Method for increasing fermentation yield of vitamin B12 by adding precursor substance
By adding new precursor substances and optimizing fermentation conditions, using transition metal oxides and specific microbial strains, the problems of increased fermentation yield and high cost of vitamin B12 were solved, and efficient and economical vitamin B12 production was achieved.
Patent Information
- Application Number
- CN202510553323.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-07-25
AI Technical Summary
The traditional vitamin B12 fermentation method has bottlenecks in yield increase and high production costs, which limits its market supply and application prospects.
New precursor substances, including transition metal oxides, methylcobalamin, hydroborate and uroporphyrinogen III, are combined with specific microbial strains and optimized fermentation medium, and promote the efficient synthesis of vitamin B12 by participating in cell redox reactions, regulating enzyme activities and synthesis pathways.
It significantly improves the fermentation yield and production efficiency of vitamin B12, reduces production costs, and achieves efficient and orderly synthesis of vitamin B12.
Smart Images
Figure CN120366405A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of vitamin fermentation, and in particular to a method for increasing the fermentation yield of vitamin B12 by adding precursor substances. Background Art
[0002] Vitamin B12, as one of the indispensable water-soluble vitamins for the human body, plays a core role in maintaining the healthy functioning of the nervous system and promoting blood production. Its application range covers a wide range of fields including pharmaceutical manufacturing, food processing and feed production.
[0003] Although vitamin B12 is currently produced mainly through microbial fermentation, traditional fermentation methods face difficulties such as production bottlenecks and high production costs, which seriously hinder the expansion of its market supply and wider application prospects. Therefore, how to overcome these limitations and improve the fermentation production efficiency of vitamin B12 has become a key technical challenge that needs to be solved in this field. Summary of the invention
[0004] The present application provides a method for increasing the fermentation yield of vitamin B12 by adding precursor substances, so as to solve the problems of difficulty in increasing yield and high production cost in related technologies.
[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present invention is as follows: The present invention provides a method for adding precursor substances to increase the fermentation yield of vitamin B12, characterized in that the preparation method comprises the following steps: S1, obtaining a microbial strain suitable for producing vitamin B12; S2, preparing a seed culture medium and a fermentation culture medium, wherein the seed culture medium comprises a carbon source, a nitrogen source, an inorganic salt and a special growth factor, and the fermentation culture medium comprises a carbon source, a nitrogen source, an inorganic salt, a plant tri-element and a novel precursor substance; S3, inoculating the microbial strain onto a slant culture medium for cultivation to obtain slant seeds; S4, inoculating the slant seeds into a seed culture medium to obtain a seed liquid; S5, inoculating the seed liquid into a fermentation culture medium, controlling the fermentation conditions for fermentation; S6, after the fermentation is completed, separating and purifying vitamin B12 from the fermentation liquid to finally obtain a finished product.
[0006] Furthermore, the three plant elements are extracted from plants rich in various nutrients such as ginseng, angelica, and ginkgo through supercritical fluid extraction and other technologies, and then inoculated into bacterial strains optimized by gene editing, and are successively produced through Triple-MAX triple fermentation, "Multi-Point" enzymatic oligopeptide, and "Bio-Synthesis" processes under precisely controlled conditions of temperature, pH value, etc.
[0007] Furthermore, the novel precursor material is composed of the following components by weight: Transition metal oxide: 30 - 50 parts; Mecobalamin: 40 - 60 parts; Hydroboric acid: 28 - 32 parts; Cobalt: 10 - 14 parts; Uroporphyrinogen III: 3 - 5 parts.
[0008] Furthermore, the transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1.
[0009] Furthermore, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide, and hafnium oxide; performing spark plasma sintering on titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1, uniformly cooling the sintered product to 1000 - 1100 °C after sintering, and then naturally cooling to room temperature to obtain a mixture, with the pressure drop being 0; performing pulverization and grinding on the mixture to obtain a mixed powder of the transition metal oxide.
[0010] Furthermore, the sintering temperature is 2000 - 2100 °C, the sintering pressure is 8.3 - 8.7 KN, and the sintering time is 5 - 10 min Furthermore, the purity of the titanium oxide is 99.5%, the purity of the zirconium oxide is 99.6%, and the purity of the hafnium oxide is 99.9%.
