Method for improving cell activity in vitamin b12 fermentation production process

Abstract

The application provides a method for supplementing nutrients required for growth of a vitamin B12 fermentation production strain, which comprises the following steps: in a fermentation operation stage, according to consumption, supplementing carbon source glucose to maintain basic metabolism, and supplementing nutrients after operation for 90 hours. The feeding method provided by the application can improve the activity of the vitamin B12 fermentation production strain, effectively prolongs the stable period of the strain, prolongs the fermentation cycle by 7.46%, and has obvious effect on the improvement of the fermentation titer, and the improvement ratio is 8.30%.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and in particular to a method for producing vitamin B12 by fermentation. Background Technology

[0002] Vitamin B12, also known as cobalamin, is the only vitamin in nature containing a metallic element and is currently the vitamin with the largest known molecular weight. As a cofactor of methyltransferases, it increases nutrient metabolism and promotes the development and maturation of red blood cells, maintaining normal hematopoietic function and thus preventing pernicious anemia and maintaining the health of the nervous system. Therefore, with the improvement of living standards, the demand for vitamin B12 will inevitably increase, and how to improve the production yield and quality of vitamin B12 is an urgent problem to be solved. In 1926, Minot and Murphy of Harvard Medical School in the United States first discovered vitamin B12 and used it to treat "Adolescent anemia." This was the first discovery of vitamin B12 and the last vitamin in the vitamin family to be discovered. In 1948, scientists in Britain and the United States almost simultaneously extracted this red crystal from the liver and named it vitamin B12. In 1955, Hodgkin of Oxford University in the United Kingdom first determined the chemical structure of VB12, including a central corrin ring, a central ring-axial Coβ ligand portion, and a Coα ligand containing a nucleotide ring. Its chemical synthesis is complex and produces many byproducts; currently, fermentation is the primary method used for vitamin B12 production. The fermentation process for vitamin B12 production mainly involves the synthesis of vitamin B12 by eubacteria and some archaea through anaerobic and aerobic pathways, involving more than 30 genes. The main producing bacteria include *Pseudomonas denitrificans* and *Propionibacterium fischeri*.

[0003] Bacillus freudenreichii, Bacillus megaterium, and Salmonella typhimurium are among the bacteria found in this product. During fermentation, the activity of the microorganisms significantly promotes vitamin B12 fermentation. This invention provides a method for controlling the addition of nutrients for microbial growth during the vitamin B12 fermentation stage, effectively improving the activity of the microorganisms and increasing production levels. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for supplementing the nutrients required for cell growth in the vitamin B12 fermentation production process. The feeding method provided by the present invention can improve the vitality of the cells in vitamin B12 fermentation production, effectively prolong the cell stability period, and increase the fermentation yield.

[0005] This invention provides a method for supplementing nutrients required for cell growth during the fermentation production of vitamin B12, comprising the following steps:

[0006] a) Ingredients: The culture medium is prepared according to the vitamin B12 fermentation production process;

[0007] Sterilization: The fermentation medium is sterilized in batches;

[0008] Inoculation: Using flame protection, inoculate the denitrified Pseudomonas aeruginosa suspension according to the inoculation amount;

[0009] b) Seed culture: After inoculation, control the operating temperature, pressure, air flow and stirring speed of the seed tank; after the culture is completed, transfer it to the fermentation tank for further culture.

[0010] c) Fermentation sterilization, inoculation and cultivation: The seed liquid is transferred into the fermenter according to the inoculation amount by pressure difference method; the operating temperature of the fermenter is controlled, and the required dissolved oxygen conditions and positive pressure environment are provided by aeration and stirring.

[0011] d) During the fermentation process, based on consumption levels, glucose is added as a carbon source to maintain basal metabolism. Nutrients are supplemented after 90 hours of operation. High-performance liquid chromatography (HPLC) is used starting at 50 hours, employing vitamin B12 as specified in the pharmacopoeia. 12 The detection method is to test the fermentation potency.

[0012] Preferably, in step a), the sterilization time is 25-35 minutes and the sterilization temperature is 120-125°C.

[0013] Preferably, in step a), the inoculation amount is 0.05-0.15%.

[0014] Preferably, in step b), the culture period is 20-60 hours, the temperature is controlled at 27.0-33.0℃, the tank pressure at 0.010-0.070 MPa, and the air flow rate at 0.5-2.5 m³ / h. 3 / h, stirring speed 100-400rpm.

