Method for producing vitamin B12 through fermentation of propionibacterium shermannii

Abstract

The invention relates to the technical field of biology, in particular to a method for producing vitamin B12 through fermentation of propionibacterium shermangii, which comprises the following steps: inoculating propionibacterium shermangii into a seed culture medium to culture a seed solution; inoculating the seed solution into a fermentation culture medium for fermentation; after fermentation is finished, thalli are removed in a centrifugal separation or filtration mode, a fermentation solution is subjected to ultrafiltration concentration, a concentrated solution is purified through ion exchange chromatography or affinity chromatography, and a vitamin B12 crude product is obtained; and further purifying by adopting a crystallization method to obtain a high-purity vitamin B12 finished product. According to the method for producing the vitamin B12 through fermentation of the propionibacterium shermannii, the yield and the purity of the vitamin B12 are remarkably improved and the production cost is reduced by accurately controlling fermentation conditions, optimizing culture medium components, introducing an intelligent regulation and control system and adopting a specific purification process, so that the method is suitable for industrial production. The problems of low yield, unstable purity, difficult production process control and the like of the vitamin B12 in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for producing vitamin B12 by fermenting Propionibacterium shermanii. Background Art

[0002] Vitamin B12 plays a vital role in the human body. It is not only essential for maintaining a healthy nervous system, but is also directly involved in key physiological processes such as red blood cell production. Traditional methods of producing vitamin B12 mainly include chemical synthesis and microbial fermentation. However, chemical synthesis is significantly limited by environmental pollution and high costs. In contrast, microbial fermentation has gained widespread popularity due to its environmental friendliness and relatively low cost. Despite this, the yield and purity of microbial fermentation are still affected by multiple factors such as strain selection, optimization of fermentation conditions, and purification process design, resulting in significant differences in yield and purity between different production batches.

[0003] In recent years, Propionibacterium shigellii has garnered significant attention for its ability to efficiently synthesize vitamin B12. However, existing fermentation methods based on this bacterium still face numerous challenges, such as relatively low yields, difficulties ensuring product purity, and difficulties in effectively controlling the production process. These issues severely restrict the industrial production and widespread application of vitamin B12. Summary of the Invention

[0004] The present application provides a method for producing vitamin B12 by fermenting Propionibacterium sieversii, so as to solve the problems of low vitamin B12 yield, unstable purity and difficult production process control in the related art.

[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 producing vitamin B12 by fermentation using Propionibacterium sieversii, the method comprising: inoculating Propionibacterium sieversii into a seed culture medium, adding 0.5-2 g / L of phytotriene, and culturing for 18-24 hours at a temperature of 30-35°C and a pH of 6.5-7.2 to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium at an inoculum amount of 5%-10%, and fermenting for 96-120 hours at a temperature of 32-37°C and a pH of 6.8-7.5; after the fermentation is completed, removing the bacteria by centrifugation or filtration, and ultrafiltration and concentration of the fermentation broth, purifying the concentrate by ion exchange chromatography or affinity chromatography to obtain a crude vitamin B12 product; crystallizing the crude vitamin B12 product, and further purifying it by crystallization to obtain a high-purity vitamin B12 finished product.

[0006] Furthermore, before obtaining the seed liquid, it includes: obtaining the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products; and automatically regulating the dynamic supplementation amount of carbon source, nitrogen source and vitamin precursors in the culture medium through an intelligent algorithm according to the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products.

[0007] Furthermore, the key nutrient concentrations include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic byproducts include organic acids, wherein the organic acid is propionic acid.

[0008] Furthermore, before the fermentation ends, the method further includes: obtaining the bacterial growth density.

[0009] Furthermore, the seed culture medium contains a carbon source, a nitrogen source, an inorganic salt and a growth factor, wherein the carbon source includes one or more of glucose, sucrose and fructose, the nitrogen source includes one or more of yeast extract, peptone and urea, the inorganic salt includes one or more of magnesium sulfate, potassium dihydrogen phosphate and calcium chloride, and the growth factor includes a cobalt ion source, and the concentration of the cobalt ion source is 0.5-2 mg / L.

[0010] Furthermore, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is selected from one or more of glucose, maltose, and starch hydrolyzate, the nitrogen source is selected from one or more of fish meal, soybean meal, and corn steep liquor, the inorganic salt includes one or more of magnesium sulfate, ferrous sulfate, and manganese sulfate, the vitamin precursor includes one or more of 5,6-dimethylbenzimidazole, adenine, and guanine, and the amount of the vitamin precursor added is 0.1-0.5 g / L, the defoaming agent is selected from one or more of polyether defoaming agents and silicone defoaming agents, and during the fermentation process, the dissolved oxygen concentration is controlled at 20%-30%.

