CN118345128A - Method for improving fermentation yield of L-amino acid - Google Patents

Abstract

The invention provides a method for improving the fermentation yield of L-amino acid. In the process of producing L-amino acid by microbial fermentation, bacterial metabolism is changed by adding a certain amount of sodium benzoate into a fermentation medium, byproducts (such as citric acid and succinic acid) are reduced, and more nitrogen sources are converted into end products; changing permeability of cell membrane, reducing energy consumption, and increasing yield of main product (L-amino acid such as L-threonine).

Description

Method for improving fermentation yield of L-amino acid

Technical Field

The invention relates to the field of fermentation engineering, in particular to a method for improving the fermentation yield of L-amino acid.

Background

At present, the L-amino acid is produced by adopting a microbial fermentation method, which mainly takes corn starch hydrolysis sugar or molasses as a carbon source and takes corn steep liquor, soybean meal hydrolysis liquid, yeast extract, peptone, ammonium salt, nitrate, urea, ammonia water, liquid ammonia and the like as nitrogen sources. The process optimization comprises dissolved oxygen, control of carbon source and nitrogen source, betaine dosage and other vitamins, etc.

At present, the transport proteins related to L-threonine in escherichia coli mainly comprise TdcC, rhtA, rhtB, rhtC and YecC, and besides TdcC, the transport proteins are export proteins. Among the export proteins, rhtA, rhtB and RhtC are currently most widely studied and used, wherein the specificity of RhtC is the highest for transporting threonine and energy is required.

Bacterial growth and production compete for the carbon source, and bacterial growth in large quantities consumes the carbon source for production, resulting in a decrease in the carbon source flow to the amino acids. In addition, during the fermentation process, along with the production of L-threonine, there is also a certain yield of hetero acids, such as glutamic acid, glycine, isoleucine and the like, and other organic acids, such as non-metabolized pyruvic acid, succinic acid and the like, wherein the related respiratory metabolism of the hetero acids is not easy to control.

Sodium benzoate is generally used as an additive in foods to play a role in preserving and preserving, and is also used for preparing a bacterial suspension so as to be convenient for long-term storage and transportation. Sodium benzoate has not been reported for use in increasing L-amino acid fermentation levels.

Disclosure of Invention

The invention aims to provide a method for improving the fermentation yield of L-amino acid.

To achieve the object of the present invention, in a first aspect, the present invention provides the use of sodium benzoate for increasing the fermentation yield of L-amino acids, preferably L-threonine, L-glutamic acid, L-lysine or L-isoleucine and the like.

In the present invention, the species used for fermentation include, but are not limited to, escherichia coli (ESCHERICHIA COLI), corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes) or Corynebacterium ammoniagenes (Corynebacterium aminogenes), preferably Escherichia coli MHZ-0216-5. Strain MHZ-0216-5 can be found in CN113846132A.

In a second aspect, the present invention provides a method for improving the fermentation yield of L-amino acids, wherein Escherichia coli is used as a fermentation strain, and a proper amount of sodium benzoate is added to a fermentation medium, so that the fermentation yield of L-amino acids is improved.

The method comprises the following steps: at the beginning of fermentation, 0.05-0.5g/L sodium benzoate is added to the fermentation medium.

The fermentation medium is as follows: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄5g/L and betaine 1g/L.

The fermentation culture method comprises the following steps: inoculating the seed solution into a 50L fermentation tank containing 20L fermentation medium, introducing air at an air flow rate of 0.8vvm and a rotation speed of 300-600rpm, controlling pH7.0, and controlling the temperature at 37 ℃ and dissolved oxygen at 30%; the total fermentation time is 28-32h (preferably 30 h).

Feeding a carbon source when the residual sugar content in the fermentation liquid is 0.1g/L, controlling the glucose content in the fermentation liquid to be 0-5g/L, and simultaneously feeding 25% ammonia water to control the pH7.0 of the fermentation system; preferably, the carbon source is a 50% glucose solution.

The preparation method of the seed liquid comprises the following steps: the glycerol frozen strain is firstly coated on LB culture medium for activation culture overnight, a certain amount of bacterial colony is selected and inoculated in a 5L triangular flask filled with 1000mL of seed culture medium, the rotation speed of a shaking table is 120rpm, the temperature is 37 ℃, and the shaking culture is carried out for 8-12h until the strain concentration is OD ₆₀₀ =10, so as to be used as seed liquid.

The seed culture medium is as follows: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate, 20g/L,MgSO₄·7H₂O0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 10mg/L,MnSO₄ 10mg/L,(NH₄)₂SO₄ 5g/L g/L glucose and 0.5g/L betaine.

Or the method comprises the following steps: during fermentation for 12-20 h (preferably 16 h), 0.05-0.5g/L sodium benzoate is added into the fermentation medium.

