CN106755159A - A kind of liquor fermentation culture medium and the method for improving L tryptophan yield - Google Patents

Abstract

The invention provides a kind of liquor fermentation culture medium and the method for improving L tryptophan yield, the liquor fermentation nutrient media components includes：Glucose 10g/L, (NH₄)₂SO₄1 5g/L, the 1g/L of inosine 0.1, the 1g/L of guanosine 0.1, the 1g/L of adenosine 0.01, glycine, tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, leucine and each 0.1 1g/L of serine, isoleucine, threonine, alanine and each 0.2 2g/L of phenylalanine, glutamic acid and each 0.5 2g/L of lysine, citric acid 0.1 2g/L, KH₂PO₄·3H₂O 4 8g/L, MgSO₄·7H₂O 0.5 3g/L, FeSO₄·7H₂O 1 50mg/L, V_B11 10mg/L, V_B31 5mg/L, V_B51 5mg/L, the 2mL/L of trace element solution 0.5, the 10mg/L of biotin 1, the 2g/L of Choline Chloride 0.1, tryptophan-producing Strain is fermented by the culture medium can obtain high yield L tryptophans.

Description

A kind of supernatant liquid fermentation culture medium and the method for improving L-tryptophan production

technical field

The invention relates to the field of L-tryptophan production, in particular to a clear liquid fermentation medium and a method for increasing the production of L-tryptophan.

Background technique

L-Tryptophan is one of the essential amino acids in human and animal life activities. It plays an important role in the growth, development and metabolism of humans and animals. Because of its irreplaceable physiological role, it is known as the second essential amino acid. In recent years, as the commercial value of L-tryptophan has been more and more recognized, it has been widely used in medicine, food and feed, etc., and has gradually become a product with good development prospects in the international market and a large demand in the Chinese market. Especially since the beginning of the 20th century, this demand has shown a steady annual growth trend. Statistics show that the global demand for L-tryptophan was 1180 tons in 2003 and 5600 tons in 2011, with a compound annual growth rate of 21.5%. . In 2003, the total domestic consumption of L-tryptophan was about 300 tons. In 2011, the total domestic demand was about 1,200 tons, with a compound annual growth rate of 18.9%. In 2014, the global demand for L-tryptophan was 20,000 tons, with a compound annual growth rate of 53.85%; in 2015, the total domestic consumption of L-tryptophan was about 6,000 tons, a year-on-year increase of 9.1%. Although L-tryptophan has been industrially produced, the current output still cannot meet the demand. Therefore, it is of great significance to further optimize the production process of L-tryptophan. Compared with the foreign advanced level, my country's L-tryptophan production has problems such as low fermentation acid production, low conversion rate and many by-products.

At present, the production methods of L-tryptophan mainly include: protein hydrolysis method, chemical synthesis method, fermentation method and enzymatic method. In the early days, L-tryptophan was mainly produced by protein hydrolysis and chemical synthesis. Protein hydrolysis requires simple production equipment and low cost, but has the disadvantages of serious pollution, low product yield, and more impurities. The chemical synthesis method is limited by the source of raw materials, and the synthesis steps are complicated. In addition to the synthesis process conditions, the resolution of isomers and the racemization of D-tryptophan isomers must also be considered, which is not conducive to industrial production. With the continuous advancement of science and technology, the production of L-tryptophan by microbial methods has become practical and is in a dominant position. Microbial methods can be roughly divided into enzymatic methods, microbial transformation methods and direct fermentation methods. The enzymatic method utilizes the catalytic function of tryptophan biosynthetic enzymes in microorganisms to produce tryptophan, including tryptophanase, tryptophan synthetase, serine racemase, etc.; this method can directly add cell wall dissolving enzymes to destroy cells The wall can be reused, or the required enzymes can be immobilized before use, which generally consists of several stages of cell culture, cell separation and washing, immobilization and reaction; the microbial conversion method uses glucose as a carbon source, and at the same time Add the precursors required for the synthesis of tryptophan, such as anthranilic acid, indole, etc., and use the tryptophan synthase system of microorganisms to synthesize tryptophan; the direct fermentation method uses cheap raw materials such as glucose and sugarcane molasses as carbon sources , using excellent tryptophan-producing strains to produce tryptophan. With the application of recombinant DNA technology in microbial breeding, it provides a reliable technical guarantee for the screening of excellent tryptophan production strains and the improvement of acid production level, making the production of L-tryptophan by direct microbial fermentation a mainstream industrial production methods.

