阅读说明：本技术 一种提高l-异亮氨酸产量的方法 (method for improving L-isoleucine yield ) 是由 包鑫 李江涛 张宗华 田辉 冯世红 李江雷 于 2019-09-21 设计创作，主要内容包括：本发明属于L-异亮氨酸生产技术领域,公开了一种提高L-异亮氨酸产量的方法,包括：其包括添加H<Sub>2</Sub>O<Sub>2</Sub>和葡萄糖营养液的步骤。本发明通过对发酵方法的优化,提高了L-异亮氨酸的产量。(the invention belongs to the technical field of L-isoleucine production, and discloses a method for improving the yield of L-isoleucine, which comprises the step of adding H 2 O 2 and glucose nutrient solution.)

1. A method for increasing the production of L-isoleucine comprising the step of adding H ₂ O ₂ and a glucose nutrient solution.

2. The method of claim 1, further comprising the step of adding chitosan.

3. the method of claim 2, wherein the H ₂ O ₂ is added in batch at the middle and late stages of fermentation, and the chitosan is added at the middle and late stages of fermentation.

4. A method according to any one of claims 1-3, characterized in that the method comprises the steps of:

transferring the corynebacterium glutamicum seed liquid into a 50L fermentation tank containing 30L of fermentation medium according to the inoculation amount of 6-10% for culturing, controlling the pH of the culture medium to be 6.8-7.0 by automatically feeding ammonia water, controlling the dissolved oxygen to be 20-30% by stirring and ventilating, and feeding glucose nutrient solution to control the content of residual sugar in the fermentation liquid to be not less than 1g/L, wherein the temperature is 30 ℃ and the culture time is 60 hours;

In the fermentation process, 0.2L of H ₂ O ₂ is injected in 40H, 42H, 44H, 46H, 48H and 50H respectively, and chitosan is added in 48H to the fermentation tank, so that the concentration of the chitosan is controlled to be 20-60 mg/L.

5. The method of claim 4, wherein the composition of the glucose nutrient solution is: glucose is 100-200g/L, and alpha-hydroxybutyric acid is 10-30 g/L.

6. The method of claim 4, wherein the fermentation medium comprises 80g/L glucose, (NH ₄) ₂ SO ₄ 20g/L corn steep liquor 10g/L KH ₂ PO ₄ 5g/L MgSO ₄ & 7H ₂ O2 g/L MnSO ₄ & H ₂ O10 mg/L FeSO ₄ & 7H ₂ O0.1 g/L V _B1 5mg/L V _H 20. mu.g/L.

7. The method of claim 4, wherein the chitosan is present at a concentration of 40 mg/L.

8. The method of claim 5, wherein the composition of the glucose nutrient solution is: 100g/L glucose and 20g/L alpha-hydroxybutyric acid.

Technical Field

The invention belongs to the technical field of L-isoleucine production, and particularly relates to a method for improving the yield of L-isoleucine.

Background

In 1901, Fischer discovered substances with higher optical rotation than leucine in an L-leucine component separated from a protein hydrolysate, which was first reported about L-isoleucine. L-isoleucine is an important component of living organisms and is an essential amino acid because its specific structure and function play an important role in human body's vital metabolism. If the human body lacks L-isoleucine for a long time, the physiological functions of the body are affected, and metabolic disturbance, resistance reduction and the like are caused. The L-isoleucine has unique effect and is widely applied to industries of food health care, biomedicine, medical treatment, beauty treatment and the like.

the production scale of isoleucine in China is small, the acid yield of the strain is low, the batch fermentation level of a fermentation tank is about 25g/L, the extraction rate is about 50%, the production process, the production level and the production equipment are far behind those of Japan and other countries, and the yield cannot meet the market demand. At the end of the last century, the annual yield of L-isoleucine is about 400t, and most of China depends on imports. In recent years, with the increase of the yield of each domestic manufacturer, the yield of isoleucine in China gradually meets the domestic requirements, and part of products are exported. The application of biochemical technology is highly emphasized in the domestic amino acid industry, the problems of long fermentation period, low acid yield and low product quality of the existing L-isoleucine are solved as soon as possible, and the application research of isoleucine in a new field is developed to promote the development of the L-isoleucine industry in China.

Disclosure of Invention

In order to solve the problems and overcome the defects of the existing fermentation process, the invention provides the method for improving the yield of the L-isoleucine, and the fermentation method is easy to operate and greatly improves the fermentation efficiency.

the purpose of the invention is realized by the following technical scheme:

A method for increasing the production of L-isoleucine comprising the step of adding H ₂ O ₂ and a glucose nutrient solution.

