A seed medium and a fermentation medium for increasing glutamic acid production

Abstract

The present invention provides a seed culture medium and a fermentation culture medium for increasing glutamic acid production, belonging to the field of fermentation engineering technology. The solutes and their concentrations in the seed culture medium are: glucose, corn steep liquor, soybean meal hydrolyzate, potassium chloride, magnesium sulfate, succinic acid, naphthalene acetic acid, antifoaming agent, biotin, ferrous sulfate, manganese sulfate, and the rest is water. The solutes and their concentrations in the fermentation culture medium are: naphthalene acetic acid, glucose, corn steep liquor, soybean meal hydrolyzate, betaine, potassium chloride, magnesium sulfate, antifoaming agent, biotin, ferrous sulfate, manganese sulfate, and the rest is water. Adding naphthalene acetic acid to the culture medium of the present invention can promote cell division and cell expansion, and promote growth. It can reduce the cell wall density, promote the excretion of glutamic acid, and the reduction of intracellular glutamic acid weakens its feedback inhibition on glutamate dehydrogenase, ultimately promoting the synthesis of glutamic acid.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation engineering, and particularly relates to a seed culture medium and a fermentation culture medium for improving the yield of glutamic acid. Background Art

[0002] Glutamic acid is an acidic amino acid containing one amino group and two carboxyl groups in the molecule, also known as α-aminoglutaric acid. It is a colorless crystal, has a fresh taste, is slightly soluble in water, easily soluble in hydrochloric acid solution, with a molecular weight of 147.1, an isoelectric point of 3.22, and is abundantly present in cereal proteins and also in relatively large amounts in animal brains. It plays an important role in the process of protein metabolism in organisms, participates in the synthesis of proteins, polypeptides and fatty acids, and regulates the ammonia level in the body together with glutamine; it can also participate in information transmission as an excitatory neurotransmitter. Glutamic acid has a levorotatory form, a dextrorotatory form and a racemic form. The levorotatory form is L-glutamic acid. Naturally occurring glutamic acid is all L-glutamic acid. Glutamic acid has a wide range of applications in food, medicine, cosmetics and agriculture. L-glutamic acid is the precursor for synthesizing monosodium glutamate (sodium glutamate). Sodium L-glutamate has a strong umami taste of meat and is the most consumed flavoring agent in the world after sugar and salt. L-glutamic acid hydrochloride can be used to improve the bitterness of beer and can also be used as a salt substitute, a nutritional supplement and a flavor enhancer.

[0003] The current production method of glutamic acid is fermentation production. The microbial synthesis pathway of glutamic acid is that glucose generates pyruvate through the Embden-Meyerhof-Parnas pathway (EMP) and the hexose monophosphate shunt (HMP), and pyruvate is decarboxylated to form acetyl coenzyme A; then, under the catalysis of aldolase, oxaloacetic acid and acetyl coenzyme A synthesize citric acid, which is further converted into isocitric acid and α-ketoglutaric acid, and α-ketoglutaric acid generates glutamic acid in the presence of glutamic dehydrogenase and NH 4+ In the presence of, glutamic acid synthesized in the cells permeates through the cell membrane, and a large amount of glutamic acid can accumulate in the fermentation broth. Theoretically, without considering microbial growth and respiratory consumption, one molecule of six-carbon glucose finally produces one molecule of five-carbon glutamic acid, and the theoretical sugar-acid conversion rate is 81.7%.

[0004] Through more than 50 years of development, great progress has been made in the production technology of fermentation. The glutamic acid fermentation method has gone through several stages such as the sub-optimal biotin method, the high biotin method and temperature-sensitive strains for producing glutamic acid. The fermentation production intensity has been significantly improved, and the fermentation acid production level has been increased from 9-13% of the sub-optimal method to 15-23% of the temperature-sensitive method, and the conversion rate level has been increased from 58-61% to 65-72%. At present, industrial production mainly focuses on the production of glutamic acid by temperature-sensitive strains. There is a certain gap between the domestic technical level and that of the international counterparts, and there is still a large room for improvement compared with the theoretical conversion rate level.

