CS203769B1 - Process for ferment preparing l-lysine - Google Patents

Description

. The invention relates to a process for the fermentative preparation of L-lysine by culturing production strains Brevibacterium flavum on liquid culture media containing assimilable carbon and nitrogen sources, mineral nutrients and growth factors.

The fermentative preparation of L-lysine, an amino acid constituting an essential component of rational nutrition of livestock in modern farms, using various production microorganisms, is known mainly from patent literature, for example glucose has been described as the main source of assimilatable carbon (U.S. Patent No. 1,258,380). or molasses (Swiss Patent No. 535,831) using strains that are dependent on certain amino acids and resistant to specific analogs (for example, lysine or threontine). Further, a fermentation preparation (US Patent No. 3,687,810) of molasses L-lysine as the main carbon source has been described, with production strains that are resistant to certain concentrations of certain antibiotics (e.g. penicillin, bacitracin, etc.). The combination of various carbon sources in the fermentation of L-lysine using molasses and peat hydrolyzate is also known (Soviet Patent No. 496,300).

The best yields in the fermentation preparation of L-1-ysine are obtained using nutrient media containing sucrose as the main source of assimilable carbon. As sucrose is becoming more expensive and a deficient raw material, a limiting factor in the economy of L-lysine biosynthetic production, efforts to replace this raw material at least partly by other sources are becoming more common

- affordable and cheaper, even at the cost of lower fermentation yields.

This object is achieved by a process for the fermentative preparation of L-lysine by culturing Brevibacterium flavum production strains on liquid nutrient media containing assimilable carbon and nitrogen sources, mineral nutrients and growth factors according to the invention. The principle of this process is that the fermentation is started on a culture medium with an initial content of 10 to 18% by weight. % saccharose, wherein 40 to 50 wt. of this amount, the sucrose is present in molasses, during which a mixture of molasses and a 30 to 40% sucrose solution in a volume ratio of 1: 1 to 1: 2 is metered into the culture medium during fermentation after reaching a pH of 7.8 to 8.0; in an amount of 1 to 2% by volume, based on the initial volume of the soil while adjusting the pH of the soil to 7.0 to 7.2 by adding ammonia water, each successive dose of molasses and sucrose solution being added to the soil each time the concentration decreases.

203768 reducing sugars to a value of 3 to 5 mg / ml until the end of the production phase.

In the process according to the invention, the ratio between molasses and sucrose solution in the mixture fed into the fermentation medium is controlled both by the sucrose content of the molasses and by the varying content of ballasts in the raw material which could negatively affect the production microorganism. Accordingly, the method of dosing this combination into the soil during fermentation is also adjusted.

In the fermentation process of L-lysine according to the invention, yields of L-lysine in the range of 48-5.3 g / liter of medium are obtained, depending on the strain used, the molasses quality and the molasses to sucrose solution ratio.

Further details of the methods of the invention will be apparent from the following examples, which are intended to illustrate but not limit the invention. .

Examples

Example 1

The Brevibacterium flavum production strain, which requires homoserine for growth and which is resistant to S-aminoethyl-L-cysitein, is inoculated with 60 ml of inoculum medium in 500 ml beakers having the following composition (g / liter):

glucose 40 urea 3 primary potassium phosphate 0.5 sec potassium phosphate 1.5 magnesium sulphate crystal. 0.5 peptone 15 beef extract 5 defect to 1000 ml pH after sterilization 7.2 to 7.5.

. After inoculation, the flasks are cultured at 29 ° C on a rotary shaker (240 rpm) for 18 hours. The grown culture was inoculated with 600 ml of fermentation medium in a 2 liter laboratory fermentation tank. The medium has the following composition (g / liter):

Wt ': ·' molasses 100 sucrose 20 urea 3 magnesium sulphate crystals. 0.2 sec. Potassium phosphate 0.9 peanut flour hydrolyzate 300 ml calcium carbonate ground 30 thiamine 100 µg biotin, 50 µg tap water up to 1000 ml pH after sterilization 7.0 to 7.2.

After inoculation, the cultures are performed in tanks at 29 ° C, aerated with 1 volume of air, with stirring at 800 rpm. After 18 hours of cultivation, a mixture of 60% molasses and 35% sucrose solution in a 1: 1 volume ratio is added to the fermentation tank. at three-hour intervals in an amount of 10 ml / tank and at the same time adjusting the pH of the fermentation broth with ammonia water to a value of 7.0 to 7.2. L-lysine production reaches 52.8 g / liter of medium at 96 hours of fermentation.

. Example 1 a d 2

An inoculum is prepared from a Brevibacterium flavum strain analogously to Example 1. Cultures grown in se flasks. used for preparation of seed material for fermentation. Inoculation medium composition (inoculation tank, g / 10 liters):

glucose. 400 primary potassium phosphate 5 sec. Potassium phosphate 15 urea ...... 30 magnesium sulphate cry. 5 peptone 200 bovine extract 50 biotin (as 1% solution) 50 mg tap water up to 10 liters pH after sterilization 7.0 - 7.2.

Cultivation is carried out at 29 ° C, for 18 hours, at 400 rpm. and aeration with 1 volume of air. Inoculate 10 liters of fermentation medium in a 20-liter fermentation tank at 10% by volume; medium has the following composition (g / 10 liters)

molasses 1000 sucrose 200 urea 30 magnesium sulfate crystals. 3 primary potassium phosphate 7 ground calcium carbonate 300 Peanut flour hydrolyzate 3000 ml. biotin (as a 1% solution) • 50 mg tap water up to 10 liters pH after sterilization 7.2.

Fermentation takes place under the following conditions: temperature 29 ° C, stirring 470 rpm, aeration 1 air volume. When the pH drops to 6.7, the pH is adjusted to 7.0-7.2 with ammonia water. During fermentation, a mixture of 400 ml conc. molasses and 600 ml of 35% sucrose solution at 200 ml / tank. L-lysine production reached 48.6-51.4 g / l of medium at 96 hours of fermentation.

Example 1 a d 3

The Brevibacterium flavum production strain of Example 1 exposed to nitrosomethylurea cultured under the conditions of Example 1 gave a lysine production of 43.7 g / liter of medium. When cultivated under the conditions of Example 2, production varied between 44 and 46 g / liter of medium, in both cases after 96 hours of fermentation.

Claims (1)

SUBJECT A process for fermentation of L-lyisin by culturing production strains of Brevibacterium glavum on liquid culture media containing assimilable carbon and nitrogen sources, mineral nutrients and growth factors, characterized in that fermentation is initiated on culture media with an initial content of 10 to 18% by weight of sucrose, wherein 40-50% by weight of this amount is sucrose contained in molasses, during which a pH of 7.8-8.0 medium of molasses and 30-40% sucrose solution is added to the culture medium during the fermentation after reaching the pH of the medium. 1: 1 to 1: 2, in an amount of 1 to 2% by volume based on the initial soil volume, while adjusting the pH of the soil to 7.0 to 7.2 by addition of ammonia water, each successive batch of molasses and sucrose solution being adds to the soil each time the concentration of reducing sugars decreases to a value of 3 to 5 mg / ml, up to end of production phase.