Culture medium for producing vitamin B2 through fermentation of bacillus subtilis engineering bacteria

Abstract

The invention relates to a culture medium for producing vitamin B2 through fermentation of bacillus subtilis engineering bacteria, the culture medium comprises a first-stage seed culture medium, a second-stage seed culture medium and a fermentation culture medium, disaccharide is used in the culture medium to replace glucose, and the repression effect of high-concentration sugar on metabolites is avoided; according to the culture medium, sorbitol is used for replacing antibiotics, so that the culture medium has a bacteriostatic effect, but does not cause gene mutation of bacillus subtilis engineering bacteria, and is beneficial to ecological environment safety and human health; the sorbitol can improve the cell membrane osmotic pressure of the bacillus subtilis engineering bacteria, so that the metabolite vitamin B2 in the cells can be quickly leaked out of the cells, the feedback inhibition effect of end products is relieved, and the yield of the fermentation product vitamin B2 is improved. The average fermentation titer of the culture medium reaches 35.7 g / L, and compared with the traditional technology, the fermentation level is improved by 11% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a culture medium for producing vitamin B2 by fermenting engineering bacteria of Bacillus subtilis. Background Art

[0002] Vitamin B2 (VB2), also known as riboflavin, has a chemical name: 7,8-dimethyl-10 (1, -D-ribitol)-isoalloxazine. VB2 is a component of the cofactor of flavonoids in humans and animals (flavonoids play a role in transferring hydrogen in biological oxidation and reduction). It is one of the indispensable vitamins in biological life activities. It has a close relationship with the metabolism of carbohydrates, fats and amino acids, and plays an important role in the growth and development of organisms. VB2 cannot be synthesized in humans and animals and must be supplied by food. When VB2 is lacking, it affects the biological oxidation of the body, causing metabolic disorders and causing a variety of diseases, such as cardiovascular disease, angular cheilitis, migraine, angina pectoris, cataracts, malaria, etc. Therefore, this product can be used for the prevention and treatment of the above diseases. In recent years, multi-dimensional preparations used for anti-aging and health care at home and abroad contain VB2. This type of medicine is not only limited to treating diseases but also used for health care, improving the body's disease resistance and prolonging life. VB2 can also be used as feed additives and cosmetics.

[0003] VB2 chemical formula: C 17 H 20 N4O6, molecular weight: 376.37

[0004] Structural formula (oxidation state):

[0005]

[0006] Currently, the industrial production of vitamin B2 mainly includes two methods: microbial fermentation and chemical semi-synthesis.

[0007] The semi-synthetic production method is to produce D-ribose by microbial fermentation, and then use D-ribose as raw material to produce vitamin B2 by synthesis; the microbial fermentation method is divided into traditional yeast fermentation method and new genetically engineered bacteria fermentation method. The yeast fermentation method mainly uses strains including Ashbya gossyp ii and Eremothecium ashbyii.

[0008] Microbial fermentation has become the mainstream of industrial production due to its advantages such as simple production process, cheap raw materials, environmental friendliness, and renewable resources.

[0009] Although the yeast fermentation method for producing vitamin B2 has a long fermentation cycle (6-7 days), a low fermentation unit (5-6 g / L), and a lower production efficiency than the semi-synthetic method, it is widely used in the industrial production of riboflavin because of its economical and effective production process, easy automation control, non-toxic products, and high purity.

[0010] With the rapid development of life science and technology, a new method of producing vitamin B2 by fermentation using genetically engineered bacteria has emerged since the 1990s, that is, using DNA recombination technology to construct genetically engineered bacteria that can over-synthesize vitamin B2. The fermentation cycle of genetically engineered bacteria is short (2 to 3 days) and the fermentation unit is high (more than 10 grams / liter). It has the characteristics of good product quality of yeast fermentation method and the advantages of high production efficiency of chemical semi-synthesis method. It represents the development direction of vitamin B2 production technology. Among them, genetically engineered bacteria with Bacillus subtilis as the receptor bacteria have a relatively mature process and have been widely used in the industrial production of vitamin B2.