[0011] Furthermore, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii, and Pseudomonas denitrificans.
[0012] Furthermore, the seed culture medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salts are potassium dihydrogen phosphate, and the special growth factor is an amino acid.
[0013] Furthermore, the fermentation culture medium includes a carbon source, a nitrogen source, inorganic salts, and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salts are zinc sulfate.
[0014] Furthermore, the components of the slant culture medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0015] The beneficial effects obtained by adopting the above-mentioned present invention are as follows: 1. The present invention prepares a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance. In the transition of the novel precursor substance, transition metal oxides act as part of the electron transport chain, participate in the redox reaction within the cell, generate ATP to provide the necessary energy for the synthesis of vitamin B12. They can also regulate the intracellular redox potential, ensure that the redox reaction in the synthesis pathway proceeds in a suitable environment, and direct the metabolic intermediates to flow towards the synthesis direction of vitamin B12. In addition, transition metal oxides interact with the enzymes in the vitamin B12 synthesis pathway, exist as cofactors or activators, change the spatial structure of the enzymes, promote the binding of the active center of the enzymes to the substrates, thereby improving the catalytic efficiency. During the enzymatic reaction process, transition metal oxides can also provide or accept electrons, accelerate the conversion of the substrates, increase the reaction rate, and ensure the efficient and orderly synthesis of vitamin B12 within the microbial cells; 2. In the present invention, mecobalamin, as the active form of vitamin B12, participates in the synthesis both as a direct precursor and as an intermediate, and regulates the activity of the enzymes in the synthesis pathway through a feedback mechanism to maintain the synthesis balance. Catalytic cobalt participates in the formation and subsequent modification of the corrin ring, is a key component of the core structure of vitamin B12, and also affects the enzyme activity; 3. In the present invention, hydroboric acid undergoes a condensation reaction with uroporphyrinogen III to jointly construct the basic skeleton of the corrin ring under the catalysis of an enzyme. Hydroboric acid converts linear uroporphyrinogen III into an intermediate product with a cyclic structure. At the same time, during the subsequent modification process, hydroboric acid also acts synergistically with other precursor substances to chemically modify the corrin ring, making it gradually approach the final structure of the corrin ring in the vitamin B12 molecule; 4. The present invention clarifies the effective components of the seed medium and the fermentation medium. In the seed medium, glucose serves as a carbon source to provide energy and precursors for synthesizing cellular substances, promoting the rapid growth and reproduction of microorganisms; peptone serves as a nitrogen source to meet the microbial demand for protein synthesis and maintain cell structure and physiological functions; potassium dihydrogen phosphate provides inorganic salts to ensure the transmission of genetic information of cells, the integrity of the membrane structure, and the stability of the physiological environment; specific amino acids serve as growth factors to promote the microbial metabolism to bias towards the growth and reproduction direction. In the fermentation medium, glucose continues to provide energy for microorganisms and participates in the chemical reactions in the vitamin B12 synthesis pathway; corn steep liquor serves as a nitrogen source to meet the microbial demand for nitrogen elements and regulate the metabolic pathway to improve the synthesis efficiency and yield of vitamin B12; zinc sulfate serves as an inorganic salt to maintain the activity of key enzymes and promote the chemical reactions during the vitamin B12 synthesis process. Specific microbial strains such as Propionibacterium shermanii, Propionibacterium freudenreichii, and Pseudomonas denitrificans, etc., have unique metabolic pathways and utilization modes of nutrients. After adding plant tricontanol, the matching degree between nutrient supply and microbial demand is further optimized, and the fermentation efficiency and the yield of vitamin B12 are improved. Description of the Drawings
[0016] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description of embodiments in conjunction with the accompanying drawings, where: Figure 1 It is the molecular structure diagram of the transition metal oxide provided in the embodiment of the present invention. Detailed implementation manners
[0017] 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 combination 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 stated, the numbers of the method steps are only convenient tools for identifying the method steps, rather than limiting the arrangement order of the method steps or the scope in which the present invention can be implemented. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope in which the present invention can be implemented.
[0018] To better understand the above technical solutions, the exemplary embodiments of the present invention are 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 to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.