[0011] Furthermore, the three plant elements are at least one of the biocolloids, wherein the preparation process of the three plant elements includes: through the Triple-MAX triple fermentation process, fermentation is carried out in sequence under aerobic, anaerobic and deep fermentation conditions to obtain a fermentation basic product; the fermentation basic product is subjected to a "Multi-Point" enzymatic oligopeptide process, enzyme preparations are added, and multi-point enzymatic cleavage reaction is carried out under mild reaction conditions; the "Bio-Synthesis" process is adopted, and the microbial metabolic pathway is regulated by genes to synthesize and transform the three plant elements; wherein, in the Triple-MAX triple fermentation process, the aerobic fermentation temperature is 28-32°C, the pH value is 6.5-7.2, and the time is 36-48 hours; the anaerobic fermentation temperature is 30-35°C, the pH value is 6.8-7.5, and the time is 48-60 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 24-36 hours.

[0012] Furthermore, the crystallization treatment temperature is 4-8°C and the time is 12-24 hours. The ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin. The affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

[0013] Furthermore, the Propionibacterium sieversii and Bacillus pumilus are co-cultured, wherein the inoculation ratio of the Bacillus pumilus to the Propionibacterium sieversii is 1:(5-10).

[0014] The present invention also provides a device for producing vitamin B12 by fermenting Propionibacterium sieversii, comprising: an acquisition module for inoculating Propionibacterium sieversii into a seed culture medium, adding 0.5-2 g / L of phytotriazine, and culturing for 18-24 hours at a temperature of 30-35°C and a pH of 6.5-7.2 to obtain a seed liquid; a fermentation module for inoculating the seed liquid into a fermentation culture medium at an inoculum rate of 5%-10%, adding 1-5 g / L of phytotriazine, and fermenting for 96-120 hours at a temperature of 32-37°C and a pH of 6.8-7.5; a purification module for removing the bacteria by centrifugation or filtration after the fermentation is completed, and ultrafiltration and concentration of the fermentation liquid, and purifying the concentrate by ion exchange chromatography or affinity chromatography to obtain a crude vitamin B12 product; and a crystallization module for crystallizing the crude vitamin B12 product and further purifying it by crystallization to obtain a high-purity finished vitamin B12 product.

[0015] The present invention also provides a system for producing vitamin B12 by fermenting Propionibacterium schreiberi. The system includes the above-mentioned device for producing vitamin B12 by fermenting Propionibacterium schreiberi.

[0016] The beneficial effects achieved by adopting the above-mentioned present invention are as follows: 1. The present invention has prepared a device for producing vitamin B12 by fermenting Propionibacterium shiehii. During the seed culture and fermentation process, online sensors and intelligent algorithms are introduced to monitor bacterial growth density, key nutrient concentrations, and the accumulation of metabolic byproducts in real time. Based on this information, the supplementation of carbon source, nitrogen source, and vitamin precursors in the culture medium is dynamically regulated. This ensures that Propionibacterium shiehii obtains appropriate nutritional conditions at each growth stage, preventing the adverse effects of nutrient deficiency or excess on bacterial growth and vitamin B12 synthesis. 2. The present invention selects Propionibacterium sieversii and Bacillus pumilus for co-cultivation. Bacillus pumilus plays a positive promoting role in the co-cultivation system. The various beneficial substances it secretes, such as proteases and growth factors, provide a more favorable growth environment for Propionibacterium sieversii. Proteases help to decompose large molecular proteins in the culture medium and release more nitrogen sources that can be used by Propionibacterium sieversii; growth factors can stimulate the expression of genes related to vitamin B12 synthesis in Propionibacterium sieversii and enhance its synthesis capacity. Through this synergistic effect, the production of vitamin B12 can be further improved on the basis of intelligent regulation, and at the same time, the product quality is further optimized and the impurity content is reduced; 3. The post-fermentation extraction and purification process of the present invention utilizes a series of techniques, including centrifugation, ultrafiltration concentration, ion exchange chromatography or affinity chromatography, and crystallization. Centrifugation or filtration effectively removes bacterial impurities, ultrafiltration concentration enriches vitamin B12, ion exchange chromatography and affinity chromatography achieve highly selective purification based on different principles, and crystallization further removes residual impurities. The synergistic effect of these techniques allows for highly purified crude vitamin B12, ultimately yielding a high-purity finished vitamin B12 product. 4. The online monitoring and intelligent control system of the present invention ensures precise control of key fermentation conditions, reduces the impact of environmental and nutrient fluctuations, ensures fermentation stability and reliability, and facilitates quality control and management of industrial large-scale production. Secondly, intelligent control reduces raw material waste and improves raw material utilization; shortens the fermentation cycle, reduces energy consumption and labor costs; and the optional resource utilization of bacterial waste further improves resource utilization efficiency, reduces production costs, and enhances product market competitiveness. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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 A flow chart of a method for producing vitamin B12 by fermentation using Propionibacterium sieversii provided in one embodiment of the present invention; Figure 2This is a schematic diagram of an apparatus for producing vitamin B12 by fermentation using Propionibacterium sieversii, provided in one embodiment of the present invention. DETAILED DESCRIPTION

[0018] 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.