[0011] At present, the main problems of using genetically engineered bacteria fermentation in China are as follows:

[0012] 1) In order to inhibit the growth of foreign bacteria, a small amount of antibiotics is often added to the fermentation medium when using Bacillus subtilis engineered bacteria to ferment and produce vitamin B2. This can easily cause the Bacillus subtilis engineered bacteria to produce resistance genes or carry unknown mutant genes. If the fermentation liquid and bacterial residue are not handled properly, environmental microorganisms can easily develop drug resistance, which will have a harmful impact on the ecological environment and human health.

[0013] 2) In the fermentation medium of Bacillus subtilis engineered bacteria, the main carbon source is glucose, which is easy to form a high-concentration glucose solution, which can easily cause overflow metabolism of Bacillus subtilis engineered bacteria, inhibit vitamin B2 metabolism, affect the fermentation level of vitamin B2, and reduce the fermentation yield of vitamin B2. Summary of the invention

[0014] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide a culture medium for producing vitamin B2 by fermentation of Bacillus subtilis engineered bacteria, which can effectively increase fermentation biomass, enhance enzyme activity and reduce production costs.

[0015] The technical solutions adopted to achieve the above objectives are:

[0016] A culture medium for producing vitamin B2 by fermentation of Bacillus subtilis engineered bacteria, characterized in that it comprises a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein

[0017] The primary seed culture medium is composed of: corn steep liquor (in dry form) 10-30 g / L, yeast powder 3-6 g / L, disaccharide 6-8 g / L, (NH4)2SO4 5-10 g / L, magnesium sulfate 0.3-1 g / L, light calcium carbonate 6-9 g / L, and dipotassium hydrogen phosphate 1-3 g / L;

[0018] The secondary seed culture medium is composed of: corn steep liquor (in dry form) 10-30 g / L, yeast powder 6-8 g / L, disaccharide 6-8 g / L, (NH4)2SO4 5-10 g / L, magnesium sulfate 0.3-1 g / L, light calcium carbonate 6-9 g / L, and dipotassium hydrogen phosphate 1-3 g / L;

[0019] The fermentation medium comprises: 30-50 g / L corn steep liquor (in dry form), 6-8 g / L yeast powder, 0.5-1.5 g / L betaine, 6-8 g / L disaccharide, 5-10 g / L (NH4)2SO4, 0.3-1 g / L magnesium sulfate, 6-9 g / L light calcium carbonate, 1-3 g / L dipotassium hydrogen phosphate, and 0.08-0.12 g / L polyether modified silicon;

[0020] Wherein: the first-level seed culture medium, the second-level seed culture medium and the fermentation culture medium also include sorbitol or isosorbide.

[0021] In some embodiments of the present invention, the disaccharide is selected from one of sucrose, lactose and maltose;

[0022] Preferably, the disaccharide is selected from maltose.

[0023] In some embodiments of the present invention, the weight percentage of sorbitol or isosorbide is 1 to 5 g / L;

[0024] Preferably, the weight percentage of the sorbitol or isosorbide is 2 to 4 g / L;

[0025] More preferably, the weight percentage of the sorbitol or isosorbide is 3 g / L.

[0026] In some embodiments of the present invention, the quality requirements of the primary seed culture medium after sterilization are: amino nitrogen 100-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.5.

[0027] In some embodiments of the present invention, the quality requirements of the secondary seed culture medium after sterilization are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.5.

[0028] In some embodiments of the present invention, the quality requirements of the fermentation medium after sterilization are: amino nitrogen 150-300 mg / 100 ml, total sugar 2.0-3.6 g / 100 ml, pH 6.8-7.5.

