Method for producing L-alanine through fermentation

Abstract

The invention belongs to the technical field of biological fermentation, and discloses a method for producing L-alanine through fermentation. The method adopts two-stage fermentation, namely aerobic stage fermentation and anaerobic fermentation after 6 hours, and comprises the following steps: inoculating an escherichia coli seed solution into a fermentation culture medium, controlling the fermentation temperature to be 35-40 DEG C, the tank pressure to be 0.02-0.05 Mpa, the PH to be 6.5-7.0 and the residual sugar to be 0.2-0.5%, controlling the dissolved oxygen to be more than 50% in the aerobic stage in the fermentation process, controlling the dissolved oxygen to be 0 after anaerobic fermentation, replacing soy peptone with bone peptone, and fermenting for 1-2 hours. The fermentation process is optimized, the fermentation efficiency of the L-alanine is improved, and the method is simple in process and suitable for large-scale industrial production.

Description

Technical Field

[0001] The invention relates to a method for producing L-alanine by fermentation, belonging to the technical field of biological fermentation. Background Art

[0002] L-alanine, also known as La-aminopropionic acid, is one of the more than 20 amino acids that make up human protein and is also the amino acid with the highest content in human blood. The molecular formula of L-alanine is CH3CH(NH2)COOH, with a molecular mass of 89.09 and a density of 1.432 g / cm 3 , melting point is 297°C, appearance is colorless to white crystalline powder, soluble in water, slightly soluble in ethanol, insoluble in ether and acetone, odorless and non-toxic, with umami and sweet taste, of which the sweetness is 1.2 times that of sucrose. It is mainly used in biochemical research, tissue culture, liver function test, flavor enhancer, can increase the flavoring effect of condiments, and can also be used as a sourness corrector to improve the sourness of organic acids.

[0003] L-alanine is a non-essential amino acid for the human body and the most widely used alanine variety. It is mainly used in many fields such as daily chemicals, pharmaceuticals and health products, food additives and feed. The demand in the daily chemical field accounted for 55% in 2019. It is mainly used to synthesize the new environmentally friendly chelating agent MGDA, which is a patented green chelating agent developed by BASF. At present, the mainstream process of L-alanine is fermentation, which has significant cost advantages over the enzyme method, and the production process is more environmentally friendly. Process innovation and cost reduction have promoted the large-scale expansion of the application of L-alanine from the fields of medicines, health products and food additives to the field of daily chemicals. Summary of the invention

[0004] The present invention adopts a fermentation method to produce L-alanine, optimizes fermentation conditions, improves the fermentation production efficiency of L-alanine, reduces production costs, and further promotes the wide application of L-alanine in daily chemicals and medical treatment.

[0005] The present invention is achieved through the following technical solutions.

[0006] A method for producing L-alanine by fermentation, characterized in that the method comprises the following steps: Step 1) Seed culture: inoculating the L-alanine-producing Escherichia coli seed solution into the seed culture medium for culture to obtain the seed solution;

[0007] Step 2) Fermentation culture: The seed liquid obtained in step 1) is inoculated into the fermentation medium, and the fermentation conditions are controlled to carry out L-alanine fermentation, using aerobic-anaerobic two-stage fermentation, growing to OD 20-30, and switching to anaerobic fermentation after 6 hours, and the fermentation ends after 48 hours to obtain the fermentation liquid.

[0008] Preferably, in step 1), the inoculation concentration of the Escherichia coli seed solution is 10-20%.

[0009] Preferably, in step 1), the formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixture.

[0010] Preferably, in step 1), the cultivation parameters are: air volume control 2-3m 3 / h, dissolved oxygen controlled above 50%, fermentation pH 6.5-7.0, rotation speed 350-450rpm, tank pressure 0.01-0.1Mpa.

[0011] Preferably, the formula of the fermentation medium in step 2) is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixture.