[0019] The present invention is further described below in conjunction with the following embodiments.
[0020] Embodiment 1 The present invention provides a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance, and the preparation method includes the following steps: S1. Obtain a microbial strain suitable for producing vitamin B12; S2. Prepare a seed medium and a fermentation medium, where the seed medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors, and the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, plant hormones and a novel precursor substance; S3. Inoculate the microbial strain onto a slant medium for culture to obtain slant seeds; S4. Inoculate the slant seeds into the seed medium to obtain a seed liquid; S5. Inoculate the seed liquid into the fermentation medium and control the fermentation conditions for fermentation; S6. After fermentation, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
[0021] In the embodiments of the present application, the novel precursor substance is composed of the following components by weight: Transition metal oxide: 30 parts; Mecobalamin: 40 parts; Hydroboric acid: 28 parts; Cobalt: 10 parts; Uroporphyrinogen III: 3 parts.
[0022] In the embodiments of the present application, the transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:1.
[0023] In the embodiments of the present application, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide and hafnium oxide; performing spark plasma sintering on titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:1, and after sintering, uniformly cooling to 1000 - 1100 °C, and then naturally cooling to room temperature to obtain a mixture, with the pressure drop being 0; performing crushing and grinding on the mixture to obtain a mixed powder of the transition metal oxide.
[0024] In the embodiments of the present application, the sintering temperature is 2000 - 2100 °C, the sintering pressure is 8.3 - 8.7 KN, and the sintering time is 5 - 10 min In the embodiments of the present application, the purity of titanium oxide is 99.5%, the purity of zirconium oxide is 99.6%, and the purity of hafnium oxide is 99.9%.
[0025] In the embodiments of the present application, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii and Pseudomonas denitrificans.
[0026] In the embodiments of the present application, the seed medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is an amino acid.
[0027] In the embodiments of the present application, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
[0028] In the embodiments of the present application, the components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0029] Example 2 The present invention provides a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance, and the preparation method includes the following steps: S1. Obtain a microbial strain suitable for producing vitamin B12; S2. Prepare the seed medium and the fermentation medium. The seed medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors. The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, the three major plant nutrients, and a novel precursor substance. S3. Inoculate the microbial strain onto a slant medium for cultivation to obtain slant seeds. S4. Inoculate the slant seeds into the seed medium to obtain a seed solution. S5. Inoculate the seed solution into the fermentation medium and control the fermentation conditions for fermentation. S6. After fermentation, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
[0030] In the embodiment of the present application, the novel precursor substance is composed of the following components by weight: Transition metal oxide: 35 parts; Methylcobalamin: 45 parts; Hydroboric acid: 29 parts; Cobalt: 11 parts; Uroporphyrinogen III: 3.5 parts.
[0031] In the embodiment of the present application, the transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1.
[0032] In the embodiment of the present application, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide, and hafnium oxide; performing spark plasma sintering on titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1, and after sintering, uniformly cooling to 1000 - 1100 °C, then naturally cooling to room temperature to obtain a mixture, with the pressure drop being 0; performing pulverization and grinding on the mixture to obtain a mixed powder of the transition metal oxide.
[0033] In the embodiment of the present application, the sintering temperature is 2000 - 2100 °C, the sintering pressure is 8.3 - 8.7 KN, and the sintering time is 5 - 10 min. In the embodiment of the present application, the purity of titanium oxide is 99.5%, the purity of zirconium oxide is 99.6%, and the purity of hafnium oxide is 99.9%.
[0034] In the embodiment of the present application, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii, and Pseudomonas denitrificans.
[0035] In the embodiment of the present application, the seed medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is amino acid.
[0036] In the embodiments of the present application, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
[0037] In the embodiments of the present application, the components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0038] Example 3 The present invention provides a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance. The preparation method includes the following steps: S1. Obtain a microbial strain suitable for producing vitamin B12; S2. Prepare a seed medium and a fermentation medium. Among them, the seed medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors, and the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, phytohormones, and a novel precursor substance; S3. Inoculate the microbial strain onto the slant medium for cultivation to obtain slant seeds; S4. Inoculate the slant seeds into the seed medium to obtain a seed liquid; S5. Inoculate the seed liquid into the fermentation medium and control the fermentation conditions for fermentation; S6. After fermentation, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
[0039] In the embodiments of the present application, the novel precursor substance is composed of the following components by weight: Transition metal oxide: 40 parts; Methylcobalamin: 50 parts; Hydroboric acid: 30 parts; Cobalt: 12 parts; Uroporphyrinogen III: 4 parts.