[0029] The present invention achieves at least one of the following beneficial effects:

[0030] 1. Disaccharides are used in the fermentation medium of the present invention to replace glucose, thereby avoiding the inhibitory effect of high-concentration sugar on metabolites and increasing the fermentation unit by more than 11% compared with the traditional process.

[0031] 2. Sorbitol or isosorbide is used in the fermentation medium of the present invention to replace antibiotics, which has antibacterial effect and can inhibit the growth of miscellaneous bacteria. However, sorbitol or isosorbide has low toxicity and will not cause gene mutation of Bacillus subtilis engineering bacteria, which is beneficial to ecological environment safety and human health; sorbitol can increase the osmotic pressure of the cell membrane of Bacillus subtilis engineering bacteria, so that the metabolite vitamin B2 in the cell quickly leaks out of the cell. The feedback inhibition of the end product is eliminated, and the yield of the fermentation product vitamin B2 is increased.

[0032] 3. The present invention confirms the optimal compatibility ratio of carbon source and nitrogen source of primary seeds, secondary seeds and fermentation tank culture medium for fermentation and production of vitamin B2 by Bacillus subtilis engineered bacteria. The growth and metabolic environment of the bacteria is stable, which is conducive to the fermentation yield and quality stability. DETAILED DESCRIPTION

[0033] The present invention is described below with examples. It should be understood that the examples are used to illustrate the present invention rather than to limit the present invention. The scope and core content of the present invention are determined according to the claims.

[0034] The bacterial strain process of the following embodiment is:

[0035] First-level seed cultivation process:

[0036] Under flame protection, the cultured Bacillus subtilis engineered bacteria mother bottle mycelium is inoculated at a volume of about 0.8L into a sterilized, sterile air-pressurized, 38±0.5°C primary seed culture medium for primary seed culture.

[0037] The first-level seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 38±0.5℃; air flow 50-100m3 / h; stirring speed 120-200r / min, culture time about 20h, transplanting. The transplanting ratio is 1:0.08-0.12 (V / V), and the seeds are transferred to the sterilized, sterile air pressure-maintained, temperature 38±0.5℃ second-level seed culture medium for second-level seed culture.

[0038] Secondary seed cultivation process:

[0039] The conditions for the secondary seed culture are: tank pressure 0.03-0.05MPa; tank temperature 38±0.5℃; air flow 1500-1000m3 / h; stirring speed 100-200r / min, and the culture time is about 10h. The ratio of seed transfer is 1:0.08-0.12 (V / V), and the seeds are transferred into the sterilized fermentation medium with sterile air pressure and temperature of 38±0.5℃ for fermentation culture.

[0040] Fermentation culture process:

[0041] The fermentation tank culture conditions are: fermentation temperature: 38±0.5℃; tank pressure 0.03~0.05MPa; air flow rate: 1:0.6~1.2V / V / min (fermentation liquid volume m3: sterile air volume m3 per minute); stirring speed 140-160r / min; pH controlled at 6.5-7.0.

[0042] Fermentation feeding process: Feeding starts 10 hours after the fermentation tank is transplanted, and stops about 2 hours before the end of fermentation. The feeding method is adopted, and the feeding liquid is a sterilized 40-50% (g / g) liquefied glucose solution at a temperature of 30-35°C, and a sterile filtered ammonia solution with a concentration of 20-25% (g / g).

[0043] Fermentation process monitoring and control: sugar concentration 1.0-3.0g / 100ml, amino nitrogen 100-200mg / 100ml

[0044] Requirements for the end of fermentation culture:

[0045] The fermentation cycle is controlled at 55 to 65 hours.

[0046] Example 1

[0047] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0048] Seed culture medium: corn syrup (dry powder) 11kg, yeast powder 3.3kg, maltose 6.6kg, sorbitol 1.1kg, (NH4)2SO4 5.5kg, magnesium sulfate 0.33kg, light calcium carbonate 6.6kg, dipotassium hydrogen phosphate 1.1kg. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 100mg / 100ml, total sugar 3.0g / 100ml, pH 6.5.

