Method for improving fermentation stability of vitamin B12
By optimizing the culture medium composition and fermentation control methods, the problems of poor fermentation stability and low yield of vitamin B12 were solved, and an efficient and stable fermentation process and improved product quality were achieved.
Patent Information
- Application Number
- CN202510765636.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-10
- Publication Date
- 2025-09-19
AI Technical Summary
The poor fermentation stability and low yield of vitamin B12 lead to problems such as low production efficiency and high production costs.
A multiple synergistic mechanism is adopted, including the use of hyaluronic acid hydrogel, nano-vanadium oxide, 4-hydroxyethylpiperazineethanesulfonic acid and borax-boric acid buffer to optimize the culture medium, combined with optimized nitrogen source combination and complex carbon source system, and through the online monitoring system and neural network model to achieve precise control of the fermentation process.
It significantly improves the stability and output of the fermentation system, reduces production costs, improves fermentation efficiency and product quality, and ensures the reliability and controllability of the fermentation process.
Smart Images
Figure CN120665972A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of biological fermentation, and in particular to a method for improving the fermentation stability of vitamin B12. Background Art
[0002] Vitamin B12 plays a vital role in various fields, including medicine, food, and feed. It is a water-soluble vitamin essential for maintaining normal physiological functions in the human body. In particular, in the pharmaceutical field, vitamin B12 is used to treat anemia, neurological diseases, and enhance immunity, demonstrating its unique medical value. In the food and feed industries, vitamin B12, as a nutritional additive, can enhance the nutritional value of products and meet the daily needs of animals and humans.
[0003] Despite its widespread application and high demand, traditional microbial fermentation production of vitamin B12 faces numerous challenges. Among them, poor fermentation stability and low yield are key factors hindering the industrialized, large-scale production of vitamin B12. This poor fermentation stability can lead to low production efficiency and inconsistent product quality, while low yield directly increases production costs and reduces market competitiveness. Summary of the Invention
[0004] The present application provides a method for improving the fermentation stability of vitamin B12 to solve the problems of poor fermentation stability and low yield in related technologies.
[0005] In order to achieve the above-mentioned purpose, the technical solution adopted by the present invention is as follows: The present invention provides a method for improving the fermentation stability of vitamin B12, and the method comprises the following steps: obtaining a microbial strain of vitamin B12 and activating it; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor the key parameters of the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0006] Furthermore, the modified buffer comprises, in parts by weight: Hyaluronic acid hydrogel: 30-50 parts; Nano vanadium oxide: 5-15 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 20-40 parts; Polyvinylpyrrolidone: 5-10 parts Borax-boric acid buffer pair: 5-15 parts.
[0007] Furthermore, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0008] Furthermore, the optimized nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0009] Furthermore, the ratio of the yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0010] Furthermore, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0011] Furthermore, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 35%-40%, in the middle stage of fermentation, sucrose accounts for 20%-30%, and maltose accounts for 10%-20%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained in the range of 10%-30%, 20%-40% and 10%-30% according to the growth of the bacteria and the synthesis of vitamin B12.
[0012] Furthermore, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0013] Furthermore, the other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0014] Furthermore, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0015] The beneficial effects achieved by adopting the above-mentioned present invention are as follows: 1. The present invention enhances the buffering performance and overall stability of the fermentation system through multiple synergistic mechanisms. Wherein, 4-hydroxyethylpiperazineethanesulfonic acid and borax-boric acid buffer play complementary buffering effects in different pH ranges, broaden the buffering range of the whole system, and effectively resist the accumulation of acid-base substances that may occur during the fermentation process. Secondly, hyaluronic acid hydrogel accommodates these buffer components through physical adsorption and embedding, and its degradation products can also react with certain intermediates produced by microbial metabolism to form small molecules with biological activity. These substances can be used as signal molecules to feedback the metabolic pathways of microorganisms so that vitamin B12 is synthesized more efficiently. At the same time, nano vanadium oxide specifically binds to certain proteins or enzymes on the surface of microbial cells, changes the conformation and activity of these biomolecules, enhances the cell's ability to absorb nutrients and energy metabolism efficiency, and this combination cooperates with the buffering enhancement effect of 4-hydroxyethylpiperazineethanesulfonic acid to form a synergistic promotion network from the extracellular environment to intracellular metabolism. At the same time, polyvinyl pyrrolidone and the borax-boric acid buffer pair stabilize and modify the nano-vanadium oxide, preventing its aggregation and optimizing its interaction with other components. Finally, the hyaluronic acid hydrogel and polyvinyl pyrrolidone jointly provide physical support and mechanical property enhancement for the system, ensuring the structural integrity and functional stability of the buffer system during the fermentation process. The borax-boric acid buffer pair also interacts with other components to regulate the system's performance in multiple ways and maintain its overall stability. 