DE2401519A1 - MICROBIOLOGICAL PROCESS FOR THE PRODUCTION OF LIQUID AND DRY FEED CONCENTRATES OF L-LYSINE AND CRYSTALLINE L-LYSINE - Google Patents

Description

Institut Mikrobiologli imeni Avgusta Kirchenltejna Akaderail Nauk Latviskoj S8R,

Riga / USSR _{p 52 565}

January 14, 1974 L / Br

MICROBIOLOGICAL PROCESS FOR THE PRODUCTION OF LIQUID AND DRY FEED CONCENTRATES OF L-LYSINE AND \ KRISTAt-

LINEN L-LYSINS

The present invention relates to the field microbiology, especially microbiological processes for the production of liquid or dry feed concentrates of L-lysine or crystalline L-lysine.

The crystalline L-lysine and the cutter concentrates of L-lysine, which is an essential amino acid, are widely used in animal and poultry breeding as additives to feed. The crystalline L-lysine is also used in the food industry to increase the nutritional value and improve the taste properties of baked goods, in the pharmaceutical industry and in medicine.

It is a microbiological process for the production of liquid or dry feed concentrate from L-lysine or . crystalline L-lysine by cultivating producer strains Brev! Bacterium late after the periodic or continuous submerged process on a nutrient medium, wel-

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before s ammonium salts or urea, carbon sources, phosphorus, magnesium, imino acids, which are necessary for the growth of the biomass, and stimulators of the biosynthesis of L-lysine, at a temperature of 28 to 33 ⁰ C with aeration and stirring of the culture liquid known, at the end of the cultivation process, the culture liquid is either evaporated to a dry substance content of 35 to percent by weight to achieve the liquid feed concentrate of L-lysine or evaporated and dried until the dry putter concentrate of L-lysine is achieved, or separated L-lysine is removed from the culture fluid in crystalline form. (See, for example, USSR copyright JSr, 171727, Bulletin for Inventions, No. 11, 1965 »Journal" Angewandte Biochemie und Mikrobilogie ", 1966, Volume 2, Issue 5, page 519).

In the process described, the yield is an L-lysine (converted to L-lysine monochlorohydrate) up to 30% to Weight of sugar used.

When carrying out the cultivation process according to the known method occurs as a result of the utilization ^ by cn the producer strains / the components of the culture medium impoverishment this medium, especially those components such as imino acids, which are necessary for the growth of the biomass, and stimulators of L-lysine biosynthesis. The impoverishment The nutrient medium causes the biomass to age; autolysis processes take place in this process and it is reduced

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the number of living cells of the producer strains and the activity of the fermentations decreases. In connection with it the yield of L-lysine decreases.

The addition of the necessary amino acids before the cultivation process / to the Fährmed ium ^ in increased amounts leads to Inhibition of some ferments, whereby the biosynthesis of L-lysine is interrupted. For example, leads in cultivation of the producer strain Brevibacterium sp, 22 die Addition of threonine in increased amounts to inhibit aspartate kinase, which is the first ferment in the branched off path is the biosynthesis of L-lysine.

The purpose of the present invention is to provide the aforementioned ii eight hurry to avoid.

The invention & _e gt the object in the

microbiological process for the production * · liquid or dry putter concentrates of L-lysine or crystalline L-Lysine the processes of the formation of the biomass and the biosynthesis of L-Lysine to maintain the culture of the Producer strains in an active state for the entire duration to regulate the cultivation process and consequently to increase the yield of L-lysine

if

This problem is solved by the fact that one, the Prodzentstämter Brevibacterium sp. according to the periodic or continuous submerging process on a nutrient medium which Ammonium salts or urea, carbon sources, phosphorus,

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iiagnesiua, amino acids, which are necessary for the growth of the biomass, and ßiiuulatoren the biosynthesis of L-lysine hold, at a temperature of 28 to 33 ° C with aeration and stirring of the cooling liquid and with subsequent evaporation of the culture liquid to a content of Dry substances of 3S '^ i ^s 55 percent by weight, in that liquid feed concentrate is obtained from L-lysine, or with subsequent evaporation of the culture liquid and drying it until the dry putter concentrate of L-lysine is obtained or with subsequent separation of L-lysine from the Culture fluid cultivated in crystalline foria. After reaching the optimal concentration of the biomass for the yield of L-lysine (fiiyfy. According to the invention the culture liquid periodically or continuously amino acids that are necessary for the growth of the biomass, and stimulators of the biosynthesis of L-lysine or sources of the named Amino acids and the stimulators of the biosynthesis of L-lysine in!.! Narrow to / that the concentration of the biomass in the culture liquid is maintained at a level which is optical for the yield of L-lysine, or the said level is not more than Exceeds 5 g / l culture fluid.

