CN115109806A

CN115109806A - Seed culture medium and fermentation culture medium for increasing glutamic acid yield

Info

Publication number: CN115109806A
Application number: CN202210620584.7A
Authority: CN
Inventors: 龙泉伟; 李必金; 程国平; 杨帆; 张金龙; 李岩
Original assignee: Jilin Meihua Amino Acid Co ltd
Current assignee: Jilin Meihua Amino Acid Co ltd
Priority date: 2022-06-02
Filing date: 2022-06-02
Publication date: 2022-09-27

Abstract

The invention provides a seed culture medium and a fermentation culture medium for improving the yield of glutamic acid, belonging to the field of fermentation engineering technology, wherein solutes and the concentrations thereof in the seed culture medium are as follows: glucose, corn steep liquor, soybean meal hydrolysate, potassium chloride, magnesium sulfate, succinic acid, naphthylacetic acid, a defoaming agent, biotin, ferrous sulfate, manganese sulfate and the balance of water. The solutes in the fermentation medium and their concentrations are: naphthylacetic acid, glucose, corn steep liquor, soybean meal hydrolysate, betaine, potassium chloride, magnesium sulfate, a defoaming agent, biotin, ferrous sulfate, manganese sulfate and the balance of water. The naphthylacetic acid is added into the culture medium, so that cell division and cell expansion can be promoted, and growth can be promoted. The cell wall density can be reduced, the glutamic acid discharge is promoted, the feedback inhibition effect of the reduction of intracellular glutamic acid on glutamate dehydrogenase is weakened, and the synthesis of the glutamic acid is promoted finally.

Description

Seed culture medium and fermentation culture medium for increasing glutamic acid yield

Technical Field

The invention relates to the technical field of fermentation engineering, in particular to a seed culture medium and a fermentation culture medium for improving the yield of glutamic acid.

Background

Glutamic acid is an acidic amino acid containing one amino group and two carboxyl groups in the molecule, also known as alpha-aminoglutaric acid. Is colorless crystal, has delicate flavor, is slightly soluble in water, is easily soluble in hydrochloric acid solution, has a molecular weight of 147.1 and an isoelectric point of 3.22, is abundantly present in cereal protein, and has high animal brain content. Plays an important role in the protein metabolism process in a living organism, participates in the synthesis of protein, polypeptide and fatty acid, and regulates the ammonia level in the body together with glutamine; can also be used as excitatory neurotransmitter to participate in information transmission. Glutamic acid includes levorotatory isomer, dextrorotatory isomer and racemic body. The levorotatory form is L-glutamic acid. The naturally occurring glutamic acids are all L-glutamic acid. Glutamic acid is widely used in food, medicine, cosmetics and agriculture. L-glutamic acid is a precursor for the synthesis of monosodium glutamate (sodium glutamate). The sodium salt of L-glutamic acid has strong meat flavor and is a flavoring agent which is consumed most in the world except for sugar and common salt. The L-glutamate hydrochloride can be used for improving the bitterness of beer, and can also be used as salt substitute, nutritional supplement and flavoring agent.

The current production method of the glutamic acid is fermentation production, the pathway for synthesizing the glutamic acid by microorganisms is that glucose generates pyruvic acid through glycolysis pathway (EMP) and hexose phosphate branch (HMP), and the pyruvic acid is desinsected to generate phthalein coenzyme A; then oxaloacetate and acetyl coenzyme A are synthesized into citric acid under the catalysis of aldolase to further generate isocitric acid and alpha-ketoglutaric acid, and the alpha-ketoglutaric acid is subjected to glutamate dehydrogenase and NH ⁴⁺ In the presence of glutamic acid, the glutamic acid synthesized in the thallus permeates cell membranes, so that a large amount of glutamic acid can be accumulated in the fermentation liquor. Theoretically one and five molecules of glucose are ultimately produced from one six carbon sub-batch, without considering microbial growth and respiratory consumptionCarbon glutamate, theoretical saccharic acid conversion 81.7%.