2. The unique optimized nitrogen source combination of the present invention significantly improves the utilization efficiency of nitrogen sources. Yeast extract is rich in various growth factors and vitamins, providing abundant nutrients for the initial growth of microbial strains and promoting rapid cell proliferation. Ammonium sulfate, as a stable and easily absorbed inorganic nitrogen source, continuously provides nitrogen elements to the bacteria during the fermentation process, ensuring its basic growth and metabolic needs. The addition of silicon dioxide helps to improve the physical structure of the culture medium, increase its air permeability and water permeability, and facilitate full contact between the bacteria and nutrients. Bioactive peptides have high bioavailability and special biological activity, which can stimulate the metabolic pathways of microorganisms and enhance their resistance to environmental stress, thereby improving the synthesis capacity of vitamin B12 and fermentation yield. 3. Certain components of the yeast extract of the present invention can synergize with the bioactive peptides to activate specific gene expression in microorganisms and upregulate the expression of enzymes related to vitamin B12 synthesis. While ammonium sulfate provides nitrogen, the ammonium ions produced by its hydrolysis and other ions in the culture medium create an ionic environment that helps maintain the potential balance of the microbial cell membrane and promotes the transport of nutrients across the membrane. This effect, combined with the silica's ability to improve the culture medium's physical structure, enables the bacteria to more efficiently absorb and utilize nitrogen sources and other nutrients, thereby significantly improving the fermentation yield and quality of vitamin B12. 4. The present invention precisely controls the composite carbon source system in stages, so that microorganisms can obtain the most suitable carbon source supply at different fermentation stages. In the initial stage of fermentation, a higher proportion of glucose can quickly start the growth metabolism of the bacteria, laying a good foundation for the subsequent fermentation process; in the middle stage of fermentation, the reasonable combination of sucrose and maltose not only meets the energy demand for rapid growth of the bacteria, but also helps to maintain a relatively stable osmotic pressure environment, avoiding substrate inhibition caused by excessive carbon source concentration; in the late stage of fermentation, the ratio of the three sugars is flexibly adjusted according to the growth of the bacteria and the synthesis of vitamin B12, which can accurately regulate the metabolic flow of the bacteria, making it more inclined to the direction of vitamin B12 synthesis, further improving the efficiency of fermentation and the output of products, while reducing production costs; 5. The present invention establishes an online monitoring system and a neural network model to achieve intelligent and precise control of the fermentation process. By real-time monitoring of key parameters in the fermentation process, such as temperature, dissolved oxygen, pH value, bacterial concentration, substrate concentration, etc., the dynamic changes of the fermentation process can be grasped in a timely and accurate manner. The neural network model established by big data analysis and intelligent algorithms can be used to deeply mine and analyze these monitoring data, predict trends and abnormal conditions in the fermentation process, and automatically adjust fermentation conditions, such as temperature control, dissolved oxygen regulation, nutrient feeding, etc., to ensure that the fermentation process is always in the best operating state, improve the stability and reliability of fermentation, reduce the risk of fermentation failure due to manual operation errors and changes in environmental factors, and also provide strong technical support for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] 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 accompanying drawings, in which: Figure 1 The present invention provides a schematic flow chart of a method for improving the fermentation stability of vitamin B12 according to an embodiment of the present invention. DETAILED DESCRIPTION
[0017] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.
[0018] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although 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] A method for improving the fermentation stability of vitamin B12 in the present application is described below with reference to the accompanying drawings. In view of the poor fermentation stability mentioned in the above-mentioned background technology, the present application provides a method for improving the fermentation stability of vitamin B12, in which the buffer range of the system is broadened by 4-hydroxyethylpiperazineethanesulfonic acid and borax-boric acid buffering components, the overall stability is enhanced, and by the interaction of substances such as hyaluronic acid hydrogel and nano vanadium oxide, a synergistic promotion network from the extracellular environment to intracellular metabolism is formed, which effectively regulates the metabolic pathway of microorganisms. Secondly, the present invention uses a unique optimized nitrogen source combination, including yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides, which significantly improves the utilization efficiency of nitrogen source, promotes the rapid proliferation of microorganisms and efficient synthesis of vitamin B12. In addition, by accurately controlling the composite carbon source system in stages, microorganisms can obtain the most suitable carbon source supply at different fermentation stages, further improving fermentation efficiency and product yield. Finally, the present invention also establishes an online monitoring system and a neural network model, realizes intelligent and precise control of the fermentation process, and reduces the risk of fermentation failure caused by manual operation errors and changes in environmental factors. Thus, the problems of poor fermentation stability and low yield in related technologies are solved.