Further, the method further comprises the step of adding chitosan.

further, the H ₂ O ₂ is added in batches in the middle and later stages of fermentation, and the chitosan is added in the middle and later stages of fermentation.

Further, the method comprises the steps of:

transferring the corynebacterium glutamicum seed liquid into a 50L fermentation tank containing 30L of fermentation medium according to the inoculation amount of 6-10% for culturing, controlling the pH of the culture medium to be 6.8-7.0 by automatically feeding ammonia water, controlling the dissolved oxygen to be 20-30% by stirring and ventilating, and feeding glucose nutrient solution to control the content of residual sugar in the fermentation liquid to be not less than 1g/L, wherein the temperature is 30 ℃ and the culture time is 60 hours;

In the fermentation process, 0.2L of H ₂ O ₂ is injected in 40H, 42H, 44H, 46H, 48H and 50H respectively, and chitosan is added in 48H to the fermentation tank, so that the concentration of the chitosan is controlled to be 20-60 mg/L.

Further, the glucose nutrient solution comprises the following components: glucose is 100-200g/L, and alpha-hydroxybutyric acid is 10-30 g/L.

Preferably, the fermentation medium comprises 80g/L glucose, (NH ₄) ₂ SO ₄ 20g/L, 10g/L corn steep liquor, KH ₂ PO ₄ 5g/L, MgSO35 ₄.7H ₂ O2 g/L, MnSO ₄. H ₂ O10 mg/L, FeSO ₄.7H ₂ O0.1 g/L, V _B1 5mg/L and V _H 20. mu.g/L.

Preferably, the concentration of the chitosan is 40 mg/L.

Preferably, the glucose nutrient solution comprises the following components: 100g/L glucose and 20g/L alpha-hydroxybutyric acid.

Compared with the prior art, the invention has the advantages that the following aspects are mainly included but not limited:

the invention improves two aspects of a biological metabolism regulation and control approach and an abiotic metabolism regulation and control approach, improves the acid production efficiency, and further improves the yield of isoleucine.

The invention selects H ₂ O ₂ as the oxygen carrier, and has the advantages that (1) H ₂ O ₂ is combined with ventilation and oxygen supply, and the appropriate H ₂ O ₂ adding concentration and adding mode can improve the cell density of the fermentation system, (2) H ₂ O ₂ is decomposed to release oxygen under the catalysis of catalase, and under the condition that the reaction is not very high, the oxygen is directly transmitted to cells in a molecular form, so that the gas-liquid mass transfer resistance cannot be formed, the oxygen mass transfer rate is improved, and the oxygen transmission can greatly improve the economic benefit if the gas-liquid transmission resistance is completely eliminated, (3) the fermentation system which is sensitive to shearing force and very high in viscosity is added with H ₂ O ₂, so that an oxygen supply method for improving oxygen supply is provided, (4) the oxygen supply and supply method can be more effective for synthesizing thallus products, and the thallus metabolism can be promoted;

Compared with single injection, the oxygen carrying agent H ₂ O ₂ is added in the middle and later stages of fermentation in a multi-injection mode, the yield of isoleucine is greatly improved on the premise that the total addition amount is not changed, and no other harmful substances are generated in the whole fermentation process of decomposition of H ₂ O ₂, so that the sanitation requirement of materials is ensured, and the environmental protection requirement is ensured;

Isoleucine fermentation belongs to a coupled growth part, and isoleucine cannot be produced in the early stage, so isoleucine is produced in the middle and later stages of fermentation, feedback regulation exists between threonine and L-isoleucine in the later stage of fermentation along with the increase of acid production, and alpha-hydroxybutyric acid is added in the later stage of fermentation, so that the feedback regulation of threonine and L-isoleucine can be bypassed, the biological metabolism regulation and control effect is relieved, and the aim of improving isoleucine is fulfilled;

In the middle and later stages of fermentation, a proper amount of chitosan is added, amino groups on the chitosan are combined with teichoic acid or lipopolysaccharide with negative charges in bacterial cell walls, and cations such as Mg2+, Ca2+ and the like are chelated, so that the permeability of the cell walls is changed, isoleucine is promoted to be secreted to the outside of the cells, and the yield of isoleucine is increased.

Drawings

FIG. 1: the effect of α -hydroxybutyrate on L-isoleucine content;

FIG. 2: the influence of alpha-hydroxybutyric acid on the concentration of the bacteria;

FIG. 3: the effect of chitosan on L-isoleucine production;

FIG. 4: influence of chitosan on the cell concentration.

Detailed Description

In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be described more clearly and completely below with reference to specific embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, 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.