The fermentation method has been developed for more than 50 years, the production technology has made great progress, the glutamic acid fermentation method has undergone several stages of producing glutamic acid by using biotin sub-quantitative method, high biotin content and temperature-sensitive type bacterial strain, the fermentation production intensity is obviously improved, the fermentation acid production level is promoted from 9-13% of the sub-quantitative method to 15-23% of the temperature-sensitive method, and the conversion rate level is promoted from 58-61% to 65-72%. At present, the industrial production is mainly the glutamic acid production of temperature sensitive strains, the domestic technical level has a certain gap compared with the international and the same industries, and the method has a larger space for improving the conversion rate compared with the theoretical conversion rate.

The main problems influencing the glutamic acid industry at present are that on one hand, a seed culture medium adopts carbon source for mixed digestion and high-temperature sterilization, Maillard reaction is easy to generate, the nutrient loss is large, and simultaneously, the generated byproducts of acrylamide and 5-hydroxymethylfurfural can inhibit the growth of thalli, so that the seed culture period is long and the strain activity is low; on the other hand, the high concentration of the fermentation medium causes the glutamic acid cells to be smaller, the cell wall density is high, and the intracellular glutamic acid accumulation inhibits the synthesis of the glutamic acid.

Disclosure of Invention

The invention aims to provide a seed culture medium and a fermentation culture medium for improving the yield of glutamic acid, improve the production process of glutamic acid, upgrade the product grade and improve the fermentation yield.

The technical scheme of the invention is realized as follows:

the invention provides a seed culture medium for improving the yield of glutamic acid, wherein solutes and concentrations thereof in the culture medium are as follows: 35-45g/L of glucose, 40-50g/L of corn steep liquor, 10-20g/L of soybean meal hydrolysate, 0.2-0.6g/L of potassium chloride, 0.6-1.0g/L of magnesium sulfate, 2-3g/L of succinic acid, 5-10ug/L of naphthylacetic acid, 0.3-0.5mL/L of defoamer, 0.4-0.5mg/L of biotin, 0.1-0.2mg/L of ferrous sulfate, 0.1-0.2mg/L of manganese sulfate and the balance of water. The improved secondary seed culture medium formula can improve the activity of strains and shorten the seed period.

As a further improvement of the invention, the concentration of the naphthylacetic acid in the culture medium is 10 ug/L.

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

As a further improvement of the invention, the total nitrogen content of the soybean meal hydrolysate is 20-30 g/L.

The invention further protects a fermentation medium for improving the yield of glutamic acid, wherein solutes and concentrations in the medium are as follows: 50-200ug/L of naphthylacetic acid, 40-50g/L of glucose, 60-80g/L of corn steep liquor, 20-30g/L of soybean meal hydrolysate, 1-2g/L of betaine, 1-2g/L of potassium chloride, 1-2g/L of magnesium sulfate, 0.3-0.5mL/L of defoaming agent, 0.4-0.5mg/L of biotin, 0.1-0.2mg/L of ferrous sulfate, 0.1-0.2mg/L of manganese sulfate and the balance of water. The improved fermentation medium formula can improve the activity of thalli, improve acid production and shorten the fermentation period.

As a further improvement of the invention, the concentration of the naphthylacetic acid in the culture medium is 50-200 ug/L.

The invention further provides a method for improving the yield of glutamic acid, which comprises the steps of carrying out seed liquid culture on a strain by using the seed culture medium, and then carrying out fermentation culture by using the fermentation culture medium to obtain a product.