[0020] Specifically, Figure 1 This invention provides a method for improving the fermentation stability of vitamin B12.
[0021] like Figure 1 As shown, the method for improving the fermentation stability of vitamin B12 comprises the following steps: In step S101, a microbial strain producing vitamin B12 is obtained and activated.
[0022] The microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii, the activation temperature is 30-32° C., the pH value is 6.8-7.2, and the culture time is 12-16 hours.
[0023] It is understood that the present application examples obtain strains with good performance and potential in vitamin B12 fermentation production, which can efficiently synthesize vitamin B12 under suitable environments and are relatively adaptable to fermentation conditions, laying the foundation for subsequent fermentation processes. Through specific activation procedures, such as culturing in a suitable culture medium and controlling parameters such as temperature, pH, and incubation time, the strain is restored from a dormant state to a state with vigorous metabolic activity, so as to better carry out the subsequent fermentation process.
[0024] In step S102, the microbial strains are prepared into a culture medium suitable for vitamin B12 fermentation according to the complex carbon source system, optimized nitrogen source combination, key elements and other nutrients, three phytonutrients and modified buffers.
[0025] Among them, other nutrients include glutamate and trace elements, among which trace elements include iron, manganese and zinc.
[0026] It can be understood that the embodiments of the present application successfully formulated a culture medium suitable for vitamin B12 fermentation by combining microbial strains with a complex carbon source system, an optimized nitrogen source combination, key elements, other nutrients, and modified buffers through a carefully designed culture medium formula. This not only provides comprehensive and balanced nutritional support for microbial strains, but also significantly enhances the stability and efficiency of the fermentation system through the synergistic effect between the various components.
[0027] Specifically, the composite carbon source system ensures that microorganisms have access to an appropriate carbon source supply at different growth stages. The optimized nitrogen source combination improves nitrogen utilization efficiency, promoting rapid microbial proliferation and efficient vitamin B12 synthesis. Furthermore, the precise addition of key elements and the supplementation of other nutrients further enhance the quality and yield of fermentation products. The addition of modified buffers effectively broadens the system's buffering range, enhancing overall stability and providing a more stable environment for microbial growth and metabolism.
[0028] It's important to note that the "Plant Trio" includes 38 targeted intermediates, 32 more active premiums, and 28 nutritionally comprehensive gelatin. These ingredients are produced using advanced technologies such as microbial engineering, enzyme engineering, synthetic biology, and gene editing, utilizing patented strains through a combination of Triple-MAX fermentation, "Multi-Point" enzymatic oligopeptides, and "Bio-Synthesis" processes. Intermediate ingredients are targeted, allowing them to precisely target specific plant parts or physiological processes; premiums are more active, helping to improve plant growth performance and stress resistance; and gelatin is nutritionally comprehensive, providing a variety of essential nutrients for plant growth and development.
[0029] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 30-50 parts; Nano vanadium oxide: 5-15 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 20-40 parts; Polyvinylpyrrolidone: 5-10 parts Borax-boric acid buffer pair: 5-15 parts.
[0030] The average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0031] It is understandable that the present application embodiment enhances the buffering performance and overall stability of the fermentation system through multiple synergistic mechanisms. Wherein, 4-hydroxyethylpiperazineethanesulfonic acid and borax-boric acid buffer play complementary buffering effects in different pH ranges, broaden the buffering range of the whole system, effectively resist the accumulation of acid-base substances that may occur during the fermentation process. Secondly, hyaluronic acid hydrogel accommodates these buffer components by physical adsorption and embedding, and its degradation products can also react with certain intermediate products produced by microbial metabolism to form small molecules with biological activity, which can be used as signal molecules to feedback the metabolic pathways of microorganisms so that they can synthesize vitamin B12 more efficiently. Meanwhile, nano vanadium oxide specifically binds to certain proteins or enzymes on the surface of microbial cells, changes the conformation and activity of these biomolecules, enhances the cell's ability to absorb nutrients and energy metabolism efficiency, and this combination cooperates with the buffering enhancement effect of 4-hydroxyethylpiperazineethanesulfonic acid to form a synergistic promotion network from the extracellular environment to intracellular metabolism. Simultaneously, polyvinyl pyrrolidone and a borax-boric acid buffer pair stabilize and modify the nanovanadium oxide, preventing its aggregation and optimizing its interactions with other components. Finally, the hyaluronic acid hydrogel and polyvinyl pyrrolidone together provide physical support and mechanical enhancement to the system, ensuring the structural integrity and functional stability of the buffer system during fermentation. The borax-boric acid buffer pair also interacts with other components, modulating the system's performance in multiple ways and maintaining its overall stability.