[0012] Preferably, it is characterized in that in step 2), the fermentation temperature is 30-45°C, the fermentation pH is 6.5-7.0, and the tank pressure is 0.01-0.1Mpa.

[0013] Preferably, in step 2), the air volume in the aerobic stage is controlled at 2-3m 3 / h, dissolved oxygen controlled above 50%, speed 350-450rpm.

[0014] Preferably, in step 2), the air volume in the anaerobic stage is adjusted to 0, the dissolved oxygen is controlled to 0, and the stirring speed is adjusted to 250-500 rpm.

[0015] Preferably, the peptone is bone peptone or soybean peptone, more preferably bone peptone.

[0016] Preferably, the residual sugar content of the fermentation broth is controlled to be 0 or 0.2-0.5%, more preferably 0.2-0.5%.

[0017] The beneficial effects achieved by the present invention mainly include but are not limited to the following aspects: In the fermentation process of the present invention, the dissolved oxygen is controlled to be above 50% in the aerobic stage, and is controlled to be 0 after switching to anaerobic stage. Bone peptone is used instead of soybean peptone to optimize the fermentation process, thereby improving the fermentation efficiency of L-alanine, and the acid production of L-alanine during fermentation is increased from 120 g / L to 140 g / L. The method is simple in process and suitable for large-scale industrial production. DETAILED DESCRIPTION

[0018] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The products and methods of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products and methods described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments. Example

[0019] A method for producing L-alanine by fermentation, comprising the following steps: Step 1) Seed cultivation:

[0020] The Escherichia coli seed liquid is inoculated into the seed culture medium for culturing to obtain the seed liquid.

[0021] The formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixed solution. Step 2) Fermentation

[0022] The formula of the fermentation medium is: 10g / L glucose, 1.5g / L magnesium sulfate, 5g / L yeast powder, 15g / L corn steep liquor, 2g / L potassium dihydrogen phosphate, 7.5g / L bone peptone, 24ml defoamer, and 2g / L trace element mixed solution.

[0023] The seed liquid was inoculated into the fermentation medium at an inoculation rate of 15-20%, the dissolved oxygen was controlled at 50%, the pH was 6.5-7.0, and the fermentation was carried out at a temperature of 30-40°C to obtain L-alanine fermentation liquid. The acid production of L-alanine was tested to be 140 g / L. Example

[0024] A method for producing L-alanine by fermentation, comprising the following steps: Step 1) Seed cultivation:

[0025] The Escherichia coli seed liquid is inoculated into the seed culture medium for culturing to obtain the seed liquid.

[0026] The formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixed solution. Step 2) Fermentation

[0027] The formula of the fermentation medium is: 10g / L glucose, 1.5g / L magnesium sulfate, 5g / L yeast powder, 15g / L corn steep liquor, 2g / L potassium dihydrogen phosphate, 7.5g / L soy peptone, 24ml defoamer, and 2g / L trace element mixed solution.

[0028] The seed liquid was inoculated into the fermentation medium at an inoculation rate of 15-20%, the dissolved oxygen was controlled at 50%, the pH was 6.5-7.0, and the fermentation was carried out at a temperature of 30-40°C to obtain L-alanine fermentation liquid. The acid production of L-alanine was tested to be 120 g / L. Example

[0029] A method for producing L-alanine by fermentation, comprising the following steps: Step 1) Seed cultivation:

[0030] The Escherichia coli seed liquid is inoculated into the seed culture medium for culturing to obtain the seed liquid.

[0031] The formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixed solution. Step 2) Fermentation

[0032] The formula of the fermentation medium is: 10g / L glucose, 1.5g / L magnesium sulfate, 5g / L yeast powder, 15g / L corn steep liquor, 2g / L potassium dihydrogen phosphate, 7.5g / L bone peptone, 24ml defoamer, and 2g / L trace element mixed solution.