Further, sodium benzoate is added to the fermentation medium (preferably for 16 h), the fermentation temperature is reduced to 34-36 ℃, dissolved oxygen is reduced to 15-25%, and glycerin and betaine are added to the final concentrations of 1-2g/L and 0.5g/L respectively, so as to further improve acid production.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

In the process of producing L-amino acid by microbial fermentation, a certain amount of sodium benzoate is added into a fermentation medium to change bacterial respiratory metabolism, reduce the production of byproducts (such as citric acid and succinic acid) and convert more nitrogen sources into end products; changing permeability of cell membrane, reducing energy consumption, and increasing yield of main product (L-amino acid such as L-threonine).

Detailed Description

The invention provides a method for improving the fermentation yield of L-amino acid (comprising optimization of culture medium and fermentation conditions), which adopts the following technical scheme:

1. Effect of sodium benzoate addition to fermentation Medium

(1) Preparing a seed culture medium and culturing seeds;

(2) Preparing a fermentation medium, adding sodium benzoate, and fermenting and culturing;

(3) Controlling fermentation conditions, adding 0.05-0.5g/L sodium benzoate into initial fermentation medium, and increasing L-amino acid (preferably L-threonine) yield.

2. Sodium benzoate is added during fermentation culture

Controlling fermentation conditions, and adding sodium benzoate after fermentation for 16 hours; the citric acid and the succinic acid are reduced, the content of pyruvic acid is increased, and the yield of L-threonine is improved.

3. And (3) fermenting for 16 hours, adding sodium benzoate, reducing the fermentation temperature to 34-36 ℃, reducing dissolved oxygen to control the dissolved oxygen to 15-25%, adding 1-2g/L glycerol as a carbon source, and simultaneously adding 0.5g/L betaine to further improve acid production.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.

The strain used in the following examples was E.coli MHZ-0216-5, supplied by Langfang plum blossom biotechnology development Co., ltd. Strain MHZ-0216-5 can be found in CN113846132A.

Example 1 production method of L-threonine with sodium benzoate added to fermentation bed charge

The method comprises the following steps:

(1) L-threonine seed medium: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate and 0.5g/L glucose 20g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 10mg/L,MnSO₄ 10mg/L,(NH₄)₂SO₄;5g/L, betaine.

(2) Seed culture: the strain adopts escherichia coli for production, the glycerol pipe frozen strain is firstly coated on an LB culture medium for activation culture overnight, a certain amount of bacterial colonies are selected and inoculated in a 5L triangular flask filled with 1000mL of seed culture medium, the bacterial colonies are put into a constant temperature reciprocating shaking table for shaking culture at the rotation speed of 120rpm and the temperature of 37 ℃ for 8-12 hours until the concentration of the strain in the seed liquid is OD ₆₀₀ = 10, and then the culture is finished to be used as the seed liquid.

(3) L-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L, betaine 1g/L, and sodium benzoate 0.05 g/L, 0.1 g/L, 0.2 g/L, 0.3 g/L, 0.5g/L, respectively.

(4) Fermentation culture: the cultured seed liquid is inoculated into a 50L fermentation tank filled with 20L fermentation medium, the aeration rate is 0.8vvm, the rotating speed is 300-600rpm, the pH value is 7.0, the temperature is 37 ℃, and the dissolved oxygen is 30%.

Adding a carbon source into the fermentation liquor after the residual sugar content is 0.1g/L, controlling pH to 7.0 by taking ammonia water as a pH regulator, preparing 50% glucose solution as the added carbon source, controlling the glucose content in the fermentation liquor to be 0.1g/L, and taking 25% ammonia water as the added nitrogen source. The total fermentation time is 30 hours.

(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC. The results are shown in Table 1.

TABLE 1 fermentation results of sodium benzoate addition to initial fermentation Medium

It can be seen that small amounts of sodium benzoate have a positive effect on bacterial production and that increased amounts affect the growth of the bacteria themselves and thus the production of L-threonine.

Example 2 production of L-threonine by fed-batch sodium benzoate during fermentation

The method comprises the following steps:

(1) L-threonine seed medium: as in example 1.

(2) Seed culture: as in example 1.

(3) L-threonine fermentation medium: l-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄0.05mg/L,(NH₄)₂SO₄ 5g/L, betaine 1g/L.

(4) Fermentation culture: as in example 1.

Sodium benzoate at 0.2g/L was added at 12h, 16h, 20h of fermentation, respectively.

(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC. The results are shown in Table 2.

