阅读说明：本技术 一种发酵生产l-亮氨酸的方法 (method for producing L-leucine by fermentation ) 是由 冯世红 包鑫 史静东 韦树高 边恩来 于 2019-10-06 设计创作，主要内容包括：本发明属于L-亮氨酸生产工艺领域,公开了一种发酵生产L-亮氨酸的方法,其包括如下步骤：采用产L-亮氨酸的黄色短杆菌发酵44h以上；当发酵至30h时,往发酵罐中流加醋酸钠水溶液,控制发酵液中醋酸钠的浓度为0.1-1g/L,直至发酵结束,所述醋酸钠水溶液的浓度为50-100g/L；当发酵至30h时,往发酵罐中流加丙二酸水溶液,控制发酵液中丙二酸的浓度为0.1-0.5 ml/L,直至发酵结束；所述丙二酸水溶液的浓度为10-20%；当发酵至40h时,往发酵罐中添加壳聚糖,控制发酵液中壳聚糖的浓度为20-80mg/L。本发明方法发酵效率高,操作简单,成本低廉。(The invention belongs to the field of L-leucine production process, and discloses a method for producing L-leucine by fermentation, which comprises the following steps: fermenting for more than 44h by using Brevibacterium flavum for producing L-leucine; when the fermentation is carried out for 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.1-1g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 50-100 g/L; when the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.1-0.5 ml/L until the fermentation is finished; the concentration of the malonic acid aqueous solution is 10-20%; when the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquor to be 20-80 mg/L. The method has the advantages of high fermentation efficiency, simple operation and low cost.)

1. A method for producing L-leucine by fermentation, comprising the steps of: fermenting for more than 44h by using Brevibacterium flavum for producing L-leucine;

When the fermentation is carried out for 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.1-1g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 50-100 g/L;

When the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.1-0.5 ml/L until the fermentation is finished; the concentration of the malonic acid aqueous solution is 10-20%;

when the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquid to be 20-80mg/L until the fermentation is finished.

2. The method according to claim 1, wherein the concentration of sodium acetate in the fermentation broth is controlled to be 0.4-0.5 g/L.

3. The method according to claim 1, wherein the concentration of malonic acid in the fermentation broth is controlled to be 0.2 to 0.3 ml/L.

4. The method according to claim 1, wherein the concentration of chitosan in the fermentation broth is controlled to be 40-50 mg/L.

5. Method according to claim 1, characterized in that it comprises the following steps:

inoculating the L-leucine-producing Brevibacterium flavum seed solution into a fermentation tank filled with a fermentation tank culture medium according to the inoculation amount of 8-10%, and stirring at the rotation speed of 300-; controlling the dissolved oxygen to be 20-30% by stirring and ventilating; controlling the pH value to be 7.0 by adding ammonia water in a flowing manner; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period was 44 h.

6. the method of claim 5, wherein the components of the fermentor medium are: 50g/L glucose, (NH4)2SO 410 g/L, 10g/L corn steep liquor, KH2PO 44 g/L, MgSO4 & 7H2O 1.5.5 g/L, citric acid 0.5g/L, MnSO4 & H2O 10mg/L, FeSO4 & 7H2O 0.1.1 g/L, VB 15 mg/L and biotin 50 mu g/L.

7. the method according to claim 5, wherein the nutrient solution has a composition: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.

Technical Field

The invention belongs to the field of L-leucine production processes, and particularly provides a method for producing L-leucine by fermentation.

Background

l-leucine is also called leucine, has a chemical name of alpha-aminoisocaproic acid, a molecular formula of C6H13O2N, a relative molecular weight of 131.18, is a nonpolar amino acid, tastes slightly bitter, is soluble in water, has an isoelectric point of 5.98, and is widely applied to various industries such as the manufacture of medicines, foods, flavoring agents, animal feeds, cosmetics and the like. The research of China on L-leucine starts relatively late, the L-leucine production level of partial enterprises by fermentation methods is relatively low, the research on downstream pretreatment and extraction processes is relatively less, the industrial production is difficult to realize, and the problems of high acid and alkali consumption, serious pollution, low product yield and purity and the like exist.