[0032] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0033] Among them, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0034] It is understandable that the unique optimized nitrogen source combination of the embodiment of the present application significantly improves the utilization efficiency of the nitrogen source. Yeast paste is rich in various growth factors and vitamins, which provide rich nutrients for the initial growth of microbial strains and promote rapid cell proliferation; ammonium sulfate, as a stable and easily absorbed inorganic nitrogen source, continuously provides nitrogen elements to the bacteria during the fermentation process, ensuring its basic needs for growth and metabolism; the addition of silicon dioxide helps to improve the physical structure of the culture medium, increase its air permeability and water permeability, and facilitate full contact between the bacteria and nutrients; bioactive peptides have high bioavailability and special biological activity, which can stimulate the metabolic pathways of microorganisms and enhance their resistance to environmental stress, thereby improving the synthesis capacity of vitamin B12 and fermentation yield; Certain components of yeast extract work synergistically with bioactive peptides to activate specific gene expression in microorganisms and upregulate the expression of enzymes involved in vitamin B12 synthesis. While ammonium sulfate provides nitrogen, the ammonium ions produced by its hydrolysis interact with other ions in the culture medium to create an ionic environment that helps maintain the potential balance of the microbial cell membrane and promotes the transport of nutrients across the membrane. This effect, combined with the ability of silica to improve the physical structure of the culture medium, enables the bacteria to more efficiently absorb and utilize nitrogen and other nutrients, significantly improving the fermentation yield and quality of vitamin B12.
[0035] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 35%-40%, sucrose accounts for 20%-30% in the middle stage of fermentation, and maltose accounts for 10%-20%. In the late fermentation, the ratio of glucose, sucrose and maltose is maintained in the range of 10%-30%, 20%-40% and 10%-30% according to the growth of the bacteria and the synthesis of vitamin B12.
[0036] It is understandable that the embodiment of the present application achieves precise control of microbial strain growth and vitamin B12 synthesis by adopting a composite carbon source system comprising glucose, sucrose and maltose and accurately regulating their proportions at different stages of fermentation. In the initial stage of fermentation, a high proportion of glucose quickly starts the growth metabolism of the bacteria; in the middle stage, the combination of sucrose and maltose meets the energy demand for rapid growth of the bacteria while maintaining a stable osmotic pressure environment; in the later stage, the proportion of sugar is flexibly adjusted according to the growth of the bacteria and the synthesis of vitamin B12, and the metabolic flow is accurately regulated, thereby improving the fermentation efficiency and product yield.
[0037] In the embodiment of the present application, the precise addition of key elements includes: adding an inorganic cobalt source, cobalt chloride, in the early stage of fermentation, and adding an organic cobalt source in the middle and late stages of fermentation.
[0038] Among them, organic cobalt sources include cobalt vitamins and cobalt amino acid chelates.
[0039] It is understandable that the embodiments of the present application effectively promote the synthesis process of vitamin B12 by accurately adding the key element cobalt, specifically using inorganic cobalt source cobalt chloride in the early stage of fermentation, and switching to adding organic cobalt sources in the middle and late stages of fermentation. Inorganic cobalt sources provide microorganisms with rapidly available cobalt resources in the early stage, which helps to start the biosynthetic pathway of vitamin B12; and the addition of organic cobalt sources in subsequent stages further supports the efficient synthesis of vitamin B12 due to its good bioavailability and stability. This method of phased and precise addition of cobalt sources not only improves the utilization efficiency of the cobalt element, but also significantly enhances the production efficiency and product purity of vitamin B12.
[0040] In step S103, the fermentation conditions are precisely controlled in stages according to the fermentation stage.
[0041] Among them, the fermentation conditions include fermentation temperature and dissolved oxygen level. The fermentation temperature is 25-35°C, and the dissolved oxygen level is 10-30%.
[0042] It is understood that the embodiments of the present application significantly optimize the growth environment and metabolic process of the microbial strains by precisely controlling fermentation conditions, such as fermentation temperature and dissolved oxygen level, according to the fermentation stage, ensuring that the microorganisms can obtain the most suitable conditions at different growth stages, thereby improving the synthesis efficiency of vitamin B12 and the quality of the product. By precisely controlling the fermentation conditions, the embodiments of the present application not only achieve refined management of the fermentation process, but also reduce production costs and improve overall production efficiency.