TABLE 2 post-fermentation results of sodium benzoate addition during fermentation

Sodium benzoate, g/L	12h	16h	20h
				L-threonine, g/L	111.5	114.8	109.1
Conversion, percent	56.1	57.1	55.5
				Succinic acid, g/L	0.1	0.1	0.1
Citric acid, g/L	0.1	0.1	0.1
				Pyruvic acid, g/L	0.2	0.3	0.3
OD600	36.1	37.6	38.4

Example 3 production method of L-threonine by fed-batch sodium benzoate during fermentation

The method comprises the following steps:

(1) L-threonine seed medium: as in example 2.

(2) Seed culture: as in example 2.

(3) L-threonine fermentation medium: l-threonine fermentation medium: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄0.05mg/L,(NH₄)₂SO₄ 5g/L, betaine 1g/L.

(4) Fermentation culture: as in example 2, 0.2g/L sodium benzoate was added during 16h of fermentation, and the temperature was lowered to 35℃and 0.5g/L betaine and 1.5g/L glycerol were fed in.

(5) Glucose content was determined using an SBA biosensor and L-threonine content was determined by HPLC at 118.5g/L. The conversion was 58.2%.

(6) Fermentation liquor OD ₆₀₀ =36.5, succinic acid content 0.1g/L, citric acid content 0.1g/L and pyruvic acid content 0.3g/L.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The application of sodium benzoate in improving the fermentation yield of L-amino acid;

preferably, the L-amino acid is L-threonine, L-glutamic acid, L-lysine or L-isoleucine.

2. Use according to claim 1, characterized in that the species used for fermentation is escherichia coli (ESCHERICHIA COLI), corynebacterium glutamicum (Corynebacterium glutamicum), corynebacterium validum (Corynebacterium efficiens), corynebacterium crenatum (Corynebacterium crenatum), corynebacterium ammoniagenes thermophilum (Corynebacterium thermoaminogenes) or corynebacterium ammoniagenes (Corynebacterium aminogenes).

3. A method for improving the fermentation yield of L-amino acid is characterized in that escherichia coli is used as a fermentation strain, and a proper amount of sodium benzoate is added into a fermentation culture medium, so that the fermentation yield of L-amino acid is improved;

preferably, the L-amino acid is L-threonine, L-glutamic acid, L-lysine or L-isoleucine.

4. A method according to claim 3, characterized in that the method comprises: at the beginning of fermentation, 0.05-0.5g/L sodium benzoate is added to the fermentation medium.

5. The method of claim 4, wherein the fermentation medium is: corn steep liquor 5g/L, soybean meal hydrolysate 5g/L, glucose 40g/L,MgSO₄·7H₂O 0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 20mg/L,MnSO₄ 20mg/L,ZnSO₄ 0.05mg/L,(NH₄)₂SO₄ 5g/L and betaine 1g/L.

6. A method according to claim 3, wherein the method of fermentation culture comprises: inoculating the seed solution into a 50L fermentation tank containing 20L fermentation medium, introducing air at an air flow rate of 0.8vvm and a rotation speed of 300-600rpm, controlling pH7.0, and controlling the temperature at 37 ℃ and dissolved oxygen at 30%; the total fermentation time is 28-32h;

Feeding a carbon source when the residual sugar content in the fermentation liquid is 0.1g/L, controlling the glucose content in the fermentation liquid to be 0-5g/L, and simultaneously feeding 25% ammonia water to control the pH7.0 of the fermentation system;

Preferably, the carbon source is a 50% glucose solution.

7. The method of claim 6, wherein the seed liquid preparation method comprises: the glycerol frozen strain is firstly coated on LB culture medium for activation culture overnight, a certain amount of bacterial colony is selected and inoculated in a 5L triangular flask filled with 1000mL seed culture medium, the rotation speed of a shaking table is 120rpm, the temperature is 37 ℃, and the shaking culture is carried out for 8-12h until the strain concentration is OD ₆₀₀ =10, so as to be used as seed liquid;

The seed culture medium is as follows: 15g/L corn steep liquor, 5g/L soybean meal hydrolysate, 20g/L,MgSO₄·7H₂O0.5g/L,KH₂PO₄ 2g/L,FeSO₄ 10mg/L,MnSO₄ 10mg/L,(NH₄)₂SO₄ 5g/L g/L glucose and 0.5g/L betaine.

8. A method according to claim 3, characterized in that the method comprises: and adding 0.05-0.5g/L sodium benzoate into the fermentation medium when fermenting for 12-20 h.

9. The method according to claim 8, wherein sodium benzoate is added to the fermentation medium while the fermentation temperature is lowered to 34-36 ℃ and dissolved oxygen is reduced to 15-25% and glycerol and betaine are added to a final concentration of 1-2g/L, 0.5g/L, respectively.

10. The method according to any one of claims 3 to 9, wherein the fermentation broth is escherichia coli MHZ-0216-5.