[0040] In the embodiments of the present application, the transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1.
[0041] In the embodiments of the present application, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide, and hafnium oxide; performing spark plasma sintering on titanium oxide, zirconium oxide, and hafnium oxide in a mass ratio of 1:1:1, uniformly cooling to 1000-1100 °C after sintering, and then naturally cooling to room temperature to obtain a mixture, with the pressure drop being 0; performing pulverization and grinding treatment on the mixture to obtain a mixed powder of the transition metal oxide.
[0042] In the embodiments of the present application, the sintering temperature is 2000 - 2100 °C, the sintering pressure is 8.3 - 8.7 KN, and the sintering time is 5 - 10 min In the embodiments of the present application, the purity of titanium oxide is 99.5%, the purity of zirconium oxide is 99.6%, and the purity of hafnium oxide is 99.9%.
[0043] In the embodiments of the present application, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii, and Pseudomonas denitrificans.
[0044] In the embodiments of the present application, the seed medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is amino acid.
[0045] In the embodiments of the present application, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
[0046] In the embodiments of the present application, the components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0047] Example 4 The present invention provides a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance. The preparation method includes the following steps: S1. Obtain a microbial strain suitable for producing vitamin B12; S2. Prepare a seed medium and a fermentation medium. Among them, the seed medium includes a carbon source, a nitrogen source, inorganic salts, and special growth factors, and the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, phytohormones, and a novel precursor substance; S3. Inoculate the microbial strain onto a slant medium for cultivation to obtain slant seeds; S4. Inoculate the slant seeds into the seed medium to obtain a seed solution; S5. Inoculate the seed solution into the fermentation medium and control the fermentation conditions for fermentation; S6. After fermentation, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
[0048] In the embodiments of the present application, the novel precursor substance is composed of the following components by weight: Transition metal oxide: 45 parts; Methylcobalamin: 55 parts; Hydroboric acid: 31 parts; Cobalt: 13 parts; Uroporphyrinogen III: 4.5 parts.
[0049] In the embodiment of the present application, the transition metal oxide is a mixture composed of titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:1.
[0050] In the embodiment of the present application, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide and hafnium oxide; subjecting titanium oxide, zirconium oxide and hafnium oxide to spark plasma sintering at a mass ratio of 1:1:1, and uniformly cooling to 1000-1100 °C after sintering, and then naturally cooling to room temperature to obtain a mixture, the pressure drop is 0; pulverizing and grinding the mixture to obtain a mixed powder of the transition metal oxide.
[0051] In the embodiment of the present application, the sintering temperature is 2000-2100 °C, the sintering pressure is 8.3-8.7 KN, and the sintering time is 5-10 min In the embodiment of the present application, the purity of titanium oxide is 99.5%, the purity of zirconium oxide is 99.6%, and the purity of hafnium oxide is 99.9%.
[0052] In the embodiment of the present application, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii and Pseudomonas denitrificans.
[0053] In the embodiment of the present application, the seed medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is amino acid.
[0054] In the embodiment of the present application, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
[0055] In the embodiment of the present application, the components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0056] Example 5 The present invention provides a method for increasing the fermentation yield of vitamin B12 by adding a precursor substance. The preparation method includes the following steps: S1. Obtain a microbial strain suitable for producing vitamin B12; S2. Prepare a seed medium and a fermentation medium. Among them, the seed medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors, and the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, plant tri-elements and a novel precursor substance; S3. Inoculate the microbial strain on the slant medium for culture to obtain slant seeds; S4. Inoculate the slant seeds into the seed medium to obtain a seed solution; S5. Inoculate the seed liquid into the fermentation medium and control the fermentation conditions for fermentation; S6. After the fermentation is completed, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
[0057] In the embodiment of the present application, the novel precursor substance is composed of the following components by weight: Transition metal oxide: 50 parts; Methylcobalamin: 60 parts; Hydroboric acid: 32 parts; Cobalt: 14 parts; Uroporphyrinogen III: 5 parts.
