Method for preparing vitamin B12 by fermenting wheat bran with propionibacterium freudenreichii
By fermenting Propionibacterium Filipino in wheat bran matrix, optimizing the culture conditions and processes, the problem of low fermentation efficiency of vitamin B12 is solved, and high yield and low cost vitamin B12 production is achieved, suitable for food, feed and pharmaceutical fields.
Patent Information
- Application Number
- CN202510553318.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-29
- Publication Date
- 2025-07-25
AI Technical Summary
In the prior art, the vitamin B12 fermentation process is inefficient, insufficient resource utilization, complex process and high cost, and the vitamin B12 content in plant-based foods is extremely low, resulting in a global health problem.
Propionibacterium fermentation in the unsterilized wheat bran matrix was used to optimize strain culture conditions, wheat bran pretreatment and fermentation technology, the yield of vitamin B12 was improved and the high-value utilization of wheat bran was achieved.
It significantly increases the production of vitamin B12 and reduces production costs. The vitamin B12 products produced meet food and pharmaceutical grade standards, have high purity and high activity, and are suitable for the food, feed and pharmaceutical fields, meet the nutritional needs of specific groups and reduce the risk of anemia and neurological diseases.
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Figure CN120366136A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of fermentation technology, and particularly relates to a method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii. Background Art
[0002] Vitamin B12, also known as cobalamin, is the most chemically complex of all vitamins. It is a cofactor for two important enzymes in human metabolism: methionine synthase in the cytoplasm, which is involved in nucleotide synthesis, and methylmalonyl-CoA mutase in the mitochondria, which is involved in the metabolism of odd-chain fatty acids and branched-chain amino acids. Deficiency of vitamin B12 can lead to serious health problems such as megaloblastic anemia and various neurological diseases. According to the recommendations of the Institute of Medicine of the United States, adults should consume 2.4 μg of vitamin B12 per day.
[0003] Vitamin B12 can only be synthesized by a few microorganisms, such as Pseudomonas, Bacillus megaterium, and Propionibacterium freudenreichii. Animal foods are currently the main source of vitamin B12 in the diet. However, since the content of vitamin B12 in plant foods is extremely low, it is usually necessary to increase its content through chemical supplementation or fermentation enhancement. Vitamin B12 deficiency, mainly due to insufficient dietary intake or absorption disorders, has become a global health problem.
[0004] Developing plant-based foods rich in vitamin B12 is an important way to solve this problem. Wheat, as one of the most important cereals in the human diet, is an ideal carrier for vitamin B12. Studies have shown that although some lactic acid bacteria (such as specific Lactobacillus strains) have been reported to produce vitamin B12, the main product is pseudo-vitamin B12, in which 5,6-dimethylbenzimidazole in its active form is replaced by adenine and has no biological activity for humans. In contrast, Propionibacterium freudenreichii can synthesize DMBI and produce physiologically relevant amounts of active vitamin B12 under food conditions.
[0005] Due to its recognized safety, Propionibacterium freudenreichii has been widely used in food fermentation, such as the production of exopolysaccharides and antifungal compounds. Existing studies have shown that Propionibacterium freudenreichii can produce 37 ng / g (fresh weight) of active vitamin B12 after fermenting in a sterilized barley matrix for 7 days. However, there has been no study to explore the ability of Propionibacterium freudenreichii to produce vitamin B12 in an unsterilized cereal matrix (such as wheat bran), nor has there been any report on its application potential in a wheat matrix. Summary of the Invention
[0006] The present invention provides a method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, aiming to solve the problems of low efficiency, insufficient resource utilization, complex process and high cost in the existing vitamin B12 fermentation process. By optimizing the strain culture conditions, wheat bran pretreatment and fermentation process, the present invention can significantly increase the yield of vitamin B12, and at the same time realize the high-value utilization of wheat bran as a by-product, which has important industrial application value.