[0049] Secondary seed culture: effective volume of seed culture medium 11m 3 .

[0050] Seed culture medium: corn syrup (dry powder) 110kg, yeast powder 66kg, maltose 66kg, sorbitol 11kg, (NH4)2SO4 55kg, magnesium sulfate 3.3kg, light calcium carbonate 66kg, dipotassium hydrogen phosphate 11kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 110mg / 100ml, total sugar 3.0g / 100ml, pH 6.8.

[0051] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0052] 1. Corn syrup (dry powder) 3300kg, yeast powder 660kg, betaine 55kg, maltose 660kg, sorbitol 110kg, (NH4)2SO4 550kg, magnesium sulfate 33kg, light calcium carbonate 660kg, dipotassium hydrogen phosphate 110kg, polyether modified silicon 8.8kg. The quality of the fermentation medium after sterilization is: amino nitrogen 150mg / 100ml, total sugar 2.0g / 100ml, pH 6.8.

[0053] The fermentation culture was completed with a fermentation titer of 35.6 g / L and a fermentation cycle of 62 h.

[0054] Example 2

[0055] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0056] Seed culture medium: corn syrup (dry powder) 16.5kg, yeast powder 4.4kg, maltose 7.7kg, sorbitol 2.2kg, (NH4)2SO4 8.25kg, magnesium sulfate 0.55kg, light calcium carbonate 7.7kg, dipotassium hydrogen phosphate 2.2kg. The quality of the first-level seed culture medium after sterilization is: amino nitrogen 135mg / 100ml, total sugar 3.9g / 100ml, pH 6.9.

[0057] Secondary seed culture: effective volume of seed culture medium 11m 3 .

[0058] Seed culture medium: corn syrup (dry powder) 165kg, yeast powder 77kg, maltose 77kg, sorbitol 22kg, (NH4)2SO4 82.5kg, magnesium sulfate 5.5kg, light calcium carbonate 77kg, dipotassium hydrogen phosphate 22kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 122mg / 100ml, total sugar 3.4g / 100ml, pH 6.5.

[0059] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0060] Corn syrup (dry powder) 3850kg, yeast powder 770kg, betaine 110kg, maltose 770kg, sorbitol 220kg, (NH4)2SO4 825kg, magnesium sulfate 55kg, light calcium carbonate 770kg, dipotassium hydrogen phosphate 220kg, polyether modified silicon 9.9kg. The quality of the fermentation medium after sterilization is: amino nitrogen 180mg / 100ml, total sugar 2.4g / 100ml, pH 7.0.

[0061] The fermentation culture was completed with a fermentation titer of 35.9 g / L and a fermentation cycle of 55 h.

[0062] Example 3

[0063] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0064] Seed culture medium: corn syrup (dry powder) 33kg, yeast powder 5.5kg, maltose 8.8kg, sorbitol 3.3kg, (NH4)2SO4 11kg, magnesium sulfate 0.77kg, light calcium carbonate 8.8kg, dipotassium hydrogen phosphate 3.3kg. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 150mg / 100ml, total sugar 5.0g / 100ml, pH 7.3.

[0065] Secondary seed culture: effective volume of seed culture medium 11m 3 .

[0066] Seed culture medium: corn syrup (dry powder) 330kg, yeast powder 88kg, maltose 88kg, sorbitol 33kg, (NH4)2SO4 110kg, magnesium sulfate 7.7kg, light calcium carbonate 88kg, dipotassium hydrogen phosphate 33kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 150mg / 100ml, total sugar 4.6g / 100ml, pH 7.2.