In dif-scm - - -a processes, by regulating the processes of the formation of biomass and the biosynthesis of L-lysine, the culture of the producer family is maintained in an alitive state ^ / during the entire duration of the cultivation process. This makes it possible to reduce the yield of L-lysine (ui-

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calculated on L-lysine monochlorohydrate) to 56 % of the weight of the sugar used, which is 20 % higher compared to the known method.

As sources to be added in addition to the culture liquid the amino acids necessary for the growth of the biomass and stimulators of the biosynthesis of L-lysine are used appropriate corn extract, molasses or yeast hydrolyzate

To increase the concentration of L-lysine in the culture liquid is expediently carried out after the optimal concentration of the biomass for the yield of L-lysine has been reached of the original culture fluid periodically or continuously assimilable carbon sources in amounts that reduce the concentration of L-lysine in the culture fluid 17 up to 40 g / l, converted to L-lysine monochlorohydrate, achieved.

Used as a completely assimilable carbon source it is advisable to use sugar or acetic acid.

The ^{n ungs} 9 ^ema microbiological process for producing liquid or dry food concentrate

of L-lysine or. crystalline L-lysine is as follows carried out:

The producer strains Brevibacterium sp », for example Brevibacterium sp. 22 and Brevibacterium sp. 22y? , is bred on Pleisch peptone - Agar slants at a temperature of 20 to 3> 0 ° C for 20 up to 24 hours. Then you rinse the cells of the producer strains with physiological solution and leads this to the

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hen or vaccinate anks. In the flask or the Tanis (pementers) there is a pre-sterilized vaccine which contains ammonium salts or urea, carbon sources, phosphorus, amino acids, which are necessary for the growth of biomass, and stimulators of the biosynthesis of L-lysine.

The ammonium salts are ammonium sulfate and ammonium chloride or ammonium nitrate in question. Various sugars, for example sucrose, glucose, Lactose, or products containing sugar, for example molasses, raw beet juice, starch hydrolyzate, are used. V? As such a component of the inoculation medium such as phosphorus, so is some of the phosphorus in some components of this medium, for example in the molasses, while the remaining necessary amount of phosphorus is in the form of the nutrient medium its mineral salts, for example the mono- and disubstituted Potassium orthophosphate is added.

Different amino acids are used as amino acids, which are necessary for the growth of the biomass, depending on the type of producer strain. For example, in the case of the breeding of the producer strain Br e ν ib act er ium sp. 22 and Brevibacterium sp. 22 f> as amino acids threonine and methionine. Various stimulators are used as stimulators for the biosynthesis of L-lysine, which are also dependent on the type of production system. One uses, for example, in the breeding of the

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- ψ- -

Producer strain Brevibacterium sp. 22 and Brevibacterium. sp. 22 ji as stimulators of the biosynthesis of L-lysine thiamine and biotin.

The amino acids necessary for the growth of the biomass and stimulators of the biosynthesis of L-lysine can be added to the culture medium in the form of pure substances or in the form of sources containing these substances.

As sources, which amino acids and stimulators of biosynthesis contain of L-lysine, come for example corn extract, Molasses, yeast hydrolyzate and groundnut flour hydrolyzate in Embossing.

Growing the inoculum on the pre-sterilized Inoculation medium of the above composition can be both according to the periodic as well as the continuous process be performed.

The cultivation of the inoculum according to the periodic method is done in flasks or inoculation tanks at a temperature of 28 to 33 ° C, a ventilation intensity of 55 to 60 mg O ₂ A-min, a stirring intensity of 200 to 800 rpm and a pH of the culture liquid carried out from 7.0 to 7.2 for 16 to 20 hours. The inoculum obtained is introduced into the fermentation tank in an amount of 5 to 8%, based on the volume of the nutrient medium in this taxus.

When cultivating the inoculation material after the continuous

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Borrowed method, pre-sterilized inoculation medium is continuously fed to the inoculation tank. The inoculation material obtained is continuously led out of the inoculation tank at the same speed and fed to the fermentation tank in a length of 15 to 25 %, based on the volume of the culture liquid in the fermentation tank. The inoculum is grown in the continuous process under the same conditions as in the periodic process.

The process of cultivating the producer strains in the fermentation tank can be carried out either after the periodic or after the continuous processes are carried out.

When cultivating the producer strains according to the periodic method, the culture medium is introduced into the fermentation tank, which ammonium salts or urea, carbon sources, Phosphorus, magnesium, amino acids necessary for biomass growth are necessary, and stimulators of the biosynthesis of Contains L-lysine.