[0058] In the embodiment of the present application, the transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:1.
[0059] In the embodiment of the present application, the preparation process of the transition metal oxide includes: weighing titanium oxide, zirconium oxide and hafnium oxide; performing spark plasma sintering on titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:1, uniformly cooling to 1000 - 1100 °C after sintering, and then naturally cooling to room temperature to obtain a mixture, with the pressure drop being 0; performing pulverization and grinding treatment on the mixture to obtain a mixed powder of the transition metal oxide.
[0060] In the embodiment of the present application, the sintering temperature is 2000 - 2100 °C, the sintering pressure is 8.3 - 8.7 KN, and the sintering time is 5 - 10 min In the embodiment of the present application, the purity of titanium oxide is 99.5%, the purity of zirconium oxide is 99.6%, and the purity of hafnium oxide is 99.9%.
[0061] In the embodiment of the present application, the microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii and Pseudomonas denitrificans.
[0062] In the embodiment of the present application, the seed medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is amino acid.
[0063] In the embodiment of the present application, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
[0064] In the embodiment of the present application, the components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.
[0065] Comparative Example 1 A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance, which is only different from Example 1 in that the component does not contain transition metal oxide, and the reduced amount of transition metal oxide is apportioned to hydroboric acid, and the other components, component contents, and preparation methods are the same as those in Example 1.
[0066] Comparative Example 2 A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance, which is only different from Example 1 in that the component does not contain uroporphyrinogen III, and the reduced amount of uroporphyrinogen III is apportioned to hydroboric acid, and the other components, component contents, and preparation methods are the same as those in Example 1.
[0067] Comparative Example 3 A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance, which is only different from Example 1 in that the component does not contain transition metal oxide and uroporphyrinogen III, and the reduced amounts of transition metal oxide and uroporphyrinogen III are apportioned to hydroboric acid, and the other components, component contents, and preparation methods are the same as those in Example 1.
[0068] Performance Test A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance prepared in Examples 1-5 and Comparative Examples 1-3 was used to accurately determine the content of vitamin B12 by a high performance liquid chromatograph. The results are shown in Table 1 below.
[0069] Table 1 Detection Results
[0070] As Figure 1 As shown in and Table 1, from Example 1 to Example 4, the yield of vitamin B12 showed a gradually increasing trend, and the highest yield was reached in Example 4. The yield in Example 5 decreased slightly, but was still higher than that in Examples 1-3. This indicates that with the adjustment of the component contents in the novel precursor substance, it is helpful to increase the fermentation yield of vitamins within a certain range. Among them, the ratio of each component in Example 4 may have reached a relatively optimal combination, which promoted the utilization of the precursor substance by microorganisms, thereby improving the synthesis efficiency of vitamins. The microbial cell density in the examples gradually increased with the improvement of the precursor substance formula, which was basically consistent with the change trend of vitamin yield. The increase in cell density means that the growth condition of microorganisms is good, and more microbial cells can participate in the vitamin synthesis process.
[0071] In Comparative Example 1, there is no transition metal oxide, and the yield is lower than that in Example 1, indicating that the transition metal oxide plays an important role in the vitamin synthesis process. In Comparative Example 2, there is no uroporphyrinogen III, and the yield is lower than that in Example 1, reflecting the positive effect of uroporphyrinogen III on the yield. In Comparative Example 3, both the transition metal oxide and uroporphyrinogen III are missing, indicating that the transition metal oxide, as part of the electron transport chain, participates in the redox reaction in the cell, provides energy for vitamin synthesis, and at the same time regulates the intracellular redox potential to direct the metabolic flow towards the vitamin synthesis direction; uroporphyrinogen III plays a key role in the process of constructing the corrin ring skeleton, etc. The lack of them will seriously affect the vitamin synthesis. The cell density of the comparative examples is lower than that in Example 1, and the cell density is the lowest in Comparative Example 3. This shows that the lack of components such as transition metal oxides and uroporphyrinogen III is not conducive to the growth and reproduction of microorganisms. The transition metal oxide indirectly affects the growth and metabolism of microorganisms by influencing the intracellular redox environment, enzyme activity, etc.; uroporphyrinogen III may participate in the synthesis or metabolic regulation of certain key substances in microbial cells. The lack of them will interfere with the normal physiological functions of microorganisms, resulting in slow cell growth and reduced density, thus affecting the vitamin yield.