As a further improvement of the invention, the strain is temperature-sensitive corynebacterium glutamicum; the culture conditions of the strain seed liquid are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation volume is 10-20L/min, the stirring speed is 200-800rpm, and the pH value is 7.0-7.2; controlling the temperature to be 33 ℃, and maintaining the dissolved oxygen at 30-50% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring; the fermentation culture conditions are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation volume is 10-30L/min, the stirring speed is 200-800rpm, and the pH value is 6.8-7.2; controlling the temperature to be 33-40 ℃, and controlling the residual sugar in the process: 65% of sugar is fed, 0-0.5% of residual sugar is controlled in the process, the feeding speed is gradually increased according to the increase of OD, and the dissolved oxygen is maintained at 30-60% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring.

The invention has the following beneficial effects:

(1) the naphthylacetic acid is added into the seed culture medium, so that cell division and cell expansion can be promoted, growth is promoted, and the seed culture period is shortened.

(2) The naphthylacetic acid is added into the fermentation medium, so that cell division and cell expansion can be promoted, and growth can be promoted. The cell wall density can be reduced, the glutamic acid discharge is promoted, the feedback inhibition effect of the reduction of intracellular glutamic acid on glutamate dehydrogenase is weakened, and the synthesis of the glutamic acid is promoted finally. The activity of the thallus and the acid production are improved, and the acid production reaches 22 percent.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments or the description of the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a graph of acid yield over time for each set of the present invention;

FIG. 2 is a graph comparing the conversion of the various groups of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The following examples and comparative columns all adopt a unified control process, the engineering strain is temperature-sensitive corynebacterium glutamicum, and the secondary seed and fermentation control process comprises the following steps:

the secondary seed culture conditions are as follows: the pot pressure is 0.07Mpa, the ventilation volume is 15L/min, the stirring is carried out at 600rpm, and the pH value is 7.1; the temperature is controlled at 33 ℃, and dissolved oxygen is maintained at 40% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring.

The fermentation conditions were: the pot pressure is 0.07Mpa, the ventilation volume is 20L/min, the stirring is 600rpm, and the pH value is 7.0; controlling the temperature to be 33-40 ℃, and controlling the residual sugar in the process: 65% of fed-batch sugar, 0.3% of residual sugar in process control, gradually increasing the fed-batch sugar rate according to OD increase, and maintaining the dissolved oxygen at 45% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring.

Example 1

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

The solute in the seed culture medium and the concentration thereof are as follows: 35g/L of glucose, 40g/L of corn steep liquor, 10g/L of soybean meal hydrolysate, 0.2g/L of potassium chloride, 0.6g/L of magnesium sulfate, 2g/L of succinic acid, 5ug/L of naphthylacetic acid, 0.3mL/L of defoaming agent, 0.4mg/L of biotin, 0.1mg/L of ferrous sulfate, 0.1mg/L of manganese sulfate and the balance of water.

The solutes in the fermentation medium and their concentrations were: 50ug/L of naphthylacetic acid, 40g/L of glucose, 60g/L of corn steep liquor, 20g/L of soybean meal hydrolysate, 1g/L of betaine, 1g/L of potassium chloride, 1g/L of magnesium sulfate, 0.3mL/L of defoaming agent, 0.4mg/L of biotin, 0.1mg/L of ferrous sulfate and 0.1mg/L of manganese sulfate, and the solvent is water.

And (3) after the seed culture is finished, measuring the OD value of the lower tank to be 1.05, determining the period to be 22.6h, operating for 34h, finishing the fermentation, and obtaining 21.8% of acid and 70% of conversion rate.

Example 2

The solute in the seed culture medium and the concentration thereof are as follows: 45g/L of glucose, 50g/L of corn steep liquor, 20g/L of soybean meal hydrolysate, 0.6g/L of potassium chloride, 1.0g/L of magnesium sulfate, 2-3g/L of succinic acid, 10ug/L of naphthylacetic acid, 0.5mL/L of defoaming agent, 0.5mg/L of biotin, 0.2mg/L of ferrous sulfate, 0.2mg/L of manganese sulfate and the balance of water.