[0043] During the initial fermentation phase, the fermentation temperature is typically controlled within a range suitable for rapid bacterial growth, typically 30-32°C. This is during the logarithmic growth phase, and higher temperatures are beneficial for accelerating bacterial growth and metabolic rates. At the same time, proper stirring and aeration maintain a certain dissolved oxygen level, ensuring that the bacteria have sufficient oxygen for aerobic respiration during growth, providing ample energy for growth and metabolism.
[0044] For example, in the fermentation experiment, when the fermentation temperature was maintained at 31 ° C, after 8 hours of culture, the bacterial concentration of Propionibacterium sieversii increased by about 30% compared to the culture conditions at 28 ° C, which fully demonstrated the promoting effect of suitable high temperature on the rapid proliferation of bacteria in the early stage. At the same time, through proper stirring and ventilation, a certain dissolved oxygen level is maintained to ensure that the bacteria have enough oxygen for aerobic respiration during the growth process, providing sufficient energy for the growth and metabolism of the bacteria. In a 50L fermenter, the initial ventilation volume is set to 0.5vvm and the stirring speed is 200rpm, so that the dissolved oxygen saturation in the fermentation liquid can be maintained at about 30%, which meets the oxygen demand of the initial growth of the bacteria, the bacteria grow vigorously, and various metabolic indicators are normal.
[0045] During the middle stages of fermentation, as bacterial populations increase and metabolic activity intensifies, the fermentation temperature may be lowered to 28-30°C to prevent excessive heat generation from overgrowth, which could affect bacterial growth and vitamin B12 synthesis. Dissolved oxygen levels are also precisely adjusted based on bacterial growth and metabolic needs. Increased aeration and agitation speed are used to ensure that dissolved oxygen meets the bacterial needs and maintain a healthy fermentation state.
[0046] For example, in a fermentation process of 10 consecutive batches, adjusting the temperature to 29°C mid-fermentation in 7 of them resulted in an average 15% increase in vitamin B12 synthesis compared to the unadjusted temperature. Furthermore, bacterial aging was significantly slowed, leading to a more stable fermentation process. Dissolved oxygen levels are also precisely adjusted based on bacterial growth and metabolic needs. By increasing aeration and stirring speed, the dissolved oxygen level is ensured to meet the bacterial needs and maintain a favorable fermentation state. For example, when the bacterial concentration reaches a certain threshold and increased metabolic activity causes the dissolved oxygen saturation to drop below 20%, the aeration rate is gradually increased to 0.8 vvm and the stirring speed is increased to 300 rpm. The dissolved oxygen level quickly recovers and stabilizes between 25% and 30%, allowing bacterial growth and vitamin B12 synthesis to proceed smoothly.
[0047] In the later stage of fermentation, the control of fermentation temperature and dissolved oxygen level is more critical, and needs to be fine-tuned according to the growth of the bacteria and the synthesis of vitamin B12, and the fermentation conditions should be further optimized so that the bacteria can use more energy and substances for the synthesis and accumulation of vitamin B12, thereby improving the yield and quality of the product.
[0048] For example, in industrial fermentation production, in the later stage of fermentation, the fermentation temperature is precisely controlled at 27°C and the dissolved oxygen saturation is maintained at around 20% based on the growth rate of the bacteria, the activity of vitamin B12 synthase, and the substrate consumption. Ultimately, the vitamin B12 production is increased by 20% compared to conventional fermentation conditions, and the product quality also meets higher standards.
[0049] In step S104, an online monitoring system is established to monitor key parameters in the fermentation process in real time, and a neural network model is established using big data analysis and intelligent algorithms to ensure the stability and efficiency of the fermentation process.
[0050] It is understood that the embodiments of the present application, by establishing an online monitoring system and integrating big data analysis and intelligent algorithms to build a neural network model, achieve real-time monitoring and accurate prediction of key parameters of the fermentation process, greatly improving the stability and efficiency of the fermentation process and ensuring the quality control and cost optimization of vitamin B12 production. Through intelligent management, not only can potential production problems be discovered and resolved in a timely manner, but also dynamic adjustment of fermentation conditions can be achieved, further promoting the intelligent upgrading of vitamin B12 fermentation technology.
[0051] The present invention will be further described below with reference to the following examples.
[0052] Example 1 The present invention provides a method for improving the fermentation stability of vitamin B12. The method comprises the following steps: obtaining a microbial strain producing vitamin B12 and activating the strain, wherein the activation temperature is 30°C, the pH value is 7.0, and the incubation time is 12 hours; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements, and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor key parameters in the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0053] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 30 parts; Nano vanadium oxide: 5 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 20 parts; Polyvinylpyrrolidone: 5 parts Borax-boric acid buffer pair: 5 parts.