At present, the acid production and conversion rate of L-tryptophan are still at a low level. One of the reasons is that the substrate glucose is used to synthesize L-tryptophan and bacteria in the early stage of fermentation. There is competition for carbon and energy sources between cell growth and L-tryptophan synthesis, and excessive biomass will inevitably lead to a decrease in sugar-acid conversion. The second reason is that in the later stage of fermentation, due to the decrease of the specific growth rate of the cells, the cells synthesize acetic acid and other by-products, which inhibit the synthesis of L-tryptophan. The third reason is that in the middle and late stages of fermentation, the enzyme activity of the bacteria decreases rapidly, resulting in a decrease in the rate of acid production in the later stage.

The composition of yeast powder is complex and there are unstable factors, which lead to fluctuations in acid production and inhibition of some unstable nutrients during the fermentation process. In addition, yeast powder contains impurities such as pigments, which are difficult to remove in the later extraction process, directly affecting product quality. Substituting various amino acids and nucleosides for yeast powder fermentation, the unstable nutrients are greatly reduced, the fermentation broth tends to be stable, and the fluctuation of yield is reduced.

Contents of the invention

The technical problem to be solved by the present invention is to provide a clear liquid fermentation medium.

Another technical problem to be solved by the present invention is to provide a method for increasing the production of L-tryptophan by using the above-mentioned clear liquid fermentation medium.

In order to solve the problems of the technologies described above, the technical solution of the present invention is:

A serum fermentation medium with the following components: glucose 10g/L, (NH ₄ ) ₂ SO ₄ 1-5g/L, inosine 0.1-1g/L, guanosine 0.1-1g/L, adenosine 0.01- 1g/L, 0.1-1g/L each of glycine, tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, leucine and serine, isoleucine Amino acid, threonine, alanine and phenylalanine each 0.2-2g/L, glutamic acid and lysine each 0.5-2g/L, citric acid 0.1-2g/L, KH ₂ PO ₄ ·3H ₂ O 4-8g/L, MgSO ₄ 7H ₂ O 0.5-3g/L, FeSO ₄ 7H ₂ O 1-50mg/L, V _B1 1-10mg/L, V _B3 1-5mg/L, V _B5 1-5mg/L, trace element solution 0.5-2mL/L, biotin 1-10mg/L, choline chloride 0.1-2g/L, wherein, the trace element solution contains 4.5g/LMnSO ₄ ·H ₂ Mixed solution of O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, 0.6g/L CuSO ₄ ·5H ₂ O; pH to 7.0.

The above serum fermentation medium was adjusted to pH 7.0 with NaOH solution and/or dilute hydrochloric acid, and the sterilization conditions were 0.1 MPa damp heat sterilization for 20 minutes.

A method for improving the production of L-tryptophan, which uses tryptophan-producing bacteria to obtain L-tryptophan by fermenting the above-mentioned clear liquid fermentation medium.

Preferably, in the method for increasing the production of L-tryptophan, the Escherichia coli is inserted into the above-mentioned clear liquid fermentation medium at an inoculum size of 10% to obtain L-tryptophan for fermentation.

Preferably, the above-mentioned method for increasing the production of L-tryptophan, the specific steps are: at 37°C, pH 7.0 and dissolved oxygen 20%-30%, insert the fermented culture containing clear liquid according to the inoculum size of 10% In the base 30L automatic control fermenter, cultivate in the automatic control fermenter to the middle and late logarithm, introduce appropriate air, adjust the appropriate stirring speed, control the dissolved oxygen to 30-50%, and control it by adding 25% ammonia water automatically. When the pH is 6.7-7.0, the residual sugar is controlled at about 15% by feeding an appropriate amount of foam to defoam, and the glucose solution with a concentration of 800g/L is fed, and the fermentation is completed for 38 hours.

