the selenium source is sodium selenite.

9. A method for producing vitamin B12 by aerobic fermentation of Pseudomonas denitrificans according to claim 6, characterized in that, The eluent is PBS buffer.

10. A method for producing vitamin B12 by aerobic fermentation using Pseudomonas denitrificans according to claim 1, characterized in that, The purification includes ion exchange resin and gel filtration.

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