JPH0349556B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing monosodium L-glutamate by a fermentation method, and its purpose is to directly accumulate monosodium L-glutamate into a culture solution during culture, thereby producing monosodium L-glutamate by a conventional fermentation method. - Reduce the amount of ammonia and hydrochloric acid used in the manufacturing method of sodium glutamate, omit the complicated process of converting ammonium L-glutamate to sodium L-glutamate, and manufacture sodium L-glutamate for industrial use. It's about doing. Conventionally, as a method for directly accumulating monosodium L-glutamate in a culture solution by fermentation, L-glutamate-producing bacteria are cultured in a medium containing acetic acid as the main carbon source, and acetic acid and acetic acid in a feed solution supplied during the culture period are used. A method was known in which the molar ratio of ammonium acetate and the molar ratio of acetic acid to sodium acetate were cultured to accumulate L-glutamic acid sodium salt in the culture solution (Japanese Patent Publication No. 1973-
26710). According to this method, the proportion of monosodium glutamate in L-glutamate is 90%.
However, the disadvantage is that the yield of L-glutamic acid is low, and it has not yet been implemented industrially. Therefore, the present inventors conducted intensive research with the aim of developing a method for producing L-glutamic acid as a sodium salt without reducing the yield, and as a result, they found that The producing bacteria are cultivated aerobically in a medium containing acetic acid and sugars as the main carbon sources,
After the production of glutamic acid has started, sodium acetate and ammonium acetate are added to the culture solution.
The present inventors have discovered that this objective can be achieved by culturing while controlling the NH ₄ /Na ratio to 1.4 or less, leading to the completion of the present invention. The method of the present invention will be explained below. The L-glutamic acid producing bacteria used in the method of the present invention are acetic acid auxotrophic mutant strains belonging to the genus Brevibacterium, such as Brevibacterium lactofermentum AJ11515 (FERM-P5335), Brevibacterium flavum AJ11517 ( FERM−
P5337), etc. (the method for inducing the above mutant strain is described in JP-A-56-92794). When L-glutamic acid producing bacteria that do not have acetic acid requirement are used, the yield of glutamic acid is low;
In particular, if the NH ₄ /Na molar ratio in the medium is less than 1.4, the yield will drop significantly, making it impossible to achieve the object of the present invention. The medium used in the present invention is a nutrient medium containing acetic acid and sugars as main carbon sources, a nitrogen source, inorganic ions, and, if necessary, organic trace nutrients. Acetic acid is used as a substance that satisfies the acetic acid requirement of the glutamic acid producing bacteria used in the present invention, but in addition to acetic acid, lower fatty acids such as propionic acid,
Higher fatty acids such as palmitic acid and stearic acid, lower aliphatic alcohols such as ethanol and propanol, etc. can also be used. Sugars used in combination with acetic acid etc. include glucose, fructose, maltose, sucrose, starch saccharification liquid containing these, cellulose saccharification liquid, fruit juice, cane molasses,
Sugar beet molasses, etc. are used. The mixing ratio of both is 3 to 2 for the former and 2 to 1 for the latter.
Further, this mixed solution is supplied continuously or in stages during the culture period, particularly during the production period of L-glutamic acid. As nitrogen sources, for example, ammonium salts, ammonia water, ammonia gas, urea, etc. used in normal L-glutamic acid fermentation are used, and inorganic ions such as phosphates and magnesium salts are added to the medium as necessary. Ru. Additionally, micronutrients such as thiamine and biotin are used as appropriate. Furthermore, a biotin action inhibitor such as polyoxyethylene sorbitan monopalmitate or penicillin is added to the medium in which biothion is present in excess by a conventional method. Cultivation may be carried out in the same manner as normal L-glutamic acid fermentation, and the culture is carried out aerobically while adjusting the culture temperature to 24-37°C and pH to 6-9. When cultured in this manner, the bacterial cells proliferate and start producing L-glutamic acid after 8 to 10 hours. The culture is no different from normal L-glutamic acid fermentation until the production of L-glutamic acid begins, but after the production of L-glutamic acid has started, in order to increase the amount of accumulated L-glutamic acid, the carbon Continuously or stepwise feeding the source and adding sodium acetate solution and ammonium acetate solution to
Control the NH ₄ /Na ratio to 1.4 or less, and
Cultivation is continued while adjusting the pH to a range of 6.0 to 9.0, and a significant amount of monosodium glutamate is accumulated in the culture medium. The method of the present invention is characterized in that the supply of ammonia is suppressed to the minimum amount necessary for the growth of bacterial cells and the biosynthesis of L-glutamic acid.
Even when cultured under these conditions, the yield of L-glutamic acid did not decrease at all, and a significant amount of L-glutamic acid was accumulated in the culture solution, and after about 48 hours, the yield of L-glutamic acid was 57%. Above, the ratio of L-glutamic acid sodium salt to total L-glutamic acid is
Reaching over 60%. L-glutamic acid sodium salt can be collected according to a conventional method.The culture solution is centrifuged to remove bacteria, and if necessary, after decolorization, L-glutamate is collected by concentration crystallization.
- by crystallizing and separating the sodium glutamic acid salt; Example 1 Two types of media (medium A, medium B) with the compositions shown in Table 1 were used.
Prepare medium), dispense 20 ml of medium A and 25 ml of medium B into a 500 ml shoulder flask, and incubate at 115°C for 10 minutes.
Heat sterilized for minutes.