Preferably, in the above-mentioned method for increasing the production of L-tryptophan, 2 g/L choline chloride, 10 g/L KH ₂ PO ₄ ·3H ₂ O, 2 g/L L Arginine, 2g/L Lysine.

Preferably, in the above-mentioned method for increasing the production of L-tryptophan, 2 g/L citric acid, 5 g/L KH ₂ PO ₄ ·3H ₂ O, 2 g/L refined Amino acid, 2g/L lysine.

Preferably, in the above-mentioned method for increasing the production of L-tryptophan, the tryptophan-producing bacteria are Escherichia coli strains with the preservation number CGMCC NO.11073.

The structure of the present invention has the following beneficial effects:

The above-mentioned method for increasing the output of L-tryptophan has the following technical effects:

Amino acids and nucleosides do not contain pigments and other ingredients, which provide advantages for the subsequent extraction process and product color and quality. Based on this principle, this technology improves the fermentation yield of L-tryptophan by removing yeast powder in the fermentation medium and adding various amino acids and nucleosides.

Substituting various amino acids and nucleosides for yeast powder fermentation, the unstable nutrients are greatly reduced, the fermentation broth tends to be stable, and the fluctuation of yield is reduced. Moreover, amino acids and nucleosides do not contain pigments and other components, which provide advantages for the subsequent extraction process and product color and quality. Based on this principle, this technology improves the fermentation yield and acid production stability of L-tryptophan by removing yeast powder in the fermentation medium and adding various amino acids and nucleosides.

The method realizes the stability of the whole fermentation cycle, the increase of the output of L-tryptophan, and the simplification of the extraction process in the later stage without adding additional equipment and equipment, and is suitable for industrial production.

In the present invention, various amino acids and nucleosides are used to substitute yeast powder for fermentation, and unstable nutritional components are greatly reduced, so that the fermentation liquid tends to be stable and the output fluctuation is reduced. Moreover, amino acids and nucleosides do not contain pigments and other components, which provide advantages for the subsequent extraction process and product color and quality. Based on this principle, this technology achieves the purpose of increasing the stable yield of L-tryptophan fermentation by removing yeast powder in the fermentation medium and adding various amino acids and nucleosides.

deposit information

Taxonomic noun: Escherichia coli Escherichia coli

Name of depository unit: General Microbiology Center of China Committee for the Collection of Microorganisms

Address of Preservation Unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing

Deposit date: July 14, 2015

Deposit number: CGMCC NO.11073

detailed description

The technical solutions of the present invention will be further described below in conjunction with specific embodiments.

Example 1

A clear liquid fermentation medium, the components are as follows:

Glucose 10g/L (disinfection, 0.075MPa damp heat sterilization for 15min), (NH ₄ ) ₂ SO ₄ 1.8g/L, inosine 0.1g/L, guanosine 0.1g/L, adenosine 0.1g/L, glycine , tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, leucine, serine 0.1g/L each, isoleucine, threonine , alanine, phenylalanine each 0.2g/L, glutamic acid, lysine each 0.5g/L, citric acid 2g/L, KH ₂ PO ₄ 3H ₂ O 7.5g/L, MgSO ₄ . 7H ₂ O 1.5g/L, FeSO ₄ 7H ₂ O 6mg/L, V _B1 5mg/L, V _B3 2mg/L, V _B5 2mg/L, trace element solution 1.2mL/L, biotin 3mg/L, Choline chloride 0.5g/L, wherein the trace element solution contains 4.5g/L MnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O , 0.6g/L CuSO ₄ ·5H ₂ O mixed solution.

The components in the above-mentioned clear liquid fermentation medium are mixed according to the recipe amount, then adjusted to pH 7.0 with NaOH solution and/or dilute hydrochloric acid, and sterilized by 0.1MPa damp heat for 20 minutes.