[0007] To achieve the above object, the present invention proposes the following technical solutions: The present invention provides a method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, comprising the following steps: 1) Strain culture: The strain of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 cryopreserved in 15% glycerol at -70°C was cultured in yeast extract lactate (YEL) agar medium. Three separate colonies were transferred as three biological replicates to 30 ml of YEL medium for incubation, and the culture was recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculum, and then 0.1 - 0.5 g / L of phytohormones was added, and then used for inoculating the wheat bran mass.
[0008] 2) Preparation of wheat bran mass: The wheat bran mass described in step 1) above was prepared by mixing deionized water and wheat bran.
[0009] 3) Fermentation culture: The inoculum obtained in step 1) above was inoculated into 30 g of wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass was placed in a test tube, and the culture temperature was fixed at 25°C for aerobic fermentation culture.
[0010] Furthermore, the incubation temperature of the strain of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 is between 28 and 32°C.
[0011] Furthermore, the wheat bran mass in the above step comprises the following components in mass percentage: 70 - 80% of deionized water; 20 - 30% of wheat bran.
[0012] Furthermore, the initial pH of the wheat bran mass is 6.24 - 6.72, and the initial titratable acidity value of the wheat bran mass is 2.4 - 3.4 ml.
[0013] Furthermore, the inoculation solution is adjusted to a pH of 7.0 with 10% NaOH solution before being inoculated into the wheat bran mass, and the test tube is sealed for fermentation culture after inoculation.
[0014] Furthermore, after inoculation, the number of colony - forming units of Propionibacterium freudenreichii in the wheat bran mass ranges from 8.7 - 9.0 log CFU / g.
[0015] Furthermore, the fermentation culture process lasts for 7 days, and the test tube is opened once on the 3rd day to allow air to enter. The whole fermentation process is carried out under the conditions of constant - temperature shaking in a water bath.
[0016] Furthermore, the temperature of the constant - temperature shaking environment in the water bath is 25 - 30 °C, and the shaking speed is 200 rpm per minute.
[0017] The beneficial effects obtained by the above - mentioned invention of the present invention are as follows: 1. The present invention realizes the efficient utilization of agricultural and forestry waste with wheat bran as the main fermentation raw material. Wheat bran is a by - product in the grain processing process, accounting for about 14% - 19% of the weight of wheat. Its main components are cellulose, hemicellulose and protein. Wheat bran is rich in vitamins and trace elements, which helps to enhance the metabolic activity of the strains, becoming a good low - cost carbon source and nitrogen source. Through pretreatment, the nutrients in wheat bran are released and converted into fermentation substrates, effectively providing the nutrients required for the growth of Propionibacterium freudenreichii and promoting the microbial fermentation to form vitamin B12; 2. Propionibacterium freudenreichii converts the organic acids (such as propionic acid) and nucleosides released from wheat bran into the core structure of vitamin B12 through specific metabolic pathways (such as carbamoyl phosphate synthesis and adenosylcobalamin synthesis). Under optimized fermentation conditions (such as pH, temperature and ventilation volume), the metabolic pathway is activated, and the synthesis efficiency of vitamin B12 is further improved. The yield of vitamin B12 in the present invention can reach 150 - 200 µg / L, which is significantly higher than the yield of traditional fermentation substrates (such as corn steep liquor and soybean meal) (usually 80 - 120 µg / L); 3. By adjusting the medium components and culture conditions (such as increasing the glucose concentration to 10 - 15 g / L) to promote the rapid growth of the bacteria, while reducing the sugar concentration and supplementing precursor substances (such as adenosine, cobalt salts) to induce the synthesis of vitamin B12. Compared with the traditional process with a fermentation time of 120 - 140 hours, the fermentation time of this process can be shortened to 100 - 110 hours, and the productivity is increased by about 30%; 4. As an agricultural waste, wheat bran utilization not only reduces resource waste. The waste generated by fermenting each ton of wheat bran is only 60%-70% of that of traditional raw materials, effectively reducing the wastewater treatment cost and pollution emissions. At the same time, compared with traditional fermentation raw materials with higher prices (such as corn steep liquor and soybean meal), the cost of wheat bran is only 30%-50% of theirs, significantly reducing the production cost; 5. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii of the present invention. The produced vitamin B12 product meets the food additive and pharmaceutical grade standards, has high purity (≥98%) and high activity, and is widely used in the fields of food, feed and medicine. In the food field, it can be used as a fortifier to meet the nutritional needs of specific populations (such as vegetarians); in the feed field, it can enhance the growth performance of animals, especially suitable for improving the growth rate of poultry and pigs; in the medical field, it is used to treat vitamin B12 deficiency and reduce the risk of anemia and neurological diseases. In addition, the global market demand for vitamin B12 is growing at a rate of 4%-5% per year, and the popularization of wheat bran fermentation technology is expected to reduce the production cost by 15%-20%, significantly enhancing the market competitiveness of the product. Brief Description of the Drawings