[0019] 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.

[0020] The following describes a method for producing vitamin B12 by fermenting Propionibacterium sieversii according to an embodiment of the present application with reference to the accompanying drawings. In response to the problem of low yield mentioned in the above background technology, the present application provides a method for producing vitamin B12 by fermenting Propionibacterium sieversii, in which the intelligent control technology and optimized production process are used to achieve precise control of the vitamin B12 production process. The yield and purity are significantly improved through online monitoring and intelligent adjustment of the culture medium components; at the same time, the optimized separation and purification process ensures the high quality of the product. In addition, intelligent control also shortens the fermentation cycle, reduces costs, and improves resource utilization efficiency through resource utilization of fermentation waste, achieving a dual improvement in environmental friendliness and economic benefits. As a result, the problems of low vitamin B12 yield, unstable purity and difficult production process control in the related technology are solved.

[0021] Specifically, Figure 1 The present invention provides a method for producing vitamin B12 by fermenting Propionibacterium sieversii.

[0022] like Figure 1 As shown, the method for producing vitamin B12 by fermentation using Propionibacterium sieversii comprises the following steps: In step S101, Propionibacterium sieversii is inoculated into a seed culture medium, 0.5-2 g / L of phytotriazine is added, and the culture is carried out at a temperature of 30-35° C. and a pH of 6.5-7.2 for 18-24 hours to obtain a seed solution.

[0023] The seed culture medium includes a carbon source, a nitrogen source, an inorganic salt and a growth factor, wherein the carbon source includes one or more of glucose, sucrose and fructose, the nitrogen source includes one or more of yeast extract, peptone and urea, the inorganic salt includes one or more of magnesium sulfate, potassium dihydrogen phosphate and calcium chloride, and the growth factor contains a cobalt ion source, and the concentration of the cobalt ion source is 0.5-2 mg / L.

[0024] It can be understood that the embodiment of the present application successfully obtained high-quality seed liquid by culturing Propionibacterium sieversii in a seed culture medium under specific conditions. The process was carried out for 18-24 hours under an optimized environment of a temperature of 30-35°C and a pH value of 6.5-7.2, which effectively promoted the growth and reproduction of the bacteria and laid a solid foundation for the subsequent efficient production of vitamin B12.

[0025] In an embodiment of the present application, before obtaining the seed liquid, the steps include: obtaining the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products; and automatically controlling the dynamic supplementation of carbon sources, nitrogen sources and vitamin precursors in the culture medium through an intelligent algorithm based on the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products.

[0026] Among them, the concentrations of key nutrients include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic by-products include organic acids, among which the organic acid is propionic acid.

[0027] It can be understood that the embodiments of the present application monitor the growth density of the bacterial cell, the concentration of key nutrients and the accumulation of metabolic by-products in real time, and use intelligent algorithms based on these data to dynamically control the composition of the culture medium, accurately supplement the carbon source, nitrogen source and vitamin precursors, thereby optimizing the fermentation environment, improving the production efficiency and product quality of vitamin B12, and realizing intelligent and refined control of the production process.

[0028] In the examples of the present application, Propionibacterium sieversii and Bacillus pumilus are co-cultured, wherein the inoculation ratio of Bacillus pumilus to Propionibacterium sieversii is 1:(5-10).

[0029] It can be understood that the embodiment of the present application effectively promotes the biosynthesis pathway of vitamin B12, improves production efficiency and yield, and may optimize the fermentation environment through interactions between microorganisms by introducing Bacillus pumilus and Propionibacterium sieversii for co-cultivation and regulating them at a specific inoculation ratio.

[0030] In the embodiments of the present application, the phytotrium is at least one of the biocolloids, wherein the preparation process of the phytotrium includes: fermenting under aerobic, anaerobic and deep fermentation conditions in sequence through the Triple-MAX triple fermentation process to obtain a fermentation basic product; subjecting the fermentation basic product to a "Multi-Point" enzymatic oligopeptide process, adding enzyme preparations, and performing multi-point enzymatic cleavage reactions under mild reaction conditions; and adopting the "Bio-Synthesis" process to utilize genes to regulate microbial metabolic pathways to synthesize and transform the phytotrium.