[Table] On the other hand, a glucose solution (45 g/dl) and a sodium acetate solution (20 g/dl) were prepared and heat sterilized, respectively. Brevibacterium lactofermentum AJ11515 was inoculated into the above A medium and cultured with shaking at 315°C. At 10, 15, 20, 25, 35 and 44 hours after the start of culture, the above glucose solution, acetic acid, sodium acetate solution and ammonium acetate solution were supplied, and the ratio of sodium acetate to ammonium acetate was changed to improve the concentration of the culture solution. NH ₄ /Na ratio from 0.5 to
The pH of the culture solution was changed to 9.0, and the culture was continued while adjusting the pH of the culture solution to 6.5 to 9.0, and the culture was completed in 48 hours. As a control, Brevibacterium lactofermentum ATCC13869 was cultured in the same manner using B medium. Yield of L-glutamic acid accumulated in the culture medium and L-in total L-glutamate
The proportion of sodium glutamate is shown in Table 2.

[Table] As shown in Table 2, the acetate-requiring mutant strain AJ11515 not only has a high yield of L-glutamic acid, but the yield almost decreases even when the NH ₄ /Na ratio is lower than 1.4. 60-90% of the total glutamate is accumulated as L-glutamate sodium salt. Example 2 Glucose 2.3g/dl, acetic acid 1.4g/dl,
KH ₂ PO ₄ 0.2g/dl, MgSO ₄・7H ₂ O0.1g/dl,
Ammonium sulfate 0.05g/dl, MnSO ₄・7H ₂ O1.0
mg/dl, FeSO ₄・7H ₂ O1.0mg/dl, biotin 3μ
g/, soybean protein decomposition solution (total nitrogen equivalent) 36mg/
A medium for L-glutamine production containing 200 μg/dl and thiamine hydrochloride was prepared, and the PH
After adjusting the temperature to 7.0, 300 ml of the mixture was poured into a 1.5-volume jar fermentor and sterilized by heating at 120°C for 15 minutes.
Brevibacterium lactofermentum AJ11515 precultured in the A medium of Example 1 was added to this medium.
The seed culture solution was inoculated and cultured with aeration at 31.5°C (1000 rpm, 1/4 VVM). After 10 hours of culture, 17g/
dl of glucose solution and 20 g/dl of acetic acid, while supplying 20 g/dl of ammonium acetate solution and 30 g/dl of ammonium acetate solution.
G/dl sodium acetate solution was supplied at a ratio of 1:6, and culture was carried out while adjusting the NH ₄ /Na ratio in the culture solution to a range of 0.6±0.2 and the pH of the culture solution to a range of 6.5 to 8.5. The culture was terminated after 48 hours. The accumulated amount of sodium L-glutamate in the culture solution was 7.78 g/dl, the proportion of sodium L-glutamate in L-glutamate was 94%, and
The yield of glutamic acid was 57.4%.

Claims (1)

[Claims] 1 L belonging to the genus Brevibacterium and requiring acetic acid
- Glutamic acid producing bacteria are cultured aerobically in a medium containing acetic acid and sugars as carbon sources, and after the accumulation of L-glutamic acid has started, the carbon source is supplied to the culture solution and sodium acetate and ammonium acetate are added. 1. A method for producing L-glutamic acid by a fermentation method, which comprises culturing while controlling the NH ₄ /Na ratio in the culture solution to 1.4 or less, thereby accumulating a significant amount of sodium L-glutamate in the culture solution.