[0005] The main problems currently affecting the glutamic acid industry are as follows: on the one hand, when the seed culture medium uses carbon source mixed sterilization at high temperature, Maillard reaction is prone to occur, resulting in large nutritional losses. At the same time, the by-products acrylamide and 5-hydroxymethylfurfural produced will inhibit the growth of bacteria, leading to a long seed culture period and low viability of the strains; on the other hand, the high concentration of the fermentation medium makes the glutamic acid cells smaller, the cell wall density higher, and the intracellular accumulation of glutamic acid inhibits the synthesis of glutamic acid. Summary of the Invention

[0006] The purpose of the present invention is to provide a seed culture medium and a fermentation culture medium for increasing the yield of glutamic acid, improving the production process of glutamic acid, upgrading the product grade, and increasing the fermentation yield.

[0007] The technical solution of the present invention is realized as follows:

[0008] The present invention provides a seed culture medium for increasing the yield of glutamic acid. The solutes and their concentrations in the culture medium are as follows: glucose 35 - 45 g / L, corn steep liquor 40 - 50 g / L, soybean meal hydrolyzate 10 - 20 g / L, potassium chloride 0.2 - 0.6 g / L, magnesium sulfate 0.6 - 1.0 g / L, succinic acid 2 - 3 g / L, naphthaleneacetic acid 5 - 10 μg / L, antifoaming agent 0.3 - 0.5 mL / L, biotin 0.4 - 0.5 mg / L, ferrous sulfate 0.1 - 0.2 mg / L, manganese sulfate 0.1 - 0.2 mg / L, and the rest is water. This improved formula of the secondary seed culture medium can improve the viability of the strains and shorten the seed period.

[0009] As a further improvement of the present invention, the concentration of naphthaleneacetic acid in the culture medium is 10 μg / L.

[0010] As a further improvement of the present invention, the concentration of the corn steep liquor is 17 - 22 Baume degrees.

[0011] As a further improvement of the present invention, the total nitrogen content of the soybean meal hydrolyzate is 20 - 30 g / L.

[0012] The present invention further protects a fermentation culture medium for increasing the yield of glutamic acid. The solutes and their concentrations in the culture medium are as follows: naphthaleneacetic acid 50 - 200 μg / L, glucose 40 - 50 g / L, corn steep liquor 60 - 80 g / L, soybean meal hydrolyzate 20 - 30 g / L, betaine 1 - 2 g / L, potassium chloride 1 - 2 g / L, magnesium sulfate 1 - 2 g / L, antifoaming agent 0.3 - 0.5 mL / L, biotin 0.4 - 0.5 mg / L, ferrous sulfate 0.1 - 0.2 mg / L, manganese sulfate 0.1 - 0.2 mg / L, and the rest is water. This improved formula of the fermentation culture medium can improve the viability of the bacteria, increase the acid production, and shorten the fermentation period.

[0013] As a further improvement of the present invention, the concentration of naphthylacetic acid in the culture medium is 50 - 200 μg / L.

[0014] As a further improvement of the present invention, the concentration of corn steep liquor is 17 - 22 degrees Baume.

[0015] As a further improvement of the present invention, the total nitrogen content of the soybean meal hydrolyzate is 20 - 30 g / L.

[0016] The present invention further protects a method for increasing the yield of glutamic acid. After culturing the strain seed liquid using the above-mentioned seed culture medium, then performing fermentation culture using the above-mentioned fermentation culture medium to obtain the product.

[0017] As a further improvement of the present invention, the strain is temperature-sensitive Corynebacterium glutamicum; the conditions for culturing the strain seed liquid are: tank pressure 0.05 - 0.1 Mpa, ventilation volume 10 - 20 L / min, stirring 200 - 800 rpm, pH 7.0 - 7.2; the temperature is controlled at 33 °C, and by sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle, the dissolved oxygen is maintained at 30 - 50%; the conditions for the fermentation culture are: tank pressure 0.05 - 0.1 Mpa, ventilation volume 10 - 30 L / min, stirring 200 - 800 rpm, pH 6.8 - 7.2; the temperature is controlled at 33 - 40 °C, and the residual sugar control during the process: the fed-batch sugar is 65%, and the residual sugar during the process is controlled at 0 - 0.5%. According to the increase in OD, the fed-batch sugar rate is gradually increased, and by sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle, the dissolved oxygen is maintained at 30 - 60%.

[0018] The present invention has the following beneficial effects:

[0019] (1) Adding naphthylacetic acid to the seed culture medium can promote cell division and cell expansion, promote growth, and shorten the seed culture period.