[0015] Preferably, in step b), the inoculation amount is 10.00-30.00%; the seed tank culture indicators are: no contamination by other microorganisms and the bacterial count (od) reaches between 0.200-0.600, and the od detection device is a UV-Vis spectrophotometer with a wavelength of 700nm.

[0016] Preferably, in step c), the sterilization time is 25-35 minutes and the sterilization temperature is 120-125°C.

[0017] Preferably, in step c), the fermentation tank is controlled at a temperature of 28-35℃, a pressure of 0.020-0.070 MPa, and an air flow rate of 0.5-4.0 m³ / h. 3 Cultured at a stirring speed of 200-500 rpm for 1 hour.

[0018] Preferably, in step c), the fermentation tank culture cycle is 140-270 hours.

[0019] Preferably, in step d), the sugar content is controlled within the range of 1.0-7.0 g / 100 ml; the amount of nutrients added is 0.05-0.15 L / L of fermentation liquid volume.

[0020] Preferably, in step d), the nutrient is a mixed culture medium containing 3 g / L diammonium hydrogen phosphate, 1 g / L ammonium sulfate, 0.1 g / L zinc sulfate heptahydrate, 0.03 g / L ferrous sulfate, 2 g / L magnesium sulfate, 1.5 g / L calcium carbonate, and 5 g / L yeast extract.

[0021] Compared with existing technologies, this invention effectively improves the fermentation potency in the vitamin B12 fermentation production process, increases equipment utilization, and boosts fermentation yield. The fermentation potency is increased by 8.30%, and various costs are significantly reduced, enhancing the company's competitiveness in the market and possessing extremely high economic value. Detailed Implementation

[0022] The main equipment used in the following embodiments and comparative examples:

[0023] Serial Number Equipment Name Specification 1 Seed container 30L 2 Fermentation tank 50L

[0024] Example 1

[0025] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 120.0℃ for 25 minutes. After sterilization, inoculate with 10ml of seed culture (0.05% inoculum). After inoculation, control the fermentation temperature at 27.0℃, the tank pressure at 0.010MPa, and the air flow rate at 0.5-1.5m³ / h. 3 / h(0-10h 0.5m 3 / h; 10-20h 1.0m 3 / h; 1.5m after 20h 3The fermenter was run at a stirring speed of 100-200 rpm (100 rpm for 0-10 hours; 200 rpm after 10 hours). After 23 hours of operation, the od value was measured using a UV spectrophotometer (wavelength = 700 nm). If the od value reached 0.237 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 120.0℃ for 25 minutes, with a sterilization volume of 30 L. After inoculation, 3 L of seed culture (10.00% inoculum) was added. After transfer, the fermenter was operated at a temperature of 28.0℃, a pressure of 0.020 MPa, and an air flow rate of 0.5-2.0 m³ / h. 3 / h(0-10h 0.5m 3 / h; 10-50h 2.0m 3 / h; 1.5m after 50h 3 The stirring speed was controlled at 200-300 rpm (200 rpm for 0-10h; 300 rpm for 10-50h; 250 rpm after 50h). Glucose was added during operation, maintaining a sugar content range of 1.0-4.0 g / 100ml. After 90h, nutrients were added at a rate of 0.05 L / L fermentation broth volume. After 182h, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 245.3 mg / L.

[0026] Example 2

[0027] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 123.0℃ for 30 minutes. After sterilization, inoculate with 20ml of seed culture (0.10% inoculum). After inoculation, control the fermentation temperature at 30.0℃, the tank pressure at 0.040MPa, and the air flow rate at 1.0-2.0m³ / h. 3 / h(0-10h 1.0m 3 / h; 10-20h 1.5m 3 / h; 2.0m after 20h 3 The fermenter was run at a stirring speed of 200-300 rpm (200 rpm for 0-10 hours; 300 rpm after 10 hours). After 41 hours of operation, the od value (OD) was measured using a UV spectrophotometer (wavelength = 700 nm). If the OD reached 0.415 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 123.0℃ for 30 minutes, with a sterilization volume of 30 L. After inoculation, 6 L of seed culture (20.00% inoculation rate) was added. After inoculation, the fermenter was operated at a temperature of 31.0℃, a pressure of 0.040 MPa, and an air flow rate of 1.5-3.0 m³ / h. 3 / h(0-10h 1.5m 3 / h; 10-50h 3.0m3 / h; 2.5m after 50h 3 The stirring speed was controlled at 300-400 rpm (0-10h 300 rpm; 10-50h 400 rpm; after 50h 350 rpm). Glucose was added during operation, controlling the sugar content within the range of 4.0-6.0 g / 100ml. After 90 hours of operation, nutrients were added at a rate of 0.10 L / L. After 186 hours of operation, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 253.2 mg / L.