[0018] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description of the embodiments in conjunction with the drawings, wherein: Figure 1 It is the concentration (μg / g) of riboflavin in the inoculated dough on the 0th day and the 7th day in the comparative example of the present invention. Detailed Description of the Invention
[0019] The technical solutions of the present invention are illustrated by specific specific examples below. It should be understood that one or more method steps mentioned in the present invention do not exclude the existence of other method steps before and after the combined steps or the insertion of other method steps between these clearly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbers of each method step are only convenient tools for identifying each method step, rather than limiting the arrangement order of each method step or the scope of implementation of the present invention. The change or adjustment of their relative relationship, without substantial change in technical content, should also be regarded as the scope of implementation of the present invention.
[0020] To better understand the above technical solutions, the exemplary embodiments of the present invention are described in more detail below. Although the exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.
[0021] The present invention will be further described in conjunction with the following embodiments.
[0022] Example 1 The present invention provides a method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, comprising the following steps: 1) Strain cultivation: The Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain cryopreserved in 15% glycerol (-70 °C) was cultured in yeast extract lactate (YEL) agar medium. Three separate colonies were transferred as three biological replicates to 30 ml of YEL medium for incubation, and the culture was recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculum, and then 0.1 g / L of plant trinity was added, which was subsequently used for inoculating the wheat bran mass.
[0023] Among them, the plant trinity is prepared by the processes of Triple - MAX triple glycolysis, "Multi - Point" enzymatic oligopeptide, and "Bio - Synthesis biosynthesis".
[0024] 2) Preparation of wheat bran mass: The wheat bran dough described in step 1) above was prepared by mixing deionized water and wheat bran.
[0025] 3) Fermentation culture: The inoculum obtained in step 1) above was inoculated into 30 g of wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass was placed in a test tube, and the culture temperature was fixed at 25 °C for aerobic fermentation culture.
[0026] In this example, the incubation temperature of the Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain is 30 °C.
[0027] Furthermore, the wheat bran mass described in the above step contains the following components in mass percentages: 80% deionized water; 20% wheat bran.
[0028] In this example, the initial pH of the wheat bran mass is 6.24 - 6.72, and the initial titratable acidity value of the wheat bran mass is 2.4 - 3.4 ml.
[0029] In this example, the inoculation liquid was adjusted to pH 7.0 with 10% NaOH solution before being inoculated into the wheat bran mass, and the test tube was sealed for fermentation culture after inoculation.
[0030] In this example, after inoculation of the wheat bran mass, the colony forming unit number of Propionibacterium freudenreichii ranged from 8.7 - 9.0 log CFU / g.
[0031] In this example, the fermentation culture process lasted for 7 days, and the test tube was opened once on the 3rd day to allow air to enter. The whole fermentation process was carried out under the condition of constant temperature shaking in a water bath.
[0032] In this example, the temperature of the constant temperature shaking environment in the water bath was 30°C, and the shaking speed was 200 rpm per minute.
[0033] Example 2 The present invention provides a method for fermenting wheat bran with Propionibacterium freudenreichii to prepare vitamin B12, which includes the following steps: 1) Strain culture: The Propionibacterium freudenreichii (Propionibacterium freudenreichii subsp. freudenreichii DSM 20271) strain cryopreserved in 15% glycerol (-70°C) was cultured in yeast extract lactate (YEL) agar medium. Three separate colonies were transferred as three biological replicates to 30 ml of YEL medium for incubation, and the culture was recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculation liquid, and then 0.2 g / L of plant trinity was added, which was subsequently used for inoculating the wheat bran mass.