The solutes in the fermentation medium and their concentrations were: 100ug/L of naphthylacetic acid, 50g/L of glucose, 80g/L of corn steep liquor, 30g/L of soybean meal hydrolysate, 2g/L of betaine, 2g/L of potassium chloride, 2g/L of magnesium sulfate, 0.5mL/L of defoaming agent, 0.5mg/L of biotin, 0.2mg/L of ferrous sulfate and 0.2mg/L of manganese sulfate, and the solvent is water.

After the seed culture is finished, determining the OD value of the lower tank to be 1.05, the period to be 22.0h, running for 34h to finish the fermentation, and obtaining 22% of acid and 70.5% of conversion rate

Example 3

This example provides a seed medium and a fermentation medium for increasing the production of glutamic acid.

The solutes in the seed culture medium and the concentrations thereof are as follows: 40g/L of glucose, 45g/L of corn steep liquor, 15g/L of soybean meal hydrolysate, 0.4g/L of potassium chloride, 0.8g/L of magnesium sulfate, 2.5g/L of succinic acid, 20ug/L of naphthylacetic acid, 0.4mL/L of defoaming agent, 0.45mg/L of biotin, 0.15mg/L of ferrous sulfate, 0.15mg/L of manganese sulfate and the balance of water.

The solutes in the fermentation medium and their concentrations were: 200ug/L of naphthylacetic acid, 45g/L of glucose, 70g/L of corn steep liquor, 25g/L of soybean meal hydrolysate, 1.5g/L of betaine, 1.5g/L of potassium chloride, 1.5g/L of magnesium sulfate, 0.4mL/L of defoaming agent, 0.45mg/L of biotin, 0.15mg/L of ferrous sulfate, 0.5mg/L of manganese sulfate and water as a solvent.

After the seed culture is finished, determining the OD value of the lower tank to be 1.05, the period to be 22.1h, operating for 34h to finish the fermentation, and obtaining 22.2 percent of acid and 70.7 percent of conversion rate

Comparative example 1

The glutamic acid is produced by fermentation by adopting a traditional method, and the specific process is as follows:

the solutes in the seed culture medium and their concentrations were: 40g/L of glucose, 45g/L of corn steep liquor, 15g/L of soybean meal hydrolysate, 0.4g/L of potassium chloride, 0.8g/L of magnesium sulfate, 2.5g/L of succinic acid, 0.4mL/L of defoamer, 0.45mg/L of biotin, 0.15mg/L of ferrous sulfate, 0.15mg/L of manganese sulfate and the balance of water.

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

And (3) after the seed culture is finished, measuring the OD value of the lower tank to be 1.05, measuring the period to be 28h, operating for 38h, finishing the fermentation, and producing 19% of acid and having a conversion rate of 66%.

Test example 1

The experimental results of examples 1 to 3 and comparative example 1 are shown in tables 1 and 2 below;

TABLE 1 indexes of seed solutions for culturing temperature-sensitive Corynebacterium glutamicum of examples 1 to 3 and comparative example 1

Item	OD value of seed liquid	Growth cycle of seeds 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

As can be seen from Table 1, when the seed culture medium in comparative example 1 is used for culturing the seed liquid for culturing the temperature-sensitive Corynebacterium glutamicum until the OD value is 1.0, the required seed growth period is 28h, which is obviously higher than the required seed growth period when the seed culture medium in examples 1 to 3 is used for culturing the seed liquid for culturing the temperature-sensitive Corynebacterium glutamicum until the OD value is 1.0, so that the method can effectively promote the division and the growth of the temperature-sensitive Corynebacterium glutamicum by adding the naphthylacetic acid into the seed culture medium, shorten the growth period of the seeds of the temperature-sensitive Corynebacterium glutamicum, and improve the vitality of strains and the acid production level.