[0031] Among them, in the Triple-MAX triple fermentation process, the aerobic fermentation temperature is 28-32°C, the pH value is 6.5-7.2, and the time is 36-48 hours; the anaerobic fermentation temperature is 30-35°C, the pH value is 6.8-7.5, and the time is 48-60 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 24-36 hours.

[0032] It is understandable that the examples of this application utilize a combination of three processes: Triple-MAX glycolysis, "Multi-Point" enzymatic oligopeptides, and "Bio-Synthesis" to prepare plant tri-nutrients. Triple-MAX glycolysis sequentially enhances microbial activity and accumulates metabolites through different fermentation conditions; "Multi-Point" enzymatic oligopeptides utilize enzyme preparations under mild conditions to break down macromolecules into easily absorbed oligopeptides, optimizing nutritional content and enhancing biological activity; and "Bio-Synthesis" utilizes genetically regulated microbial metabolic pathways to precisely synthesize and transform plant tri-nutrient components with multiple functions, thereby improving product purity, stability, and production efficiency.

[0033] In step S102, the seed liquid is inoculated into the fermentation medium at an inoculum amount of 5%-10%, 1-5 g / L of phytotriazine is added, and fermentation is carried out at a temperature of 32-37°C and a pH value of 6.8-7.5 for 96-120 hours.

[0034] The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is selected from one or more of glucose, maltose and starch hydrolysate, the nitrogen source is selected from one or more of fish meal, soybean meal and corn steep liquor, the inorganic salt includes one or more of magnesium sulfate, ferrous sulfate and manganese sulfate, the vitamin precursor includes one or more of 5,6-dimethylbenzimidazole, adenine and guanine, and the amount of the vitamin precursor added is 0.1-0.5 g / L, the defoaming agent is selected from one or more of polyether defoaming agents and silicone defoaming agents, and during the fermentation process, the dissolved oxygen concentration is controlled at 20%-30%.

[0035] It can be understood that the embodiments of the present application effectively promote the synthesis and accumulation of vitamin B12 by inoculating the seed liquid into the fermentation medium at an appropriate inoculation ratio and conducting a long-term fermentation under optimized temperature and pH conditions.

[0036] In step S103, after the fermentation is completed, the bacteria are removed by centrifugation or filtration, and the fermentation liquid is concentrated by ultrafiltration. The concentrate is purified by ion exchange chromatography or affinity chromatography to obtain crude vitamin B12.

[0037] The ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin, and the affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

[0038] It is understandable that, in the embodiment of the present application, after the fermentation is completed, the bacteria are removed by centrifugation or filtration, and then the fermentation broth is efficiently purified by ultrafiltration concentration and ion exchange chromatography or affinity chromatography to successfully obtain crude vitamin B12.

[0039] In the embodiment of the present application, before the fermentation ends, the process further includes: obtaining the bacterial growth density.

[0040] In step S104, the crude vitamin B12 is crystallized and further purified by a crystallization method to obtain a high-purity finished vitamin B12 product.

[0041] The crystallization treatment temperature is 4-8°C and the time is 12-24 hours.

[0042] It can be understood that the embodiments of the present application successfully produced a high-purity vitamin B12 finished product by crystallizing the crude vitamin B12 and deeply purifying it using the crystallization method, which greatly improved the purity of the product, met the strict requirements of high-end fields such as medicine for vitamin B12, and ensured the quality and market competitiveness of the product.

[0043] The present invention will be further described below with reference to the following examples.

[0044] Example 1 The present invention provides a method for producing vitamin B12 by fermenting Propionibacterium sievertii. The method comprises: inoculating Propionibacterium sievertii into a seed culture medium, adding 0.5 g / L of phytotriazine, and culturing the culture medium at a temperature of 30° C. and a pH value of 7.0 for 18 hours to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium at an inoculum amount of 5%, adding 1 g / L of phytotriazine, and fermenting the culture medium at a temperature of 32° C. and a pH value of 7.1 for 96 hours; after the fermentation is completed, removing the bacteria by centrifugation or filtration, and concentrating the fermentation liquid by ultrafiltration, purifying the concentrate by ion exchange chromatography or affinity chromatography to obtain a crude vitamin B12 product; and crystallizing the crude vitamin B12 product, and further purifying the product by crystallization to obtain a high-purity finished vitamin B12 product.

[0045] In an embodiment of the present application, before obtaining the seed liquid, the steps include: obtaining the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products; and automatically controlling the dynamic supplementation of carbon sources, nitrogen sources and vitamin precursors in the culture medium through an intelligent algorithm based on the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products.

[0046] In the embodiments of the present application, the concentrations of key nutrients include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic byproducts include organic acids, wherein the organic acid is propionic acid.