[0020] (2) Adding naphthylacetic acid to the fermentation culture medium can promote cell division and cell expansion, and promote growth. It can reduce the cell wall density and promote the excretion of glutamic acid. The decrease in intracellular glutamic acid weakens its feedback inhibition on glutamate dehydrogenase, and ultimately promotes the synthesis of glutamic acid. It improves the cell viability and acid production, and the acid production reaches 22%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1Graph showing the acid production rate of each group of the present invention over time;

[0023] Figure 2 Comparison graph of the conversion rates of each group of the present invention. Detailed implementation manners

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0025] The following examples and comparative examples all adopt a unified control process. The engineering strain is temperature-sensitive Corynebacterium glutamicum. The secondary seed and fermentation control processes are as follows:

[0026] The secondary seed culture conditions are: tank pressure 0.07 Mpa, ventilation volume 15 L / min, stirring 600 rpm, pH = 7.1; temperature controlled at 33 °C, and the dissolved oxygen is maintained at 40% by sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle.

[0027] The fermentation conditions are: tank pressure 0.07 Mpa, ventilation volume 20 L / min, stirring 600 rpm, pH = 7.0; temperature controlled at 33 - 40 °C, process residual sugar control: feeding sugar 65%, process control residual sugar 0.3%, and the feeding sugar rate is gradually increased according to the OD increase. The dissolved oxygen is maintained at 45% by sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle.

[0028] Example 1

[0029] A seed culture medium and a fermentation culture medium for increasing the yield of glutamic acid.

[0030] The solutes and their concentrations in the seed culture medium are: glucose 35 g / L, corn steep liquor 40 g / L, soybean meal hydrolyzate 10 g / L, potassium chloride 0.2 g / L, magnesium sulfate 0.6 g / L, succinic acid 2 g / L, naphthaleneacetic acid 5 μg / L, antifoaming agent 0.3 mL / L, biotin 0.4 mg / L, ferrous sulfate 0.1 mg / L, manganese sulfate 0.1 mg / L, and the rest is water.

[0031] The solutes and their concentrations in the fermentation culture medium are: naphthaleneacetic acid 50 μg / L, glucose 40 g / L, corn steep liquor 60 g / L, soybean meal hydrolyzate 20 g / L, betaine 1 g / L, potassium chloride 1 g / L, magnesium sulfate 1 g / L, antifoaming agent 0.3 mL / L, biotin 0.4 mg / L, ferrous sulfate 0.1 mg / L, manganese sulfate 0.1 mg / L, and the solvent is water.

[0032] The seed culture was completed, and the OD value of the broth was measured to be 1.05. The cycle was 22.6 h, and the fermentation ended after 34 h of operation, with an acid production of 21.8% and a conversion rate of 70%.

[0033] Example 2

[0034] A seed culture medium and a fermentation medium for increasing the yield of glutamic acid.

[0035] The solutes and their concentrations in the seed culture medium are as follows: glucose 45 g / L, corn steep liquor 50 g / L, hydrolyzed soybean meal solution 20 g / L, potassium chloride 0.6 g / L, magnesium sulfate 1.0 g / L, succinic acid 2 - 3 g / L, naphthaleneacetic acid 10 μg / L, antifoaming agent 0.5 mL / L, biotin 0.5 mg / L, ferrous sulfate 0.2 mg / L, manganese sulfate 0.2 mg / L, and the rest is water.

[0036] The solutes and their concentrations in the fermentation medium are as follows: naphthaleneacetic acid 100 μg / L, glucose 50 g / L, corn steep liquor 80 g / L, hydrolyzed soybean meal solution 30 g / L, betaine 2 g / L, potassium chloride 2 g / L, magnesium sulfate 2 g / L, antifoaming agent 0.5 mL / L, biotin 0.5 mg / L, ferrous sulfate 0.2 mg / L, manganese sulfate 0.2 mg / L, and the solvent is water.

[0037] The seed culture was completed, and the OD value of the broth was measured to be 1.05. The cycle was 22.0 h, and the fermentation ended after 34 h of operation, with an acid production of 22% and a conversion rate of 70.5%

[0038] Example 3

[0039] This example provides a seed culture medium and a fermentation medium for increasing the yield of glutamic acid.