[0054] In the embodiment of the present application, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0055] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0056] In the examples of the present application, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0057] In the embodiment of the present application, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0058] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 35%, sucrose accounts for 20% in the middle stage of fermentation, and maltose accounts for 10%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained at 10%, 20% and 10% according to the growth of the bacteria and the synthesis of vitamin B12.
[0059] In the embodiments of the present application, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0060] In the embodiment of the present application, other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0061] In the examples of the present application, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0062] Example 2 The present invention provides a method for improving the fermentation stability of vitamin B12, which includes the following steps: obtaining a microbial strain of vitamin B12 and activating it, wherein the activation temperature is 31°C, the pH value is 7.0, and the incubation time is 13 hours; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements, and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor key parameters in the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0063] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 35 parts; Nano vanadium oxide: 7 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 25 parts; Polyvinylpyrrolidone: 6 parts Borax-boric acid buffer pair: 7 parts.
[0064] In the embodiment of the present application, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0065] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0066] In the examples of the present application, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0067] In the embodiment of the present application, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0068] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 36%, sucrose accounts for 22% in the middle stage of fermentation, and maltose accounts for 12%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained at 15%, 25% and 15% according to the growth of the bacteria and the synthesis of vitamin B12.
[0069] In the embodiments of the present application, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0070] In the embodiment of the present application, other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0071] In the examples of the present application, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0072] Example 3 The present invention provides a method for improving the fermentation stability of vitamin B12, which includes the following steps: obtaining a microbial strain of vitamin B12 and activating it, wherein the activation temperature is 32°C, the pH value is 7.0, and the incubation time is 14 hours; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements, and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor key parameters in the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0073] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 40 parts; Nano vanadium oxide: 10 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 30 parts; Polyvinylpyrrolidone: 7 parts Borax-boric acid buffer pair: 10 parts.
[0074] In the embodiment of the present application, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0075] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0076] In the examples of the present application, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0077] In the embodiment of the present application, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0078] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 37%, sucrose accounts for 25% in the middle stage of fermentation, and maltose accounts for 15%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained at 20%, 30% and 20% according to the growth of the bacteria and the synthesis of vitamin B12.
[0079] In the embodiments of the present application, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0080] In the embodiment of the present application, other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0081] In the examples of the present application, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0082] Example 4 The present invention provides a method for improving the fermentation stability of vitamin B12, which includes the following steps: obtaining a microbial strain of vitamin B12 and activating it, wherein the activation temperature is 32°C, the pH value is 7.2, and the incubation time is 15 hours; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements, and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor key parameters in the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0083] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 45 parts; Nano vanadium oxide: 12 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 35 parts; Polyvinylpyrrolidone: 9 parts Borax-boric acid buffer pair: 12 parts.
[0084] In the embodiment of the present application, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0085] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0086] In the examples of the present application, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0087] In the embodiment of the present application, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0088] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 38%, sucrose accounts for 28% in the middle stage of fermentation, and maltose accounts for 18%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained at 25%, 35% and 25% according to the growth of the bacteria and the synthesis of vitamin B12.
[0089] In the embodiments of the present application, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0090] In the embodiment of the present application, other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0091] In the examples of the present application, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0092] Example 5 The present invention provides a method for improving the fermentation stability of vitamin B12, which includes the following steps: obtaining a microbial strain of vitamin B12 and activating it, wherein the activation temperature is 2°C, the pH value is 7.2, and the incubation time is 16 hours; preparing a culture medium suitable for vitamin B12 fermentation based on a composite carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements, and a modified buffer; accurately controlling the fermentation conditions in stages according to the fermentation stage; establishing an online monitoring system to monitor key parameters in the fermentation process in real time, and using big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
[0093] In the embodiment of the present application, the modified buffer comprises the following components in parts by weight: Hyaluronic acid hydrogel: 50 parts; Nano vanadium oxide: 15 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 40 parts; Polyvinylpyrrolidone: 5-10 parts Borax-boric acid buffer pair: 15 parts.
[0094] In the embodiment of the present application, the average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:1.
[0095] In an embodiment of the present application, optimizing the nitrogen source combination includes: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination includes yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
[0096] In the examples of the present application, the ratio of yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:1.
[0097] In the embodiment of the present application, the microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
[0098] In the embodiment of the present application, the composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 40%, sucrose accounts for 30% in the middle stage of fermentation, and maltose accounts for 20%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained at 30%, 40% and 30% according to the growth of the bacteria and the synthesis of vitamin B12.