[0047] In the embodiment of the present application, before the fermentation ends, the process further includes: obtaining the bacterial growth density.

[0048] In the embodiment of the present application, the seed culture medium comprises a carbon source, a nitrogen source, inorganic salts and growth factors.

[0049] Among them, the carbon source is glucose 20g / L, the nitrogen source is yeast extract 10g / L, the inorganic salts are magnesium sulfate 0.5g / L, potassium dihydrogen phosphate 2g / L and calcium chloride 0.2g / L, and the growth factor is cobalt chloride 1mg / L.

[0050] In an embodiment of the present application, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is 30 g / L maltose, the nitrogen source is 15 g / L fish meal, the inorganic salt is 1 g / L magnesium sulfate, 0.1 g / L ferrous sulfate and 0.05 g / L manganese sulfate, the vitamin precursor is 0.3 mg / L 5,6-dimethylbenzimidazole, and the defoaming agent is 0.1 g / L polyether defoaming agent.

[0051] In the embodiments of the present application, the phytotrium is at least one of the biocolloids, wherein the preparation process of the phytotrium includes: fermenting under aerobic, anaerobic and deep fermentation conditions in sequence through the Triple-MAX triple fermentation process to obtain a fermentation basic product; subjecting the fermentation basic product to a "Multi-Point" enzymatic oligopeptide process, adding enzyme preparations, and performing multi-point enzymatic cleavage reactions under mild reaction conditions; and adopting the "Bio-Synthesis" process to utilize genes to regulate microbial metabolic pathways to synthesize and transform the phytotrium.

[0052] Among them, in the Triple-MAX triple fermentation process, the aerobic fermentation temperature is 30°C, the pH value is 7.0, and the time is 40 hours; the anaerobic fermentation temperature is 33°C, the pH value is 7.2, and the time is 55 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 30 hours.

[0053] In the examples of the present application, the crystallization treatment temperature is 4°C, the time is 12 hours, the ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin, and the affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

[0054] In the examples of the present application, Propionibacterium sieversii and Bacillus pumilus were co-cultured, wherein the inoculation ratio of Bacillus pumilus to Propionibacterium sieversii was 1:5.

[0055] Example 2 The present invention provides a method for producing vitamin B12 by fermenting Propionibacterium sievertii. The method comprises: inoculating Propionibacterium sievertii into a seed culture medium, adding 1.2 g / L of phytotriazine, and culturing the culture medium at a temperature of 30°C and a pH value of 7.0 for 18 hours to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium at an inoculum amount of 8%, adding 3 g / L of phytotriazine, and fermenting the culture medium at a temperature of 35°C and a pH value of 7.1 for 110 hours; after the fermentation is completed, removing the bacteria by centrifugation or filtration, and concentrating the fermentation liquid by ultrafiltration, purifying the concentrate by ion exchange chromatography or affinity chromatography to obtain a crude vitamin B12 product; and crystallizing the crude vitamin B12 product, and further purifying the product by crystallization to obtain a high-purity finished vitamin B12 product.

[0056] In an embodiment of the present application, before obtaining the seed liquid, the steps include: obtaining the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products; and automatically controlling the dynamic supplementation of carbon sources, nitrogen sources and vitamin precursors in the culture medium through an intelligent algorithm based on the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products.

[0057] In the embodiments of the present application, the concentrations of key nutrients include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic byproducts include organic acids, wherein the organic acid is propionic acid.

[0058] In the embodiment of the present application, before the fermentation ends, the process further includes: obtaining the bacterial growth density.

[0059] In the embodiment of the present application, the seed culture medium comprises a carbon source, a nitrogen source, inorganic salts and growth factors.

[0060] In the examples of the present application, the carbon source is 22 g / L glucose, the nitrogen source is 12 g / L yeast extract, the inorganic salts are 0.6 g / L magnesium sulfate, 2.2 g / L potassium dihydrogen phosphate and 0.3 g / L calcium chloride, and the growth factor is 1.2 mg / L cobalt chloride.

[0061] In an embodiment of the present application, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is 32 g / L maltose, the nitrogen source is 16 g / L fish meal, the inorganic salt is 1.2 g / L magnesium sulfate, 0.2 g / L ferrous sulfate and 0.06 g / L manganese sulfate, the vitamin precursor is 0.4 mg / L 5,6-dimethylbenzimidazole, and the defoaming agent is 0.3 g / L polyether defoaming agent.