[0040] The solutes and their concentrations in the seed culture medium are as follows: glucose 40 g / L, corn steep liquor 45 g / L, hydrolyzed soybean meal solution 15 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.8 g / L, succinic acid 2.5 g / L, naphthaleneacetic acid 20 μg / L, antifoaming agent 0.4 mL / L, biotin 0.45 mg / L, ferrous sulfate 0.15 mg / L, manganese sulfate 0.15 mg / L, and the rest is water.

[0041] The solutes and their concentrations in the fermentation medium are as follows: naphthaleneacetic acid 200 μg / L, glucose 45 g / L, corn steep liquor 70 g / L, hydrolyzed soybean meal solution 25 g / L, betaine 1.5 g / L, potassium chloride 1.5 g / L, magnesium sulfate 1.5 g / L, antifoaming agent 0.4 mL / L, biotin 0.45 mg / L, ferrous sulfate 0.15 mg / L, manganese sulfate 0.5 mg / L, and the solvent is water.

[0042] The seed culture was completed, and the OD value at the end of the tank was measured to be 1.05, the cycle was 22.1 h, and the fermentation ended after 34 h of operation, with an acid production of 22.2% and a conversion rate of 70.7%.

[0043] Comparative Example 1

[0044] Fermentation production of glutamic acid was carried out by a traditional method, and the specific process was as follows:

[0045] The solutes and their concentrations in the seed medium were: glucose 40 g / L, corn steep liquor 45 g / L, soybean meal hydrolyzate 15 g / L, potassium chloride 0.4 g / L, magnesium sulfate 0.8 g / L, succinic acid 2.5 g / L, antifoaming agent 0.4 mL / L, biotin 0.45 mg / L, ferrous sulfate 0.15 mg / L, manganese sulfate 0.15 mg / L, and the rest was water.

[0046] The solutes and their concentrations in the fermentation medium were: glucose 45 g / L, corn steep liquor 70 g / L, soybean meal hydrolyzate 25 g / L, betaine 1.5 g / L, potassium chloride 1.5 g / L, magnesium sulfate 1.5 g / L, antifoaming agent 0.4 mL / L, biotin 0.45 mg / L, ferrous sulfate 0.15 mg / L, manganese sulfate 0.5 mg / L, and the solvent was water.

[0047] The seed culture was completed, and the OD value at the end of the tank was measured to be 1.05, the cycle was 28 h, and the fermentation ended after 38 h of operation, with an acid production of 19% and a conversion rate of 66%.

[0048] Test Example 1

[0049] The experimental results of Examples 1-3 and Comparative Example 1 are compared as shown in Tables 1 and 2 below;

[0050] Table 1 Indexes of the cultured temperature-sensitive Corynebacterium glutamicum seed liquid in Examples 1-3 and Comparative Example 1

[0051] Project OD value of seed solution Seed growth cycle h Example 1 1.05 22.6 Example 2 1.05 22.0 Example 3 1.05 22.1 Comparative Example 1 1.05 28

[0052] It can be seen from Table 1 that when the temperature-sensitive Corynebacterium glutamicum seed liquid was cultured in the seed medium in Comparative Example 1 until the OD value reached 1.0, the required seed growth cycle was 28 h, which was significantly higher than that when the temperature-sensitive Corynebacterium glutamicum seed liquid was cultured in the seed medium in Examples 1-3 of the present invention until the OD value reached 1.0. Thus, it can be known that adding naphthylacetic acid to the seed medium in the method of the present invention can effectively promote the division of temperature-sensitive Corynebacterium glutamicum, promote growth, shorten the growth cycle of temperature-sensitive Corynebacterium glutamicum seeds, and improve the strain viability and acid production level.

