KR870001812B1 - Process for preparing l-glutamic acid - Google Patents

Abstract

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Description

Method for preparing L-glutamic acid

The present invention relates to a method for producing glutamic acid by an effective method of administering additional sugars when glutamic acid is prepared by batch culture while aeration with a biotin-containing carbohydrate medium.

In general, when glutamic acid is prepared under aerobic conditions in a carbohydrate medium containing a large amount of biotin, various methods have been devised to increase the amount of glutamic acid in the culture medium.

Until now, the high concentration of 15% high sugar was added to the initial medium or the additional sugar was supplied continuously or intermittently instead of lowering the sugar concentration in the initial medium to increase the amount of glutamic acid in the final culture. However, when high concentration of sugar was added to the initial medium of these methods, the addition of more than 15% of sugar resulted in the inhibition of cell growth and the decrease of glutamic acid production yield. Although it has no sex and a high yield, it was not an industrially good method due to the small amount of glutamic acid in the final culture.

On the other hand, when fermenting glutamic acid in molasses medium containing excessive amounts of biotin, the initial medium is adjusted to a relatively high concentration of 5 to 10%, and the bacterial growth is suppressed by conventional methods, and then intermittently several times during the culture. In addition, a batch culture method of continuously supplying additional sugars and inducing a high concentration of glutamic acid while maintaining the sugar concentration of the culture medium below a certain level has been widely used.

In fermentation in which glutamic acid is produced by a batch culture method in a medium containing excessive amount of biotin, the growth of bacteria inoculated into the medium is controlled to obtain an appropriate amount of cells, and the obtained cells are converted into glutamic acid-producing cells by an appropriate method. The most important method is to add antibiotics such as surfactants or penicillin to the culture, or to incubate at 30-33 ℃, which is the optimum temperature for growing bacteria, and to inhibit the growth of glutamic acid. For incubation at 35-37 ℃ was used.

The present inventors have found that a high yield of glutamic acid can be produced by changing the method of adding additional sugars through the study of various methods to more efficiently induce the conversion of cultured microorganisms into glutamic acid-producing cells. The present invention has been completed.

The difference between the present invention and the conventional invention (Japanese Patent No. 40-1992, 41-5909) is as follows.

As can be seen from the previous table, the present invention artificially delays the addition time of primary sugars so that the cells in the culture medium are depleted for a certain period of time (20-30 minutes) before adding the first additional sugars. It features.

The results of the present inventors have demonstrated that the formation of such artificial sugars may allow the cells to be converted into glutamic acid-producing cells in a shorter time, thereby allowing a large amount of glutamic acid to be accumulated in a short time. In the previous table, the sugar concentration before the first sugar injection was 0.6-0.8%, but this was not used in the conventional molasses medium by bacteria such as Brevibacterium lactopfermentum and Corynebacterium glutamicum which are used in the present invention. It is because it contains less than 10% of non-fermentable sugar, which means that the cells are in a state of sugar depletion from the initial concentration of 10% of the medium.

To determine the relationship between sugar depletion status and glutamic acid production capacity before the first sugar injection, the experiment was performed as follows.

[Cultivation medium composition]

Glucose 3%, KH ₂ PO ₄ 0.05%, K ₂ HPO ₄ 0.05%, MgSO ₄ 7H ₂ O 0.03% FeSO ₄ 7H ₂ O 0.001%, MnSO ₄ 4H ₂ O 0.001%, urea 0.3%, thiamine hydrochloride 200 μg / l, biotin 100 μg / l, pH7.0

[Main culture medium composition]

Molecular weight 7% (sugar concentration), KH ₂ PO ₄ 0.1%, MgSO ₄ · 7H ₂ O 0.05%, CSL (CORN STEEP LIQUOR) 0.3L was prepared in a total amount of 13ℓ and pH was adjusted to 7.6 with liquid ammonia. .

First, seedlings cultured in a seed culture medium for 24 hours are inoculated and cultured aseptically.

A certain amount of penicillin was injected 6 to 8 hours after inoculation, and sterilized molasses (45% sugar) was added several times until the concentration of sugar in the culture solution was lower than a certain concentration.

During the culture, the liquid ammonia was injected so that the pH was 7.6-7.8 by the automatic controller, and the temperature was maintained at 32 ° C. and 35-37 ° C. before the first injection.

Aeration in the culture was 1VVM and fermentation tube pressure was adjusted to 0.5kg / cm ² and incubated for 25 to 35 hours.

The culture results with different sugar depletion time before the first sugar injection were as follows.

As can be seen from the above results, when the sugar depletion time before the first injection was about 20 minutes, it showed good results in the concentration of glutamic acid and the incubation time.

Experimental results were as follows: the sugar depletion time before the first sugar input was 20 minutes and the sugar concentration in the medium by the added sugar was changed.

Hereinafter, the present invention will be described with reference to Examples.

Example 1

Cultivation was carried out using the above-described main culture medium and incubated for 30 hours with different input times per primary. The result is as follows.

^{* The} time point for depletion was based on the sharp drop in CO ₂ concentration in the exhaust and the sharp rise in dissolved oxygen in the culture.

Example 2

To this culture medium, 0.02% of surfactant TWEEN-40 (Polyoxy ethylene sorbitan monopalmitate) was added as one of the media components, and cultured as in Example 1. The result is as follows.

Example 3

Incubate with this culture medium, add 0.2% of surfactant TWEEN-40 instead of penicillin at 6 hours of cultivation, add primary sugar 20 minutes after the time of sugar depletion, and keep the total sugar content in the medium while maintaining the sugar concentration at 2.5-3.5%. The volume was changed to 7 ml and the temperature change during the culture was varied. Initial culture temperature was 31 ℃. The result is as follows.

Claims (1)

Batch to incubate in aerobic state in a biotin excess medium using strains of Brevi bacterium lactofermentum ATCC 13869 and Corinbacterium glutamicum ATCC 13032 A method for producing L-glutamic acid, characterized in that the glutamic acid is accumulated and recovered by a sugar addition method.