[0034] Among them, the plant trinity is prepared by the "Triple - MAX triple glycolysis", "Multi - Point" enzymatic oligopeptide, and "Bio - Synthesis biosynthesis" processes.
[0035] 2) Preparation of the wheat bran mass: The wheat bran dough described in step 1) above was prepared by mixing deionized water and wheat bran.
[0036] 3) Fermentation culture: The inoculation liquid obtained in step 1) above was inoculated into 30 g of the wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass was placed in a test tube, and the culture temperature was fixed at 25°C for aerobic fermentation culture.
[0037] In this example, the incubation temperature of the Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain is 30 °C.
[0038] In this example, the wheat bran mass contains the following components by mass percentage: 80% deionized water; 20% wheat bran.
[0039] In this example, the initial pH of the wheat bran mass is 6.24 - 6.72, and the initial titratable acidity value of the wheat bran mass is 2.4 - 3.4 ml.
[0040] In this example, the inoculation solution is adjusted to pH 7.0 with 10% NaOH solution before being inoculated into the wheat bran mass, and the test tube is sealed after inoculation for fermentation culture.
[0041] In this example, after inoculation of the wheat bran mass, the colony forming unit number of Propionibacterium freudenreichii ranges from 8.7 - 9.0 log CFU / g.
[0042] In this example, the fermentation culture process takes 3 days, and the test tube is kept sealed throughout the 3 days without allowing air to enter. The entire fermentation process is carried out under the condition of constant temperature shaking in a water bath.
[0043] In this example, the temperature of the constant temperature shaking environment in the water bath is 30 °C, and the shaking speed is 200 rpm per minute.
[0044] Example 3 The present invention provides a method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, comprising the following steps: 1) Strain culture: The Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain cryopreserved in 15% glycerol (-70 °C) is cultured in yeast extract lactate (YEL) agar medium. Three separate colonies are transferred as three biological replicates to 30 ml of YEL medium for incubation, and the culture is recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculation solution, and then 0.3 g / L of phytotriol is added, which is subsequently used for inoculation of the wheat bran mass.
[0045] Among them, the plant tricin is prepared by the processes of Triple - MAX triple fermentation, "Multi - Point" enzymatic oligopeptide, and "Bio - Synthesis".
[0046] 2) Preparation of wheat bran mass: The wheat bran dough described in step 1) above is prepared by mixing deionized water and wheat bran.
[0047] 3) Fermentation culture: The inoculation liquid obtained in step 1) above is inoculated into 30 g of wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass is placed in a test tube, and the culture temperature is fixed at 25°C for aerobic fermentation culture.
[0048] In this example, the incubation temperature of the Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain is 30°C.
[0049] Furthermore, the wheat bran mass in the above step contains the following components in mass percentages: 70% deionized water; 30% wheat bran.
[0050] In this example, the initial pH of the wheat bran mass is 6.24 - 6.72, and the initial titratable acidity value of the wheat bran mass is 2.4 - 3.4 ml.
[0051] In this example, the inoculation liquid is adjusted to pH 7.0 with 10% NaOH solution before being inoculated into the wheat bran mass, and the test tube is sealed after inoculation for fermentation culture.
[0052] In this example, after inoculation of the wheat bran mass, the number of colony - forming units of Propionibacterium freudenreichii ranges from 8.7 - 9.0 log CFU / g.
[0053] In this example, the fermentation culture process takes 7 days, and the test tube is opened once on the 3rd day to allow air to enter. The whole fermentation process is carried out under the conditions of constant - temperature water - bath oscillation.
[0054] In this example, the temperature of the constant - temperature water - bath oscillation environment is 30°C, and the oscillation speed is 200 rpm per minute.