[0053] Table 2 Summary of fermentation indexes of Examples 1-3 and Comparative Example 1

[0054] Project Acid production rate % Conversion rate % Fermentation cycle h Example 1 21.8 70 34 Example 2 22 70.5 34 Example 3 22.2 70.7 34 Comparative Example 1 19 66 38

[0055] As can be seen from Table 2, the acid production rate of glutamic acid obtained by the fermentation method in Comparative Example 1 is 19%, and the conversion rate is 66%, which are significantly lower than the acid production rate and conversion rate of glutamic acid obtained by the fermentation methods in Examples 1-3 of the present invention; the fermentation period of glutamic acid obtained by the fermentation method in Comparative Example 1 is 38 h, which is significantly higher than the fermentation period of glutamic acid obtained by the fermentation methods in Examples 1-3 of the present invention. It can be seen that adding naphthylacetic acid to the fermentation medium of the present invention can promote cell division and cell volume expansion, promote growth, reduce the cell wall density, promote the excretion of glutamic acid, and the decrease of intracellular glutamic acid weakens its feedback inhibition on glutamate dehydrogenase, ultimately promoting the synthesis of glutamic acid. It shortens the fermentation period, improves the cell viability and acid production level, and at the same time can reduce the production cost.

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A seed culture medium for increasing glutamic acid production, characterized in that, The solutes and their concentrations in the culture medium are as follows: glucose 35 - 45 g / L, corn steep liquor 40 - 50 g / L, soybean meal hydrolyzate 10 - 20 g / L, potassium chloride 0.2 - 0.6 g / L, magnesium sulfate 0.6 - 1.0 g / L, succinic acid 2 - 3 g / L, naphthaleneacetic acid 5 - 10 μg / L, antifoaming agent 0.3 - 0.5 mL / L, biotin 0.4 - 0.5 mg / L, ferrous sulfate 0.1 - 0.2 mg / L, manganese sulfate 0.1 - 0.2 mg / L, and the rest is water.

2. The seed culture medium for increasing glutamic acid production according to claim 1, characterized in that, The concentration of naphthaleneacetic acid in the culture medium is 10 μg / L.

3. The seed culture medium for increasing the glutamic acid production according to claim 1, characterized in that, The concentration of the corn steep liquor is 17 - 22 Baumé.

4. The seed culture medium for increasing glutamic acid production according to claim 1, wherein The total nitrogen content of the soybean meal hydrolyzate is 20 - 30 g / L.

5. A fermentation medium for increasing glutamic acid production, characterized in that, The solutes and their concentrations in the culture medium are as follows: naphthaleneacetic acid 50 - 200 μg / L, glucose 40 - 50 g / L, corn steep liquor 60 - 80 g / L, soybean meal hydrolyzate 20 - 30 g / L, betaine 1 - 2 g / L, potassium chloride 1 - 2 g / L, magnesium sulfate 1 - 2 g / L, antifoaming agent 0.3 - 0.5 mL / L, biotin 0.4 - 0.5 mg / L, ferrous sulfate 0.1 - 0.2 mg / L, manganese sulfate 0.1 - 0.2 mg / L, and the rest is water.

6. The fermentation medium for increasing glutamic acid production according to claim 5, characterized in that, The concentration of naphthaleneacetic acid in the culture medium is 200 μg / L.

7. The fermentation medium for increasing glutamic acid production according to claim 5, characterized in that, The concentration of the corn steep liquor is 17 - 22 Baumé.

8. The fermentation medium for increasing glutamate production according to claim 5, characterized in that, The total nitrogen content of the soybean meal hydrolyzate is 20 - 30 g / L.

9. A method for increasing glutamic acid production, characterized in that, After culturing the strain seed liquid using the seed culture medium as described in Claim 1, and then performing fermentation culture using the fermentation culture medium as described in Claim 5, the product is obtained; the strain is temperature-sensitive Corynebacterium glutamicum.

10. The method according to claim 9, characterized in that, The conditions for culturing the strain seed liquid are as follows: tank pressure 0.05 - 0.1 Mpa, ventilation volume 10 - 20 L / min, stirring 200 - 800 rpm, pH 7.0 - 7.2; the temperature is controlled at 33°C, and by sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle, the dissolved oxygen is maintained at 30 - 50%; the conditions for fermentation culture are as follows: tank pressure 0.05 - 0.1 Mpa, ventilation volume 10 - 30 L / min, stirring 200 - 800 rpm, pH 6.8 - 7.2; the temperature is controlled at 33 - 40°C, and the residual sugar during the process is controlled: the fed-batch sugar is 65%, the residual sugar during the process is controlled at 0 - 0.5%, and the fed-batch sugar rate is gradually increased according to the OD growth. By sequentially adjusting the tank pressure, ventilation volume, and stirring in a cycle, the dissolved oxygen is maintained at 30 - 60%.

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