[0067] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0068] Corn syrup (dry powder) 4400kg, yeast powder 880kg, betaine 165kg, maltose 880kg, sorbitol 330kg, (NH4)2SO4 1100kg, magnesium sulfate 77kg, light calcium carbonate 880kg, dipotassium hydrogen phosphate 330kg, polyether modified silicon 11kg. The quality of the fermentation medium after sterilization is: amino nitrogen 300mg / 100ml, total sugar 3.6g / 100ml, pH 7.2.

[0069] The fermentation culture was completed with a fermentation titer of 36.1 g / L and a fermentation cycle of 63 h.

[0070] Example 4

[0071] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0072] Seed culture medium: corn syrup (dry powder) 16.5kg, yeast powder 6.6kg, maltose 7.7kg, sorbitol 4.4kg, (NH4)2SO4 8.25kg, magnesium sulfate 1.1kg, light calcium carbonate 9.9kg, dipotassium hydrogen phosphate 2.2kg. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 140mg / 100ml, total sugar 4.2g / 100ml, pH 7.5.

[0073] Secondary seed culture: effective volume of seed culture medium 11m 3 .

[0074] Seed culture medium: corn syrup (dry powder) 165kg, yeast powder 77kg, maltose 77kg, sorbitol 44kg, (NH4)2SO4 82.5kg, magnesium sulfate 11kg, light calcium carbonate 99kg, dipotassium hydrogen phosphate 22kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 123mg / 100ml, total sugar 4.6g / 100ml, pH 7.5.

[0075] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0076] Corn syrup (dry powder) 4950kg, yeast powder 770kg, betaine 110kg, maltose 770kg, sorbitol 440kg, (NH4)2SO4 825kg, magnesium sulfate 110kg, light calcium carbonate 990kg, dipotassium hydrogen phosphate 220kg, polyether modified silicon 12.1kg. The quality of the fermentation medium after sterilization is: amino nitrogen 230mg / 100ml, total sugar 3.2g / 100ml, pH 7.5.

[0077] The fermentation culture was completed with a fermentation titer of 35.7 g / L and a fermentation cycle of 65 h.

[0078] Example 5

[0079] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0080] Seed culture medium: corn syrup (dry powder) 16.5kg, yeast powder 4.95kg, maltose 7.7kg, sorbitol 5.5kg, (NH4)2SO4 8.25kg, magnesium sulfate 0.72kg, light calcium carbonate 8.2kg, dipotassium hydrogen phosphate 2.2kg. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 136mg / 100ml, total sugar 4.8g / 100ml, pH 6.8.

[0081] Secondary seed culture: effective volume of seed culture medium 11m 3 .

[0082] Seed culture medium: corn syrup (dry powder) 165kg, yeast powder 77kg, maltose 77kg, sorbitol 55kg, (NH4)2SO4 82.5kg, magnesium sulfate 7.2kg, light calcium carbonate 82.5kg, dipotassium hydrogen phosphate 22kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 125mg / 100ml, total sugar 5.0g / 100ml, pH 7.3.

[0083] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0084] Corn syrup (dry powder) 5500kg, yeast powder 770kg, betaine 110kg, maltose 770kg, sorbitol 550kg, (NH4)2SO4 825kg, magnesium sulfate 71.5kg, light calcium carbonate 825kg, dipotassium hydrogen phosphate 220kg, polyether modified silicon 13.2kg. The quality of the fermentation medium after sterilization is: amino nitrogen 290mg / 100ml, total sugar 3.4g / 100ml, pH 7.1.

[0085] The fermentation culture was completed with a fermentation titer of 35.2 g / L and a fermentation cycle of 63 h.

[0086] Comparative Case 1

[0087] Primary seed culture: effective volume of seed culture medium 1.1m 3 .

[0088] First-level seed culture medium: corn syrup (dry powder) 22kg, yeast powder 5.5g / L, glucose 16.5kg, (NH4)2SO4 8.8kg, magnesium sulfate 0.66kg, light calcium carbonate 6.6kg, dipotassium hydrogen phosphate 2.2kg, erythromycin 0.011kg, chloramphenicol 0.011kg. The mass of the first-level seed culture medium after sterilization is: amino nitrogen 145mg / 100ml, total sugar 4.8g / 100ml, pH 6.5.