Example 2

A clear liquid fermentation medium, the components are as follows:

Glucose 10g/L, (NH ₄ ) ₂ SO ₄ 1g/L, inosine 0.1g/L, guanosine 0.1g/L, adenosine 0.01g/L, glycine, tyrosine, aspartic acid, arginine 1g/L each of acid, methionine, proline, cystine, valine, leucine and serine, 2g/L each of isoleucine, threonine, alanine and phenylalanine , each of glutamic acid and lysine 2g/L, citric acid 0.1g/L, KH ₂ PO ₄ 3H ₂ O 4g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 1mg /L, V _B1 1mg/L, V _B3 1mg/L, V _B5 1mg/L, trace element solution 0.5mL/L, biotin 1mg/L, choline chloride 0.1g/L, wherein, the trace element The solution was a mixed solution containing 4.5g/LMnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, and 0.6g/L CuSO ₄ ·5H ₂ O.

The components in the above-mentioned clear liquid fermentation medium are mixed according to the recipe amount, then adjusted to pH 7.0 with NaOH solution and/or dilute hydrochloric acid, and sterilized by 0.1MPa damp heat for 20 minutes.

Example 3

A clear liquid fermentation medium, the components are as follows:

Glucose 10g/L, (NH ₄ ) ₂ SO ₄ 5g/L, inosine 1g/L, guanosine 1g/L, adenosine 1g/L, glycine, tyrosine, aspartic acid, arginine, methyl 0.1g/L each of thionine, proline, cystine, valine, leucine and serine, 0.2g/L each of isoleucine, threonine, alanine and phenylalanine, Glutamic acid and lysine each 0.5g/L, citric acid 2g/L, KH ₂ PO ₄ 3H ₂ O 8g/L, MgSO ₄ 7H ₂ O 3g/L, FeSO ₄ 7H ₂ O 50mg/L, V _B1 10mg/L, V _B3 5mg/L, V _B5 5mg/L, trace element solution 2mL/L, biotin 10mg/L, choline chloride 2g/L, wherein, the trace element solution contains 4.5g A mixed solution of /L MnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, 0.6g/L CuSO ₄ ·5H ₂ O.

The components in the above-mentioned clear liquid fermentation medium are mixed according to the recipe amount, and then the pH is adjusted to 7.0 with NaOH solution and/or dilute hydrochloric acid, and sterilized by 0.1MPa damp heat for 20 minutes.

Example 4

A clear liquid fermentation medium, the components are as follows:

Glucose 10g/L, (NH ₄ ) ₂ SO ₄ 3g/L, inosine 0.5g/L, guanosine 0.5g/L, adenosine 0.5g/L, glycine, tyrosine, aspartic acid, arginine acid, methionine, proline, cystine, valine, leucine and serine each 0.5g/L, each of isoleucine, threonine, alanine and phenylalanine 1g/L L, glutamic acid and lysine each 1.5g/L, citric acid 1g/L, KH ₂ PO ₄ 3H ₂ O 6g/L, MgSO ₄ 7H ₂ O 2g/L, FeSO ₄ 7H ₂ O 10mg /L, V _B1 3mg/L, V _B3 2mg/L, V _B5 4mg/L, trace element solution 1mL/L, biotin 5mg/L, choline chloride 1g/L, wherein, the trace element solution is A mixed solution containing 4.5g/L MnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, and 0.6g/L CuSO ₄ ·5H ₂ O.

The components in the above-mentioned clear liquid fermentation medium are mixed according to the recipe amount, then adjusted to pH 7.0 with NaOH solution and/or dilute hydrochloric acid, and sterilized by 0.1MPa damp heat for 20 minutes.