[0055] Example 4 The present invention provides a method for fermenting wheat bran with Propionibacterium freudenreichii to prepare vitamin B12, including the following steps: 1) Strain culture: The strain of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 cryopreserved in 15% glycerol at -70 °C was cultured in yeast extract lactate (YEL) agar medium. Three separate colonies were transferred as three biological replicates into 30 ml of YEL medium for incubation, and the culture was recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculum, then 0.5 g / L of phytotriol was added, and subsequently used for inoculating the wheat bran mass.
[0056] Among them, the phytotriol is prepared by the "Triple - MAX" triple glycolysis, "Multi - Point" enzymatic oligopeptide, and "Bio - Synthesis" process.
[0057] 2) Preparation of the wheat bran mass: The wheat bran dough described in step 1) above was prepared by mixing deionized water and wheat bran.
[0058] 3) Fermentation culture: The inoculum obtained in step 1) above was inoculated into 30 g of the wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass was placed in a test tube, and the culture temperature was fixed at 25 °C for aerobic fermentation culture.
[0059] In this example, the incubation temperature of the strain of Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 was 30 °C.
[0060] In this example, the wheat bran mass in the above step contains the following components by mass percentage: 70% deionized water; 30% wheat bran.
[0061] In this example, the initial pH of the wheat bran mass was 6.24 - 6.72, and the initial titratable acidity value of the wheat bran mass was 2.4 - 3.4 ml.
[0062] In this example, the inoculum was adjusted to pH 7.0 with 10% NaOH solution before inoculating into the wheat bran mass, and the test tube was sealed for fermentation culture after inoculation.
[0063] In this example, after inoculation of the wheat bran mass, the number of colony - forming units of Propionibacterium freudenreichii ranged from 8.7 - 9.0 log CFU / g.
[0064] In this example, the fermentation culture process lasts for 3 days, and the test tube is kept sealed throughout the 3 days without allowing air to enter. The entire fermentation process is carried out under the conditions of constant temperature water bath and shaking.
[0065] In this example, the temperature of the constant temperature water bath shaking environment is 30°C, and the shaking speed is 200 rpm per minute.
[0066] Comparative Example 1 A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, which is different from Example 1 in that the wheat bran mass is replaced with wheat dough, and it contains the following components by mass percentage: 70% deionized water; 30% wheat flour.
[0067] The remaining components and their contents are the same as those in Example 1.
[0068] Comparative Example 2 A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, which is different from Example 1 in that the wheat bran mass is replaced with whole wheat dough, and it contains the following components by mass percentage: 70% deionized water; 30% whole wheat flour.
[0069] The remaining components and their contents are the same as those in Example 1.
[0070] Comparative Example 3 A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii, which is different from Example 1 in that 0.6 μg / g dw of cobalt chloride is additionally added during the fermentation process.
[0071] The remaining components and their contents are the same as those in Example 1.
[0072] Performance Test The concentration of vitamin B12 during the fermentation process is shown in Table 1.
[0073] Table 1 Yield of vitamin B12 (μg / L, dw)
[0074] The results are expressed as mean ± standard deviation (sample size = 3).