[0072] In summary, the present invention provides a method for increasing the fermentation yield of vitamin B12 by adding precursor substances. By adding novel precursor substances and optimizing the fermentation conditions, using transition metal oxides to promote the energy supply and enzymatic reactions in vitamin B12 synthesis, combining the regulatory role of methylcobalamin and the key role of catalytic cobalt, and the participation of hydroboric acid in the construction of the corrin ring, the effective components of the seed and fermentation media are clarified, and in cooperation with specific microbial strains, the efficient and orderly synthesis of vitamin B12 is achieved, and the production cost is reduced. Thus, the problems of difficult yield improvement and high production cost in the related technologies are solved.
[0073] 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 increasing the fermentation yield of vitamin B12 by adding a precursor substance, characterized in that, The preparation method comprises the following steps: S1. Obtain a microbial strain suitable for the production of vitamin B12; S2. Prepare a seed culture medium and a fermentation culture medium. Among them, the seed culture medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors, and the fermentation culture medium includes a carbon source, a nitrogen source, inorganic salts, plant hormones and a novel precursor substance; S3. Inoculate the microbial strain onto a slant medium for cultivation to obtain slant seeds; S4. Inoculate the slant seeds into the seed culture medium to obtain a seed solution; S5. Inoculate the seed solution into the fermentation culture medium and control the fermentation conditions for fermentation; S6. After fermentation, separate and purify vitamin B12 from the fermentation broth to finally obtain the finished product.
2. The method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 1, characterized in that The novel precursor substance is composed of the following components by weight: Transition metal oxide: 30-50 parts; Mecobalamin: 40-60 parts; Hydroboric acid: 28-32 parts; Cobalt: 10-14 parts; Uroporphyrinogen III: 3-5 parts.
3. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 2, characterized in that The transition metal oxide is composed of a mixture of titanium oxide, zirconium oxide and hafnium oxide in a mass ratio of 1:1:
1.
4. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 3, characterized in that, The preparation process of the transition metal oxide includes: Weigh titanium oxide, zirconium oxide and hafnium oxide; Perform spark plasma sintering on titanium oxide, zirconium oxide and hafnium oxide according to a mass ratio of 1:1:
1. After sintering, cool down evenly to 1000-1100 °C, and then naturally cool to room temperature to obtain a mixture, and the pressure drops to 0; Perform pulverization and grinding treatment on the mixture to obtain a mixed powder of the transition metal oxide.
5. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 4, characterized in that, The sintering temperature is 2000-2100 °C, the sintering pressure is 8.3-8.7 KN, and the sintering time is 5-10 min.
6. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 4, characterized in that, The purity of the titanium oxide is 99.5%, the purity of the zirconium oxide is 99.6%, and the purity of the hafnium oxide is 99.9%.
7. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 1, characterized in that, The microbial strain includes one of Propionibacterium shermanii, Propionibacterium freudenreichii and Pseudomonas denitrificans.
8. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 1, characterized in that, The seed culture medium includes a carbon source, a nitrogen source, inorganic salts and special growth factors. Among them, the carbon source is glucose, the nitrogen source is peptone, the inorganic salt is potassium dihydrogen phosphate, and the special growth factor is amino acid.
9. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 1, characterized in that, The fermentation culture medium includes a carbon source, a nitrogen source, inorganic salts and a novel precursor substance. Among them, the carbon source is glucose, the nitrogen source is corn steep liquor, and the inorganic salt is zinc sulfate.
10. A method for increasing the fermentation yield of vitamin B12 by adding a precursor substance according to claim 1, characterized in that, The components of the slant medium include: 2% glucose, 1% yeast extract, 0.2% potassium dihydrogen phosphate, 0.1% magnesium sulfate, 1.8% agar, and the balance is water.