[0062] In the embodiments of the present application, the phytotrium is at least one of the biocolloids, wherein the preparation process of the phytotrium includes: fermenting under aerobic, anaerobic and deep fermentation conditions in sequence through the Triple-MAX triple fermentation process to obtain a fermentation basic product; subjecting the fermentation basic product to a "Multi-Point" enzymatic oligopeptide process, adding enzyme preparations, and performing multi-point enzymatic cleavage reactions under mild reaction conditions; and adopting the "Bio-Synthesis" process to utilize genes to regulate microbial metabolic pathways to synthesize and transform the phytotrium.

[0063] Among them, in the Triple-MAX triple fermentation process, the aerobic fermentation temperature is 30°C, the pH value is 7.0, and the time is 40 hours; the anaerobic fermentation temperature is 33°C, the pH value is 7.2, and the time is 55 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 30 hours.

[0064] In the examples of the present application, the crystallization treatment temperature is 6° C., the time is 20 hours, the ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin, and the affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

[0065] In the examples of the present application, Propionibacterium sieversii and Bacillus pumilus were co-cultured, wherein the inoculation ratio of Bacillus pumilus to Propionibacterium sieversii was 1:8.

[0066] Example 3 The present invention provides a method for producing vitamin B12 by fermenting Propionibacterium shigellae. The method comprises: inoculating Propionibacterium shigellae into a seed culture medium, adding 2 g / L of phytotriazine, and culturing at a temperature of 30°C and a pH value of 7.0 for 35 hours to obtain a seed liquid; inoculating the seed liquid into a fermentation culture medium at an inoculum amount of 8%, adding 5 g / L of phytotriazine, and fermenting at a temperature of 35°C and a pH value of 7.1 for 120 hours; after the fermentation is completed, removing the bacteria by centrifugation or filtration, and ultrafiltration and concentration of the fermentation liquid, purifying the concentrate by ion exchange chromatography or affinity chromatography to obtain a crude vitamin B12 product; and crystallizing the crude vitamin B12 product, and further purifying it by crystallization to obtain a high-purity finished vitamin B12 product.

[0067] In an embodiment of the present application, before obtaining the seed liquid, the steps include: obtaining the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products; and automatically controlling the dynamic supplementation of carbon sources, nitrogen sources and vitamin precursors in the culture medium through an intelligent algorithm based on the bacterial growth density, the concentration of key nutrients and the accumulation of metabolic by-products.

[0068] In the embodiments of the present application, the concentrations of key nutrients include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic byproducts include organic acids, wherein the organic acid is propionic acid.

[0069] In the embodiment of the present application, before the fermentation ends, the process further includes: obtaining the bacterial growth density.

[0070] In the embodiment of the present application, the seed culture medium comprises a carbon source, a nitrogen source, inorganic salts and growth factors.

[0071] Among them, the carbon source is glucose 25g / L, the nitrogen source is yeast extract 13g / L, the inorganic salts are magnesium sulfate 0.7g / L, potassium dihydrogen phosphate 2.5g / L and calcium chloride 0.4g / L, and the growth factor is cobalt chloride 2mg / L.

[0072] In an embodiment of the present application, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is 34 g / L maltose, the nitrogen source is 17 g / L fish meal, the inorganic salt is 1.5 g / L magnesium sulfate, 0.3 g / L ferrous sulfate and 0.07 g / L manganese sulfate, the vitamin precursor is 0.5 mg / L 5,6-dimethylbenzimidazole, and the defoaming agent is 0.3 g / L polyether defoaming agent.

[0073] In the embodiments of the present application, the phytotrium is at least one of the biocolloids, wherein the preparation process of the phytotrium includes: fermenting under aerobic, anaerobic and deep fermentation conditions in sequence through the Triple-MAX triple fermentation process to obtain a fermentation basic product; subjecting the fermentation basic product to a "Multi-Point" enzymatic oligopeptide process, adding enzyme preparations, and performing multi-point enzymatic cleavage reactions under mild reaction conditions; and adopting the "Bio-Synthesis" process to utilize genes to regulate microbial metabolic pathways to synthesize and transform the phytotrium.

[0074] Among them, in the Triple-MAX triple fermentation process, the aerobic fermentation temperature is 30°C, the pH value is 7.0, and the time is 40 hours; the anaerobic fermentation temperature is 33°C, the pH value is 7.2, and the time is 55 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 30 hours.

[0075] In the examples of the present application, the crystallization treatment temperature is 6° C., the time is 20 hours, the ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin, and the affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

[0076] In the embodiment of the present application, Propionibacterium sieversii and Bacillus pumilus are co-cultured, wherein the inoculation ratio of Bacillus pumilus to Propionibacterium sieversii is 1:10.