[0075] As Figure 1As shown in Table 1, significant differences were observed in the fermentation processes of different raw materials (wheat bran, whole wheat flour, and wheat flour) in the comparative examples of the present invention, mainly reflected in aspects such as glucose content, growth dynamics of lactic acid bacteria and Propionibacterium freudenreichii, lactic acid consumption, and vitamin B12 production. These differences stem from the different characteristics of the raw materials in terms of nutrient composition, endogenous microbial communities, enzyme activities, and potential inhibitory factors. First, the glucose content increased in wheat flour and whole wheat flour at the initial stage of fermentation, mainly attributed to the hydrolysis of starch by endogenous α-amylase. However, due to the low initial available carbohydrate content in wheat bran, its glucose was depleted after the third day of fermentation. In addition, lactic acid bacteria showed rapid proliferation in all raw materials and dominated the microbial ecosystem. Especially in wheat bran, due to the strong activity of lactic acid bacteria, the lactic acid content continued to increase even after glucose was depleted, resulting in significant acidification. There were also significant differences in the growth of Propionibacterium freudenreichii in different raw materials. It proliferated rapidly at the initial stage in wheat flour but was restricted by the acidic environment (pH < 4.0). In wheat bran, although the vitamin B12 production was significantly increased (about 5 times that of wheat flour), the cell density was relatively low, which might be related to the presence of endogenous inhibitory factors (such as flavonoids) in wheat bran. Studies have shown that such substances may have an inhibitory effect on the growth of Propionibacterium. Although there were sufficient carbon sources and suitable initial pH in whole wheat flour, the growth of Propionibacterium freudenreichii was also restricted, possibly related to the competition of endogenous microorganisms or inhibitory components. The production of vitamin B12 showed a trend closely related to the characteristics of the raw materials. Among them, wheat bran, due to its high cobalt content (about 0.1 μg / g, significantly higher than < 0.01 μg / g in wheat flour) and relatively high riboflavin content (1.93 μg / g vs. 0.18 μg / g in wheat flour), demonstrated excellent potential for vitamin B12 synthesis during fermentation. In addition, riboflavin, as an important precursor for vitamin B12 synthesis, its high content in wheat bran and whole wheat flour might further promote the accumulation of vitamin B12. Due to the lack of sufficient precursor substances in wheat flour, its vitamin B12 production was relatively low. The present invention indicates that wheat bran has great potential in vitamin B12 fermentation. Especially its high cobalt and riboflavin content make it an ideal substrate, and at the same time provide an important solution for the high-value utilization of agricultural by-products.
[0076] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations of the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.
Claims
1. A Propionibacterium freudenreichii strain, characterized in that, Its classification name is Propionibacterium freudenreichii subsp. freudenreichii DSM 20271.
2. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 according to claim 1, characterized in that, It includes the following steps: 1) Strain culture: The Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain cryopreserved in 15% glycerol (-70 °C) is cultured in yeast extract lactate agar medium. Three separate colonies are transferred as three biological replicates to 30 ml of YEL medium for incubation, and the culture is recovered by centrifugation (3200 rpm, 10 minutes) after 72 hours, resuspended in 10 ml of sterile water as the inoculum, and then 0.1 - 0.5 g / L of phytohormones is added for inoculating the wheat bran mass. 2) Preparation of wheat bran mass: The wheat bran dough described in step 1 above is prepared by mixing deionized water and wheat bran. 3) Fermentation culture: The inoculum obtained in step 1 above is inoculated into 30 g of the wheat bran mass at an inoculation amount of 10%. The inoculated wheat bran mass is placed in a test tube, and the culture temperature is fixed at 25 °C for aerobic fermentation culture.
3. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 2, characterized in that, In step 1), the incubation temperature of the Propionibacterium freudenreichii subsp. freudenreichii DSM 20271 strain is between 28 and 32 °C.
4. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 2, characterized in that, The wheat bran mass in step 2 contains the following components by mass percentage: 70 - 80% deionized water; 20 - 30% wheat bran.
5. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 4, characterized in that, The initial pH of the wheat bran mass is 6.24 - 6.72, and the titratable acidity value of the wheat bran mass is 2.4 - 3.4 ml.
6. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 2, characterized in that, In step 3), the pH of the inoculum is adjusted to 7.0 with 10% NaOH solution before inoculating it into the wheat bran mass, and the test tube is sealed after inoculation for fermentation culture.
7. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 6, wherein The number of colony forming units of Propionibacterium freudenreichii in the inoculated wheat bran mass ranges from 8.7 - 9.0 log CFU / g.
8. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 2, characterized in that, In step 3), the fermentation culture time is 7 days, and the test tube is opened once on the 3rd day to allow air to enter. The whole fermentation process is carried out under the condition of constant temperature shaking in a water bath.
9. A method for preparing vitamin B12 by fermenting wheat bran with Propionibacterium freudenreichii according to claim 6, characterized in that The temperature of the constant temperature shaking environment in the water bath is 25 - 30 °C, and the shaking speed is 200 rpm per minute.