Example 5

A method for improving the output of L-tryptophan, the specific steps are as follows:

The Escherichia coli used (preserved in China General Microorganism Culture Collection Center, preservation number: CGMCCNO.11073); the culture medium is clear liquid fermentation medium [glucose 10g/L (divided and eliminated, 0.075MPa damp heat sterilization for 15min) , (NH ₄ ) ₂ SO ₄ 1.8g/L, inosine 0.1g/L, guanosine 0.1g/L, adenosine 0.1g/L, glycine, tyrosine, aspartic acid, arginine, methyl Thionine, proline, cystine, valine, leucine, serine each 0.1g/L, isoleucine, threonine, alanine, phenylalanine each 0.2g/L, Glutamic acid and lysine each 0.5g/L, citric acid 2g/L, KH ₂ PO ₄ 3H ₂ O 7.5g/L, MgSO ₄ 7H ₂ O 1.5g/L, FeSO ₄ 7H ₂ O 6mg /L, V _B1 5mg/L, V _B3 2mg/L, V _B5 2mg/L, trace elements 1.2mL/L (trace elements contain 4.5g/L MnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, 0.6g/L CuSO ₄ ·5H ₂ O mixed solution), biotin 3mg/L, choline chloride 0.5g/L, adjusted with NaOH and hydrochloric acid pH to 7.0. Sterilization conditions: 0.1MPa damp heat sterilization for 20min]; culture method: insert the bacteria into the seed medium [glucose 40g/L, (NH ₄ ) ₂ SO ₄ 3g/L, inosine 0.1g/L, guanosine 0.1 g/L, adenosine 0.1g/L, glycine, tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, leucine, serine 0.1 each g/L, isoleucine, threonine, alanine, phenylalanine each 0.2g/L, glutamic acid, lysine each 0.5g/L, citric acid 2g/L, KH ₂ PO ₄ 6g/L, MgSO ₄ 7H ₂ O 1.6g/L, FeSO ₄ 7H ₂ O 10mg/L, V _B1 5mg/L, V _B3 2mg/L, V _B5 2mg/L, trace elements 1mL/L, biological 2 mg/L], the inoculum size is 10%; at 37°C, pH is 7.0 and dissolved oxygen is 20%-30%, cultivate in a 5L automatic control fermenter for 12h to the middle and late logarithm, and inoculate with 10% Put the amount into the 30L automatic control fermenter containing the above-mentioned clear liquid fermentation medium, introduce appropriate air, adjust the appropriate stirring speed, control the dissolved oxygen to 30-50%, and control the pH at 6.7-5% by automatically adding 25% ammonia water 7.0, defoaming by adding an appropriate amount of foam enemy defoamer, and controlling the residual sugar at about 15% by feeding a glucose solution with a concentration of 800g/L, and the fermentation is over for 38 hours; two batches of parallel experiments were carried out. When putting tank, the output of L-tryptophan is respectively 38.6g/L, 38.2g/L, compared with control experiment (two batches of experiments) respectively (L-tryptophan output is respectively 34.2g/L, 35.5g/L ) increased by 12.86% and 7.61%, the acid production stability is high, and the yield fluctuation is small.

Example 6

The bacterial strain that adopts is Escherichia coli; Culture medium is that 2g/L choline chloride is added with sugar flow in the supernatant liquid fermentation culture medium (same as embodiment 5), 10g/L KH ₂ PO ₄ 3H ₂ O, 2g/L refined Amino acid, 2g/L lysine; Culture method is with embodiment 5. Two parallel experiments were performed. When putting tank, the output of L-tryptophan is respectively 40.6g/L, 41.0g/L, compared with control experiment (two batches of experiments) respectively (L-tryptophan output is respectively 34.2g/L, 35.5g/L ) increased by 18.71% and 15.49%, the stability of acid production was high, and the yield fluctuation was small.

Example 7

The bacterial strain that adopts is Escherichia coli; Culture medium is to add 2g/L citric acid, 5g/LKH ₂ PO ₄ 3H ₂ O, 2g/L arginine with sugar flow in clear liquid fermentation medium (same as embodiment 5), 2g/L lysine; Culture method is with embodiment 5. Two parallel experiments were performed. When putting tank, the output of L-tryptophan is respectively 41.6g/L, 41.9g/L, compared with control experiment (two batches of experiments) respectively (L-tryptophan output is respectively 34.2g/L, 35.5g/L ) increased by 21.64% and 18.03%, the stability of acid production was high, and the yield fluctuation was small.