[0033] The seed liquid is inoculated into the fermentation medium at an inoculation rate of 15-20%, the dissolved oxygen is controlled at 50%, the pH is 6.5-7.0, and the fermentation is carried out at a temperature of 30-40°C to obtain L-alanine fermentation liquid. When the residual sugar is controlled to 0, the acid production of L-alanine is 123 g / L. Example

[0034] A method for producing L-alanine by fermentation, comprising the following steps: Step 1) Seed cultivation:

[0035] The Escherichia coli seed liquid is inoculated into the seed culture medium for culturing to obtain the seed liquid.

[0036] The formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixed solution. Step 2) Fermentation

[0037] The formula of the fermentation medium is: 10g / L glucose, 1.5g / L magnesium sulfate, 5g / L yeast powder, 15g / L corn steep liquor, 2g / L potassium dihydrogen phosphate, 7.5g / L bone peptone, 24ml defoamer, and 2g / L trace element mixed solution.

[0038] The seed liquid is inoculated into the fermentation medium at an inoculation rate of 15-20%, the dissolved oxygen is controlled at 50%, the pH is 6.5-7.0, and the fermentation is carried out at a temperature of 30-40°C to obtain L-alanine fermentation liquid. When the residual sugar is controlled at 0.2-0.5%, the acid production of L-alanine is 138 g / L.

[0039] Although the above describes the specific implementation of the present invention in combination with the embodiments, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for producing L-alanine by fermentation, characterized in that: The method comprises the following steps: Step 1) Seed culture: inoculating the L-alanine-producing Escherichia coli seed solution into the seed culture medium for culture to obtain the seed solution; Step 2) Fermentation culture: The seed liquid obtained in step 1) is inoculated into the fermentation medium, and the fermentation conditions are controlled to carry out L-alanine fermentation, using aerobic-anaerobic two-stage fermentation, growing to OD 20-30, and switching to anaerobic fermentation after 6 hours, and the fermentation ends after 48 hours to obtain the fermentation liquid.

2. The method according to claim 1, characterized in that: In step 1), the inoculation concentration of the Escherichia coli seed solution is 10-20%.

3. The method according to claim 1, characterized in that In step 1), the formula of the seed culture medium is: 10 g / L glucose, 1.5 g / L magnesium sulfate, 5 g / L yeast powder, 15 g / L corn steep liquor, 2 g / L potassium dihydrogen phosphate, 7.5 g / L peptone, 24 ml defoamer, and 2 g / L trace element mixture.

4. The method according to claim 1, characterized in that: In step 1), the cultivation parameters are: air volume control 2-3m 3 / h, dissolved oxygen controlled above 50%, fermentation pH 6.5-7.0, rotation speed 350-450rpm, tank pressure 0.01-0.1Mpa.

5. The method according to claim 1, characterized in that The formula of the fermentation medium described in step 2) is: glucose 10g / L, magnesium sulfate 1.5g / L, yeast powder 5g / L, corn steep liquor 15g / L, potassium dihydrogen phosphate 2g / L, peptone 7.5g / L, defoamer 24ml, trace element mixed solution 2g / L.

6. The method according to claim 1, characterized in that Step 2) The fermentation temperature is 30-45°C, the fermentation pH is 6.5-7.0, and the tank pressure is 0.01-0.1Mpa.

7. The method according to claim 1, characterized in that In step 2), the air volume in the aerobic stage is controlled at 2-3m 3 / h, dissolved oxygen controlled above 50%, speed 350-450rpm.

8. The method according to claim 1, characterized in that In step 2), the air volume in the anaerobic stage is adjusted to 0, the dissolved oxygen is controlled to 0, and the stirring speed is adjusted to 250-500 rpm.

9. The method according to claims 1-5, characterized in that: The peptone is bone peptone or soybean peptone, more preferably bone peptone.

10. The method according to claims 1-5, characterized in that: The residual sugar content of the fermentation broth is controlled to be 0 or 0.2-0.5%, more preferably 0.2-0.5%.