At present, the production method of L-leucine is mainly a fermentation method, and the L-leucine fermentation liquor produced by the fermentation method can be prepared into a finished product through the steps of extraction, evaporation concentration, centrifugal drying and the like. The prior patent technology of the applicant, namely 'a fermentation process for producing L-leucine', improves the yield of leucine by feeding a compound amino acid solution and a nutrient solution in the middle and later stages of fermentation, but the feeding solution has higher cost and is not suitable for large-scale production. The applicant's previous patent technology ' a method for improving L-leucine fermentation yield ', membrane coupling intermittent dialysis fermentation is adopted, the concentration of byproducts is effectively reduced, the sugar-acid conversion rate is improved, and L-leucine yield and the sugar-acid conversion rate in fermentation liquor can be promoted by supplementing nutrient solution containing glycerol and betaine in the middle stage of fermentation; however, this method has a drawback of complicated steps. The technical problem to be solved is to provide a leucine fermentation process which is low in cost, easy to operate and capable of being developed sustainably.

Disclosure of Invention

In order to solve the common problem of L-leucine in industrial fermentation production, the invention provides a method for producing L-leucine by fermentation, which has the advantages of high fermentation efficiency, simple operation and low cost.

The invention is realized by the following technical scheme.

A method for producing L-leucine by fermentation, comprising the steps of: fermenting for more than 44h by using Brevibacterium flavum for producing L-leucine;

when the fermentation is carried out for 30 hours, adding a sodium acetate aqueous solution into the fermentation tank, and controlling the concentration of sodium acetate in the fermentation liquor to be 0.1-1g/L until the fermentation is finished, wherein the concentration of the sodium acetate aqueous solution is 50-100 g/L;

When the fermentation time is 30 hours, adding a malonic acid aqueous solution into the fermentation tank, and controlling the concentration of malonic acid in the fermentation liquid to be 0.1-0.5 ml/L until the fermentation is finished; the concentration of the malonic acid aqueous solution is 10-20%;

When the fermentation time is up to 40h, adding chitosan into the fermentation tank, and controlling the concentration of the chitosan in the fermentation liquor to be 20-80 mg/L.

preferably, the concentration of sodium acetate in the fermentation broth is controlled to be 0.4-0.5 g/L.

preferably, the concentration of malonic acid in the fermentation broth is controlled to be 0.2-0.3 ml/L.

Preferably, the concentration of chitosan in the fermentation broth is controlled to be 40-50 mg/L.

Preferably, the method comprises the steps of:

Inoculating the L-leucine-producing Brevibacterium flavum seed solution into a fermentation tank filled with a fermentation tank culture medium according to the inoculation amount of 8-10%, and stirring at the rotation speed of 300-; controlling the dissolved oxygen to be 20-30% by stirring and ventilating; controlling the pH value to be 7.0 by adding ammonia water in a flowing manner; the culture temperature is 33 ℃; defoaming with foam killer; when the fermentation time is 20 hours, feeding nutrient solution into the fermentation tank, maintaining the sugar concentration in the fermentation tank to be 1g/L, and stopping feeding 6 hours before the fermentation is finished; the fermentation period was 44 h.

Preferably, the components of the fermenter medium: 50g/L glucose, (NH4)2SO 410 g/L, 10g/L corn steep liquor, KH2PO 44 g/L, MgSO4 & 7H2O 1.5.5 g/L, citric acid 0.5g/L, MnSO4 & H2O 10mg/L, FeSO4 & 7H2O 0.1.1 g/L, VB 15 mg/L and biotin 50 mu g/L.

Preferably, the components of the nutrient solution are: 100g/L of glucose, 5g/L of glycerol and 1g/L of betaine.

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

Acetic acid is one of the main byproducts in the leucine fermentation process; the invention discovers that in the fermentation process, the sodium acetate is added, so that the feedback inhibition can be carried out on the acetic acid synthetic pathway, the acetic acid synthetic pathway is blocked, the generation amount of Val is correspondingly reduced, and more metabolic flows flow to the Leu synthetic pathway;

The malonic acid can inhibit key enzymes of TCA cycle, so that the TCA cycle is weakened, the generation amount of acetic acid and other byproducts in the TCA cycle is reduced, more carbon sources enter a Leu synthesis way, and the output of Leu is improved;

In the middle and later stages of fermentation, a certain amount of chitosan is added, amino on the chitosan is 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, and leucine is promoted to be secreted out of cells, and the yield of the leucine is improved; however, when the chitosan concentration is too high, a certain damage is caused to the strain, the strain proliferation is hindered, and death occurs.

Drawings

FIG. 1: influence of sodium acetate concentration on L-Leu yield;

FIG. 2: influence of sodium acetate concentration on Val and Hac content;

FIG. 3: the effect of malonic acid concentration on L-Leu production;

FIG. 4: effect of chitosan concentration on L-Leu production.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

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.