It can be seen that by removing the yeast powder in the existing medium and adding specific amino acids and nucleosides to the medium to achieve a substitution effect, the concentration in the medium is 0.1-0.5g/L, and in the middle and late stages of fermentation Feed feed nutrients, and finally obtain high-yield L-tryptophan, wherein,

The various amino acids include: glycine, tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, isoleucine, leucine, threonine acid, alanine, phenylalanine, glutamic acid and lysine;

The various nucleosides include: guanosine, inosine, adenosine;

The fed-in nutrients include: citric acid, KH ₂ PO ₄ ·3H ₂ O, arginine, and lysine.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (7)

1. A clear liquid fermentation medium, characterized in that: the components are as follows: glucose 10g/L, (NH ₄ ) ₂ SO ₄ 1-5g/L, inosine 0.1-1g/L, guanosine 0.1-1g/L L, adenosine 0.01-1g/L, glycine, tyrosine, aspartic acid, arginine, methionine, proline, cystine, valine, leucine and serine each 0.1- 1g/L, 0.2-2g/L each of isoleucine, threonine, alanine and phenylalanine, 0.5-2g/L of glutamic acid and lysine, 0.1-2g/L of citric acid, KH ₂ PO ₄ 3H ₂ O 4-8g/L, MgSO ₄ 7H ₂ O 0.5-3g/L, FeSO ₄ 7H ₂ O 1-50mg/L, V _B1 1-10mg/L, V _B3 1- 5mg/L, V _B5 1-5mg/L, trace element solution 0.5-2mL/L, biotin 1-10mg/L, choline chloride 0.1-2g/L, wherein, the trace element solution contains 4.5g Mixed solution of /L MnSO ₄ ·H ₂ O, 6.4g/L ZnSO ₄ , 4.9g/L Co(NO ₃ ) ₂ ·6H ₂ O, 0.6g/L CuSO ₄ ·5H ₂ O; pH to 7.0. 2. A method for increasing the output of L-tryptophan, characterized in that: the L-tryptophan is obtained by fermenting the serum fermentation medium according to claim 1 by using tryptophan-producing bacteria. 3. The method for increasing the production of L-tryptophan according to claim 2, characterized in that: Escherichia coli is inserted into the above-mentioned clear liquid fermentation medium to ferment to obtain L-tryptophan according to the inoculum size of 10%. 4. The method for improving L-tryptophan output according to claim 2, characterized in that: the specific steps are: at 37°C, pH is 7.0 and dissolved oxygen is 20%-30% condition, press 10% The inoculum amount is connected to a 30L automatic control fermenter containing clear liquid fermentation medium, cultivated in the automatic control fermenter to the middle and late logarithmic stages, injecting appropriate air, adjusting the appropriate stirring speed, and controlling the dissolved oxygen to 30-50%. The pH is controlled at 6.7-7.0 by automatically feeding 25% ammonia water, the foam is defoamed by feeding an appropriate amount, and the residual sugar is controlled at about 15% by feeding a glucose solution with a concentration of 800g/L, and the fermentation is over for 38 hours, that is have to. 5. the method for improving L-tryptophan output according to claim 4, is characterized in that: in described supernatant liquid fermentation medium, additionally add 2g/L choline chloride with sugar flow, 10g/L KH ₂ PO ₄ ·3H ₂ O, 2g/L arginine, 2g/L lysine. 6. The method for improving L-tryptophan production according to claim 4, characterized in that: additionally add 2g/L citric acid, 5g/L KH ₂ PO ₄ • _3H2O , 2g/L arginine, 2g/L lysine. 7. The method for increasing the production of L-tryptophan according to claim 2, characterized in that: the tryptophan-producing bacteria is Escherichia coli strain with preservation number CGMCC NO.11073.