[0028] Example 3

[0029] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 125.0℃ for 35 minutes. After sterilization, inoculate with 30ml of seed culture (0.15% inoculum). After inoculation, control the fermentation temperature at 33.0℃, the tank pressure at 0.070MPa, and the air flow rate at 1.5-2.5m³ / h. 3 / h(0-10h 1.5m 3 / h; 10-20h 2.0m 3 / h; 2.5m after 20h 3 The fermenter was run at a stirring speed of 300-400 rpm (300 rpm for 0-10 hours; 400 rpm after 10 hours). After 23 hours of operation, the od value was measured using a UV spectrophotometer (wavelength = 700 nm). If the od value reached 0.587 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 125.0℃ for 35 minutes, with a sterilization volume of 30 L. After inoculation, 9 L of seed culture (30.00% inoculation) was added. After transfer, the fermenter was operated at a temperature of 35.0℃, a pressure of 0.070 MPa, and an air flow rate of 2.5-4.0 m³ / h. 3 / h(0-10h 2.5m 3 / h; 10-50h 4.0m 3 / h; 3.5m after 50h 3 The stirring speed was controlled at 400-500 rpm (400 rpm for 0-10h; 500 rpm for 10-50h; 450 rpm after 50h). Glucose was added during operation, maintaining a sugar content range of 5.0-7.0 g / 100ml. After 90h, nutrients were added at a rate of 0.15 L / L. After 195h, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 261.2 mg / L.

[0030] Comparative Example 1

[0031] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 120.0℃ for 25 minutes. After sterilization, inoculate with 10ml of seed culture (0.05% inoculum). After inoculation, control the fermentation temperature at 27.0℃, the tank pressure at 0.010MPa, and the air flow rate at 0.5-1.5m³ / h. 3 / h(0-10h 0.5m 3 / h; 10-20h 1.0m 3 / h; 1.5m after 20h 3 The fermenter was run at a stirring speed of 100-200 rpm (100 rpm for 0-10 hours; 200 rpm after 10 hours). After 23 hours of operation, the od value was measured using a UV spectrophotometer (wavelength = 700 nm). If the od value reached 0.232 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 120.0℃ for 25 minutes, with a sterilization volume of 30 L. After inoculation, 3 L of seed culture (10.00% inoculum) was added. After transfer, the fermenter was operated at a temperature of 28.0℃, a pressure of 0.020 MPa, and an air flow rate of 0.5-2.0 m³ / h. 3 / h(0-10h 0.5m 3 / h; 10-50h 2.0m 3 / h; 1.5m after 50h 3 The stirring speed was controlled at 200-300 rpm (200 rpm for 0-10h; 300 rpm for 10-50h; 250 rpm after 50h). Glucose was added during operation, maintaining a sugar content range of 1.0-4.0 g / 100ml. After 176 hours of operation, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 232.5 mg / L.

[0032] Comparative Example 2

[0033] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 123.0℃ for 30 minutes. After sterilization, inoculate with 20ml of seed culture (0.10% inoculum). After inoculation, control the fermentation temperature at 30.0℃, the tank pressure at 0.040MPa, and the air flow rate at 1.0-2.0m³ / h. 3 / h(0-10h 1.0m 3 / h; 10-20h 1.5m 3 / h; 2.0m after 20h 3The fermenter was run at a stirring speed of 200-300 rpm (200 rpm for 0-10 hours; 300 rpm after 10 hours). After 41 hours of operation, the od value (OD) was measured using a UV spectrophotometer (wavelength = 700 nm). If the OD reached 0.416 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 123.0℃ for 30 minutes, with a sterilization volume of 30 L. After inoculation, 6 L of seed culture (20.00% inoculum) was added. After transfer, the fermenter was operated at a temperature of 31.0℃, a pressure of 0.040 MPa, and an air flow rate of 1.5-3.0 m³ / h. 3 / h(0-10h 1.5m 3 / h; 10-50h 3.0m 3 / h; 2.5m after 50h 3 The stirring speed was controlled at 300-400 rpm (300 rpm for 0-10h; 400 rpm for 10-50h; 350 rpm after 50h). Glucose was added during operation, maintaining a sugar content range of 4.0-6.0 g / 100ml. After 175h of operation, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 231.5 mg / L.