[0089] Secondary seed culture medium: effective volume of seed culture medium 11m 3 .

[0090] Corn syrup (dry powder) 220kg, yeast powder 66kg, glucose 220kg, (NH4)2SO4 88kg, magnesium sulfate 6.6kg, light calcium carbonate 88kg, dipotassium hydrogen phosphate 25kg, erythromycin 0.11kg, chloramphenicol 0.11kg. The quality of the secondary seed culture medium after sterilization is: amino nitrogen 134mg / 100ml, total sugar 4.6g / 100ml, pH 6.9.

[0091] Fermentation culture: effective volume of fermentation medium 110m 3 .

[0092] Corn syrup (dry powder) 4400kg, yeast powder 660kg, betaine 110kg, glucose 2200kg, (NH4)2SO4 1100kg, magnesium sulfate 66kg, light calcium carbonate 770kg, dipotassium hydrogen phosphate 220kg, polyether modified silicon 11kg, erythromycin 1.1kg, chloramphenicol 1.1kg. The quality of the fermentation medium after sterilization is: amino nitrogen 236mg / 100ml, total sugar 3.8g / 100ml, pH 7.6. The fermentation culture is completed, the fermentation titer is 31.8g / L, and the fermentation cycle is 61h.

Claims

1. A culture medium for producing vitamin B2 by fermentation of Bacillus subtilis engineered bacteria, comprising a primary seed culture medium, a secondary seed culture medium and a fermentation culture medium, wherein The primary seed culture medium is composed of: corn steep liquor (in dry form) 10-30 g / L, yeast powder 3-6 g / L, disaccharide 6-8 g / L, (NH4)2SO4 5-10 g / L, magnesium sulfate 0.3-1 g / L, light calcium carbonate 6-9 g / L, and dipotassium hydrogen phosphate 1-3 g / L; The secondary seed culture medium is composed of: corn steep liquor (in dry form) 10-30 g / L, yeast powder 6-8 g / L, disaccharide 6-8 g / L, (NH4)2SO4 5-10 g / L, magnesium sulfate 0.3-1 g / L, light calcium carbonate 6-9 g / L, and dipotassium hydrogen phosphate 1-3 g / L; The fermentation medium comprises: 30-50 g / L corn steep liquor (in dry form), 6-8 g / L yeast powder, 0.5-1.5 g / L betaine, 6-8 g / L disaccharide, 5-10 g / L (NH4)2SO4, 0.3-1 g / L magnesium sulfate, 6-9 g / L light calcium carbonate, 1-3 g / L dipotassium hydrogen phosphate, and 0.08-0.12 g / L polyether modified silicon; Features: The primary seed culture medium, the secondary seed culture medium and the fermentation culture medium also include sorbitol or isosorbide; The disaccharide is maltose.

2. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 1, wherein the weight percentage of sorbitol or isosorbide is 1 to 5 g / L.

3. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 2, wherein the weight percentage of sorbitol or isosorbide is 2-4 g / L.

4. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 3, wherein the weight percentage of sorbitol or isosorbide is 3 g / L.

5. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 1, wherein the quality requirements of the first-level seed culture medium after sterilization are: amino nitrogen 100-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.

5.

6. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 1, wherein the quality requirements of the secondary seed culture medium after sterilization are: amino nitrogen 110-150 mg / 100 ml, total sugar 3.0-5.0 g / 100 ml, pH 6.5-7.

5.

7. The culture medium for producing vitamin B2 by fermentation using Bacillus subtilis engineered bacteria according to claim 1, wherein the quality requirements of the fermentation medium after sterilization are: amino nitrogen 150-300 mg / 100 ml, total sugar 2.0-3.6 g / 100 ml, pH 6.8-7.5.

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