TABLE 2 fermentation index summarisation of examples 1-3 and comparative example 1

Item	The acid yield is%	Conversion rate%	Fermentation period 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

As can be seen from Table 2, the acid production rate of glutamic acid obtained by the fermentation method in comparative example 1 was 19%, and the conversion rate was 66%, which were significantly lower than those of glutamic acid obtained by the fermentation methods in examples 1 to 3 of the present invention; the fermentation period of the glutamic acid obtained by the fermentation method in the comparative example 1 is 38h, which is obviously higher than that of the glutamic acid obtained by the fermentation method in the examples 1-3 of the invention, so that the naphthylacetic acid added into the fermentation medium of the invention can promote cell division and cell volume expansion, promote growth, reduce cell wall density, promote glutamic acid discharge, reduce the intracellular glutamic acid, weaken the feedback inhibition effect of the intracellular glutamic acid on glutamate dehydrogenase, and finally promote the synthesis of the glutamic acid. The fermentation period is shortened, the thallus activity and the acid production level are improved, and the production cost can be reduced.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A seed culture medium for improving the yield of glutamic acid is characterized in that solutes and the concentrations thereof in the culture medium are as follows: 35-45g/L of glucose, 40-50g/L of corn steep liquor, 10-20g/L of soybean meal hydrolysate, 0.2-0.6g/L of potassium chloride, 0.6-1.0g/L of magnesium sulfate, 2-3g/L of succinic acid, 5-10ug/L of naphthylacetic acid, 0.3-0.5mL/L of defoaming agent, 0.4-0.5mg/L of biotin, 0.1-0.2mg/L of ferrous sulfate, 0.1-0.2mg/L of manganese sulfate and the balance of water.

2. The seed culture medium for increasing the production of glutamic acid according to claim 1, wherein the concentration of naphthylacetic acid in the culture medium is 10 ug/L.

3. The seed culture medium for increasing the production of glutamic acid according to claim 1, wherein the concentration of the corn steep liquor is 17 to 22 Baume degrees.

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

5. A fermentation culture medium for improving the yield of glutamic acid is characterized in that solutes and concentrations thereof in the culture medium are as follows: 50-200ug/L of naphthylacetic acid, 40-50g/L of glucose, 60-80g/L of corn steep liquor, 20-30g/L of soybean meal hydrolysate, 1-2g/L of betaine, 1-2g/L of potassium chloride, 1-2g/L of magnesium sulfate, 0.3-0.5mL/L of defoaming agent, 0.4-0.5mg/L of biotin, 0.1-0.2mg/L of ferrous sulfate, 0.1-0.2mg/L of manganese sulfate and the balance of water.

6. The fermentation medium for increasing the production of glutamic acid according to claim 5, wherein the concentration of naphthylacetic acid in the medium is 50-200 ug/L.

7. The fermentation medium for increasing the production of glutamic acid according to claim 5, wherein the concentration of the corn steep liquor is 17 to 22 Baume degrees.

8. The fermentation medium for increasing the production of glutamic acid according to claim 5, wherein the total nitrogen content of the soybean meal hydrolysate is 20-30 g/L.

9. A method for increasing the yield of glutamic acid, characterized in that, after a seed culture is performed by using the seed culture medium of claim 1, a fermentation culture is performed by using the fermentation medium of claim 5 to obtain a product.

10. The method of claim 9, wherein the bacterial species is C.glutamicum; the culture conditions of the strain seed liquid are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation volume is 10-20L/min, the stirring speed is 200-800rpm, and the pH value is 7.0-7.2; controlling the temperature to be 33 ℃, and maintaining the dissolved oxygen at 30-50% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring; the fermentation culture conditions are as follows: the pressure in the tank is 0.05-0.1Mpa, the ventilation volume is 10-30L/min, the stirring speed is 200-800rpm, and the pH value is 6.8-7.2; controlling the temperature to be 33-40 ℃, and controlling the residual sugar in the process: 65% of sugar is fed, 0-0.5% of residual sugar is controlled in the process, the feeding speed is gradually increased according to the increase of OD, and the dissolved oxygen is maintained at 30-60% by sequentially and circularly adjusting the tank pressure, the ventilation volume and the stirring.