[0099] In the embodiments of the present application, the precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
[0100] In the embodiment of the present application, other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
[0101] In the examples of the present application, the fermentation conditions include fermentation temperature and dissolved oxygen level.
[0102] Comparative Example 1 This comparative example provides a method for improving the fermentation stability of vitamin B12. The only difference from Example 1 is that the modified buffer does not contain hyaluronic acid hydrogel and nano-vanadium oxide, and the reduced amount of hyaluronic acid hydrogel and nano-vanadium oxide is distributed to 4-hydroxyethylpiperazineethanesulfonic acid. The remaining components, component contents, and preparation methods are the same as those in Example 1.
[0103] Comparative Example 2 This comparative example provides a method for improving the fermentation stability of vitamin B12. The only difference from Example 1 is that the novel nitrogen source combination does not contain silicon dioxide and bioactive peptides, and the reduced amounts of silicon dioxide and bioactive peptides are distributed to the yeast extract. The remaining components, component contents, and preparation methods are the same as those in Example 1.
[0104] Comparative Example 3 This comparative example provides a method for improving the fermentation stability of vitamin B12. The only difference between the method and Example 1 is that the modified buffer does not contain hyaluronic acid hydrogel and nano-vanadium oxide, the new nitrogen source combination does not contain silicon dioxide and bioactive peptides, the reduced amounts of hyaluronic acid hydrogel and nano-vanadium oxide are distributed to 4-hydroxyethylpiperazineethanesulfonic acid, and the reduced amounts of silicon dioxide and bioactive peptides are distributed to the yeast extract. The remaining components, component contents, and preparation methods are the same as those in Example 1.
[0105] Performance Testing The methods for improving the fermentation stability of vitamin B12 prepared in Examples 1-5 and Comparative Examples 1-3 were used to accurately measure the vitamin B12 content using high-performance liquid chromatography. The vitamin B12 content in the samples was calculated using specific chromatographic conditions and a standard curve. A precision pH meter was used to set multiple measurement points at different locations in the fermenter, and the pH data was recorded. The bacterial growth density was measured at a specific wavelength using an ultraviolet spectrophotometer. The results are shown in Table 1 below.
[0106] Table 1 Test results
[0107] As shown in Table 1, the test results of Examples 1-5 and Comparative Examples 1-3 in terms of pH stability, vitamin B12 production, and bacterial growth density are shown, which reflect the advantages of the fermentation conditions of the present invention and the relationship between various factors.
[0108] In terms of pH stability, the pH fluctuation range in Examples 1-5 gradually widened from 6.8-7.2 in Example 1 to 6.4-7.6 in Example 5, but remained within the optimal range for microbial growth and vitamin B12 synthesis. This is attributed to the effect of changes in the content of the modified buffer components on the buffer system. Overall, the pH stability in the Examples is positively correlated with the fermentation effect. The narrow pH fluctuation range is beneficial for maintaining enzyme activity within microbial cells, thereby promoting bacterial growth and vitamin B12 synthesis. This relationship is demonstrated by the comparison between Example 1 and Example 4. However, in Comparative Examples 1-3, the buffer system was disrupted due to changes in fermentation conditions, making it difficult to control the accumulation of acidic and alkaline substances, resulting in a significantly wider pH fluctuation range and decreased stability.
[0109] In terms of vitamin B12 production, Examples 1-5 showed a trend of first increasing and then slightly decreasing. In the early stage, due to the reasonable configuration of nutrients and the optimization of fermentation conditions, the ability of microbial growth and synthesis of vitamin B12 was enhanced, and the yield increased. It reached a maximum of 170 mg / L in Example 4 and then decreased slightly. It may be limited by factors such as the proportion of ingredients, but it is higher than the comparative example as a whole. The yield is closely related to the cell growth density. In the examples, as the cell growth density increases, the number of cells involved in synthesis increases, and the yield increases accordingly. However, in the comparative example, due to changes in fermentation conditions, the growth and metabolism of microorganisms are affected, the number of cells involved in synthesis is reduced, the metabolic environment is destroyed, and the synthesis efficiency is reduced. The yield is significantly lower than that of the examples.
[0110] In terms of bacterial growth density, the bacterial growth density (OD value) of Examples 1-5 first increased from 2.0 to 2.8 (Example 4) and then decreased to 2.6 (Example 5). This is closely related to the nutrient supply (energy provided by the complex carbon source system, nitrogen provided by the optimized nitrogen source combination) and the stable pH environment in the fermentation system. The rational combination of various components can achieve optimal bacterial growth. This trend is consistent with vitamin B12 production, highlighting the importance of bacterial growth to production. Comparative Examples 1-3, due to changes in key components or combinations, disrupted the favorable conditions for bacterial growth, resulting in a lower bacterial growth density than the examples, which affected vitamin B12 synthesis. This negatively confirms the role of the fermentation conditions of the present invention in promoting bacterial growth.