[0034] Comparative Example 3

[0035] Add seed culture medium to the seed tank and bring the volume to 20L. Sterilize by steam at 125.0℃ for 35 minutes. After sterilization, inoculate with 30ml of seed culture (0.15% inoculum). After inoculation, control the fermentation temperature at 33.0℃, the tank pressure at 0.070MPa, and the air flow rate at 1.5-2.5m³ / h. 3 / h(0-10h 1.5m 3 / h; 10-20h 2.0m 3 / h; 2.5m after 20h 3 The fermenter was run at a stirring speed of 300-400 rpm (300 rpm for 0-10 hours; 400 rpm after 10 hours). After 23 hours of operation, the od value was measured using a UV spectrophotometer (wavelength = 700 nm). If the od value reached 0.592 and there was no contamination, the fermenter was transferred to a sterilized fermenter. The fermenter was sterilized at 125.0℃ for 35 minutes, with a sterilization volume of 30 L. After inoculation, 9 L of seed culture (30.00% inoculum) was added. After transfer, the fermenter was operated at a temperature of 35.0℃, a pressure of 0.070 MPa, and an air flow rate of 2.5-4.0 m³ / h. 3 / h(0-10h 2.5m 3 / h; 10-50h 4.0m 3 / h; 3.5m after 50h 3The stirring speed was controlled at 400-500 rpm (400 rpm for 0-10h; 500 rpm for 10-50h; 450 rpm after 50h). Glucose was added during operation, maintaining a sugar content range of 5.0-7.0 g / 100ml. After 172 hours of operation, the vitamin B12 content was determined using high-performance liquid chromatography (HPLC), and the result was 237.5 mg / L.

[0036] Comparison results of Examples 1-3 and Comparative Examples 1-3

[0037]

[0038] Based on the implementation cases, this invention can effectively improve the fermentation cycle of vitamin B12 by 7.46%; at the same time, it also has a significant effect on improving the fermentation potency by 8.30%, which has extremely high economic value.

Claims

1. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that, Includes the following steps: a) Culture medium preparation, batch sterilization, and inoculation; b) Seed culture, after which the seed culture is completed, is transferred to a fermenter for further culture; c) Fermentation sterilization, inoculation, and culture; d) During the fermentation process, add carbon source glucose to maintain basal metabolism based on the consumption. After 90 hours of operation, add nutrients.

2. A method for improving the cell activity during the fermentation production of vitamin B12, characterized in that: The sterilization time in step a) is 25-35 min, and the sterilization temperature is 120-125℃.

3. A method for improving the cell activity during the fermentation production of vitamin B12, characterized in that: In step b), the culture period is 20-60 hours, with the temperature controlled at 27.0-33.0℃, the tank pressure at 0.010-0.070 MPa, and the air flow rate at 0.5-2.5 m³ / h. 3 / h, stirring speed 100-400rpm.

4. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step b), the inoculation amount is 10.00-30.00%.

5. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step c), the sterilization time is 25-35 minutes and the sterilization temperature is 120-125℃.

6. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step c), the fermentation tank temperature is controlled at 28-35℃, the tank pressure at 0.020-0.070 MPa, and the air flow rate at 0.5-4.0 m³ / h. 3 Cultured at a stirring speed of 200-500 rpm for 1 hour.

7. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step c), the fermentation tank culture cycle is 140-270 hours.

8. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step d), the sugar content is controlled within the range of 1.0-7.0 g / 100 ml, and the amount of nutrients added is 0.05-0.15 L / L of fermentation liquid volume.

9. A method for improving cell activity during the fermentation production of vitamin B12, characterized in that: In step d), the nutrient is a mixed culture medium containing 3 g / L diammonium hydrogen phosphate, 1 g / L ammonium sulfate, 0.1 g / L zinc sulfate heptahydrate, 0.03 g / L ferrous sulfate, 2 g / L magnesium sulfate, 1.5 g / L calcium carbonate, and 5 g / L yeast extract.

Images