[0077] Comparative Example 1 A method for producing vitamin B12 by fermenting Propionibacterium shiehii, differing from Example 1 only in that it does not employ intelligent control and a co-cultivation system. Specifically, an 8% inoculum of Propionibacterium shiehii seed liquid is inoculated into a fermentation medium, and fermentation is carried out at 35°C and a pH of 7.2. No online monitoring or intelligent control is performed, and only nutrients are added at fixed intervals. The dissolved oxygen concentration is not controlled within a specific range. After fermentation, the same extraction and purification steps as in Example 1 are employed.

[0078] Performance Testing The vitamin B12 produced by fermentation of Propionibacterium sieversii prepared in Examples 1-3 and Comparative Example 1 was respectively measured for its purity. An appropriate amount of vitamin B12 standard was accurately weighed, dissolved in methanol and diluted to form a series of standard solutions of different concentrations. The solutions were measured according to the chromatographic conditions. The results are shown in Table 1 below.

[0079] Table 1 Test results

[0080] The test results in Table 1 show that the yield of Example 1 is higher than that of Comparative Example 1. This is because the intelligent regulation can provide appropriate nutrients in a timely manner according to the growth and metabolic needs of the bacteria, avoiding the adverse effects of nutrient deficiency or excess on bacterial growth and vitamin B12 synthesis; the beneficial substances secreted by Bacillus pumilus in the co-culture system promote the growth of Propionibacterium sieversii and vitamin B12 synthesis; the optimized culture medium components and fermentation conditions provide a more favorable environment for bacterial growth and vitamin B12 synthesis.

[0081] At the same time, an embodiment of the present application provides a method for producing vitamin B12 by fermenting Propionibacterium sieversii. In this method, intelligent control technology and optimized production processes are used to achieve precise control of the vitamin B12 production process. Through online monitoring and intelligent adjustment of culture medium components, the yield and purity are significantly improved; at the same time, the optimized separation and purification process ensures the high quality of the product. In addition, intelligent control also shortens the fermentation cycle, reduces costs, and improves resource utilization efficiency through resource utilization of fermentation waste, achieving a dual improvement in environmental friendliness and economic benefits. As a result, the problems of low vitamin B12 yield, unstable purity and difficult production process control in the related technology are solved.

[0082] This application also proposes a device for producing vitamin B12 by fermenting Propionibacterium sieversii. Figure 2 As shown, the device 10 includes: an acquisition module 100, a fermentation module 200, a purification module 300 and a crystallization module 400.

[0083] Among them, the acquisition module 100 is used to inoculate Propionibacterium sieversii into the seed culture medium, add 0.5-2g / L phytotriene, and culture for 18-24 hours at a temperature of 30-35°C and a pH value of 6.5-7.2 to obtain seed liquid; the fermentation module 200 is used to inoculate the seed liquid into the fermentation culture medium at an inoculation rate of 5%-10%, add 1-5g / L phytotriene, and ferment for 96-120 hours at a temperature of 32-37°C and a pH value of 6.8-7.5; the purification module 300 is used to remove the bacteria by centrifugation or filtration after the fermentation is completed, and ultrafiltration and concentration of the fermentation liquid are performed, and the concentrate is purified by ion exchange chromatography or affinity chromatography to obtain crude vitamin B12; the crystallization module 400 is used to crystallize the crude vitamin B12 and further purify it by crystallization to obtain a high-purity finished vitamin B12 product.

[0084] The embodiment of the present application provides a device for producing vitamin B12 by fermenting Propionibacterium sieversii, and uses intelligent control technology and optimized production processes to achieve precise control of the vitamin B12 production process. Through online monitoring and intelligent adjustment of culture medium components, the yield and purity are significantly improved; at the same time, the optimized separation and purification process ensures the high quality of the product. In addition, intelligent control also shortens the fermentation cycle, reduces costs, and improves resource utilization efficiency through resource utilization of fermentation waste, achieving a dual improvement in environmental friendliness and economic benefits. As a result, the problems of low vitamin B12 yield, unstable purity and difficult production process control in the related art are solved.

[0085] The present application also proposes a system for producing vitamin B12 by fermenting Propionibacterium sieversii. The system includes the above-mentioned device for producing vitamin B12 by fermenting Propionibacterium sieversii.

[0086] 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 producing vitamin B12 by fermenting Propionibacterium sieversii, characterized in that: The method comprises: Inoculate Propionibacterium sieversii into a seed culture medium, add 0.5-2 g / L of phytotriazine, and culture at a temperature of 30-35° C. and a pH of 6.5-7.2 for 18-24 hours to obtain a seed solution; The seed liquid is inoculated into the fermentation medium at an inoculum rate of 5%-10%, 1-5 g / L of phytotriazine is added, and the fermentation is carried out at a temperature of 32-37° C. and a pH value of 6.8-7.5 for 96-120 hours; After the fermentation is completed, the bacteria are removed by centrifugation or filtration, and the fermentation broth is concentrated by ultrafiltration. The concentrate is purified by ion exchange chromatography or affinity chromatography to obtain crude vitamin B12; The crude vitamin B12 product is crystallized and further purified by a crystallization method to obtain a high-purity finished vitamin B12 product.

2. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1, wherein: Before obtaining the seed solution, include: Obtain bacterial growth density, key nutrient concentrations, and metabolic byproduct accumulation; The dynamic supplementation amount of carbon source, nitrogen source and vitamin precursor in the culture medium is automatically regulated by an intelligent algorithm according to the bacterial growth density, the concentration of the key nutrients and the accumulation of the metabolic by-products.

3. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 2, wherein: The key nutrient concentrations include carbon source concentration, nitrogen source concentration and inorganic salt concentration, and the metabolic byproducts include organic acids, wherein the organic acid is propionic acid.

4. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1, wherein: Before the end of fermentation, the process also includes: obtaining the bacterial growth density.

5. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1 or 2, characterized in that: The seed culture medium includes a carbon source, a nitrogen source, an inorganic salt and a growth factor, wherein the carbon source includes one or more of glucose, sucrose and fructose; the nitrogen source includes one or more of yeast extract, peptone and urea; the inorganic salt includes one or more of magnesium sulfate, potassium dihydrogen phosphate and calcium chloride; and the growth factor contains a cobalt ion source, and the concentration of the cobalt ion source is 0.5-2 mg / L.

6. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1 or 4, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, a vitamin precursor and a defoaming agent, wherein the carbon source is selected from one or more of glucose, maltose and starch hydrolysate; the nitrogen source is selected from one or more of fish meal, soybean meal and corn steep liquor; the inorganic salt includes one or more of magnesium sulfate, ferrous sulfate and manganese sulfate; the vitamin precursor includes one or more of 5,6-dimethylbenzimidazole, adenine and guanine, and the vitamin precursor is added in an amount of 0.1-0.5 g / L; the defoaming agent is selected from one or more of polyether defoaming agents and silicone defoaming agents; and during the fermentation process, the dissolved oxygen concentration is controlled at 20%-30%.

7. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1 or 4, characterized in that: The three phytonutrients are at least one of the biocolloids, wherein the preparation process of the three phytonutrients includes: The Triple-MAX fermentation process is used to ferment under aerobic, anaerobic and deep fermentation conditions in sequence to obtain a fermentation base product; the fermentation base product is subjected to a "Multi-Point" enzymatic oligopeptide process, in which enzyme preparations are added and multi-point enzymatic cleavage reactions are carried out under mild reaction conditions; the "Bio-Synthesis" process is used to regulate microbial metabolic pathways through gene regulation to synthesize and transform the plant trio of elements; in the Triple-MAX fermentation process, the aerobic fermentation temperature is 28-32°C, the pH value is 6.5-7.2, and the time is 36-48 hours; the anaerobic fermentation temperature is 30-35°C, the pH value is 6.8-7.5, and the time is 48-60 hours; the deep fermentation temperature changes periodically between 32-37°C, the pH value fluctuates between 7.0-7.8, and the time is 24-36 hours.

8. The method for producing vitamin B12 by fermentation using Propionibacterium sieversii according to claim 1, wherein: The crystallization treatment is carried out at a temperature of 4-8° C. for 12-24 hours. The ion exchange resin in the ion exchange chromatography is a strongly basic anion exchange resin. The affinity ligand in the affinity chromatography is a vitamin B12-specific antibody or a fragment thereof.

9. A device for producing vitamin B12 by fermenting Propionibacterium sieversii, characterized in that: include: An acquisition module is used to inoculate Propionibacterium sieversii into a seed culture medium, add 0.5-2 g / L of phytotriazine, and culture for 18-24 hours at a temperature of 30-35° C. and a pH of 6.5-7.2 to obtain a seed solution; A fermentation module is used to inoculate the seed liquid into a fermentation medium at an inoculum rate of 5%-10%, add 1-5g / L of phytotriazine, and ferment for 96-120 hours at a temperature of 32-37°C and a pH of 6.8-7.5; The purification module is used to remove the bacteria by centrifugation or filtration after the fermentation is completed, and the fermentation broth is concentrated by ultrafiltration. The concentrate is purified by ion exchange chromatography or affinity chromatography to obtain crude vitamin B12; The crystallization module is used to crystallize the crude vitamin B12 product and further purify it by crystallization to obtain a high-purity finished vitamin B12 product.

10. A system for producing vitamin B12 by fermentation using Propionibacterium sieversii, characterized in that: The system includes the device for producing vitamin B12 by fermentation using Propionibacterium sieversii as described in claim 9.

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