[0111] The present application embodiment proposes a method for improving the fermentation stability of vitamin B12, in which the buffer range of the system is broadened by 4-hydroxyethylpiperazineethanesulfonic acid and borax-boric acid buffering components, the overall stability is enhanced, and by the interaction of substances such as hyaluronic acid hydrogel and nano vanadium oxide, a synergistic promotion network from the extracellular environment to intracellular metabolism is formed, which effectively regulates the metabolic pathways of microorganisms. Secondly, the present invention adopts a unique optimized nitrogen source combination, including yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides, which significantly improves the utilization efficiency of nitrogen source, promotes the rapid proliferation of microorganisms and the efficient synthesis of vitamin B12. In addition, by accurately controlling the composite carbon source system in stages, microorganisms can obtain the most suitable carbon source supply at different fermentation stages, further improving fermentation efficiency and product yield. Finally, the present invention also establishes an online monitoring system and a neural network model, realizes intelligent and precise control of the fermentation process, and reduces the risk of fermentation failure caused by manual operation errors and environmental factors. Thus, the problems such as poor fermentation stability and low yield in the related art are solved.
[0112] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.
Claims
1. A method for improving the fermentation stability of vitamin B12, characterized in that: The method comprises the following steps: Obtaining and activating microbial strains of vitamin B12; The microbial strain is prepared into a culture medium suitable for vitamin B12 fermentation according to a complex carbon source system, an optimized nitrogen source combination, key elements and other nutrients, three plant elements and a modified buffer; According to the fermentation stage, the fermentation conditions can be precisely controlled in stages; Establish an online monitoring system to monitor key parameters in the fermentation process in real time, and use big data analysis and intelligent algorithms to establish a neural network model to ensure the stability and efficiency of the fermentation process.
2. A method for improving the fermentation stability of vitamin B12 according to claim 1, characterized in that: The modified buffer comprises, by weight: Hyaluronic acid hydrogel: 30-50 parts; Nano vanadium oxide: 5-15 parts; 4-Hydroxyethylpiperazineethanesulfonic acid: 20-40 parts; Polyvinylpyrrolidone: 5-10 parts Borax-boric acid buffer pair: 5-15 parts.
3. A method for improving the fermentation stability of vitamin B12 according to claim 2, characterized in that: The average particle size of the nano-vanadium oxide is 10-50 nm, and the mass ratio of the borax-boric acid buffer pair is 1:
1.
4. The method for improving the fermentation stability of vitamin B12 according to claim 1, wherein: The optimized nitrogen source combination comprises: preparing a novel nitrogen source combination, wherein the novel nitrogen source combination comprises yeast extract, ammonium sulfate, silicon dioxide and bioactive peptides.
5. A method for improving the fermentation stability of vitamin B12 according to claim 4, characterized in that: The ratio of the yeast extract, ammonium sulfate, silicon dioxide and bioactive peptide is 4:3:2:
1.
6. The method for improving the fermentation stability of vitamin B12 according to claim 1, wherein: The microbial strain is Propionibacterium sieversii or Propionibacterium freudenreichii.
7. The method for improving the fermentation stability of vitamin B12 according to claim 1, wherein: The composite carbon source system includes glucose, sucrose and maltose. In the initial stage of fermentation, glucose accounts for 35%-40%, in the middle stage of fermentation, sucrose accounts for 20%-30%, and maltose accounts for 10%-20%. In the late stage of fermentation, the ratio of glucose, sucrose and maltose is maintained in the range of 10%-30%, 20%-40% and 10%-30% according to the growth of bacteria and the synthesis of vitamin B12.
8. The method for improving the fermentation stability of vitamin B12 according to claim 1, wherein: The precise addition of key elements includes: adding inorganic cobalt source cobalt chloride in the early stage of fermentation, adding organic cobalt source in the middle and late stages of fermentation, and the organic cobalt source includes cobalt vitamin and cobalt amino acid chelate.
9. The method for improving the fermentation stability of vitamin B12 according to claim 1, wherein: The other nutrients include glutamic acid and trace elements, wherein the trace elements include iron, manganese and zinc.
10. The method for improving the fermentation stability of vitamin B12 according to claim 7, characterized in that: The fermentation conditions include fermentation temperature and dissolved oxygen level.