Ammonium sulfate and ammonium chloride are used as ammonium salts or ammonium nitrate in imprint. As carbon sources you can use different sugars, for example sucrose, glucose, lactose, or products containing sugar, for example leave, Eübenrohsaft, ütäricehydrolysat, serve. Phosphorus and magnesium are added to the nutrient medium ... , .... ". in the form of their mineral salts, for example the mono- and d! -substituted Potassium orthophosphate, magnesium sulphate, liagnesium chloride fed.

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The amino acids necessary for the growth of the biomass and ßimulatoren the biosynthesis of L-lysine can the iTährmediua added in i'orm pure substances or in fora from sources that contain these substances. As sources that contain amino acids and simulators of the biosynthesis of L-lysine, can, for example, llais extract, molasses, yeast hydrolyzate or peanut flour hydrolyzate can be used.

The nutrient medium brought into the fermentation tank of the above Composition is sterilized, after which in the Fermentation tank the inoculum is introduced. The cultivation of the producer strains is carried out at a temperature of 28 to 3 ° C, a ventilation intensity of 40 to 60 mg Op / l * min,

iner lVu ^ rintensity vo :. ⁷ JOO to SOO U /: .. i .: and a pH value of the culture liquid of 7> 0 to 8.0 carried out for 60 to 70 hours.

To intensify the process for the production of L-lysine and to increase its effectiveness, it is advisable to cultivate the producer strains for the first 16 to 20 hours at an aeration intensity of 50 to 60 mg 0p / l.min and a pH value of the culture fluid from 7.0 to 7 »2 and during the further 20 to 25 hours at an aeration intensity of 40 to 50 mg 0 _? / l «min and a pH value of the culture fluid of 7.8 to 8.0 and during the further 24 to 25 hours at an aeration _{intensity of 35 to 50 mg 0 2} / lrmin and a pH value of the culture fluid of 7, 0 to 7.2 through.

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Iiach. the achievement of the optimum for the yield of L-lysine Concentration of the biomass (after. 16 to 20 Hours) periodically feeds the culture fluid, or continuously the additional amount of for the growth of the Amino acids necessary for biomass and the stimulator of biosynthesis of L-lysine too. The amino acids and stimulators can be in the form of pure substances or in the culture liquid ]? form of sources containing these substances may be added. As sources, the amino acids and stimulators of L-lysine contain, for example, shark extract, molasses, yeast hydrolyzate or peanut flour hydrolyzate in embossing.

As stated above, the amino acids and the stimulators are the biosynthesis of L-lysine or the sources which

such

contain these substances, added to the culture liquid in 'J.en ~ ea that the concentration of the biomass in the culture liquid is maintained at a level which is optical for the yield of L-lysine, or the said level of not more than 5 g / l Exceeds cult liquidity.

To increase the concentration of L-lysine in the Culture fluid is expediently given after reaching the periodically or continuously for the optimal concentration of the biomass of the culture liquid for the yield of L-lysine

Look

carbon swells that can be completely eliminated, that the concentration of L-lysine in the culture fluid 17 to 40 g / l, converted to L-lysine monochlorohydrate,

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achieved. Various sugars, for example sucrose, glucose or lactose, volatile fatty acids, for example acetic acid or butyric acid, ethanol can be used as completely assimilable carbon sources. The magnitude of the increase in the concentration of L-lysine in the culture fluid depends on the type of fully assimilable carbon source used

When cultivating the pro duz ent amme according to the continuous process, the process mentioned can be carried out according to both the homogeneous and the heterogeneous principle. The heterogeneous principle of carrying out the cultivation process is preferred. In this case, the production line generally consists of three fermenters connected to one another The same (first) fermenter is continuously fed with the inoculum in an amount of 15 to 25 / o _t based on the volume of the culture liquid in the fermenter mentioned Fermenter and then into the third fermenter · The dilution coefficient in the three fermenters is in a range from 0.12 to 0.14 St "

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the ratio of the volume of the fermenter during 1 hour supplied nutrient medium or the culture liquid to the volume of the culture liquid, which is in "the Fermenter).

The cultivation process of the producer tribes according to the continuous process on the nutrient medium of the above composition is listed in the table below Conditions carried out.

Tabel

No. of Tempe 33 Ventilation p Stirring inks o / min pH the culture- , 0 to 7.2 Fermen rature, 33 intensity, sity, liquid , 8 to 8.0 ters ⁰ C 33 mg O _0/1 * min 800 , 0 to 7.1 1 28 to 55 to 60 300 to 800 7th 2 28 to 40 to 55 300 to 800 7th 3 28- to 40 to 55 300 to 7th

Under the stated conditions of the cultivation process the biomass of the producer strains grows in the first fermenter, with the biomass being responsible for biosynthesis of L-lysine reached optimal concentration. By doing The second and third fermenters essentially lead to the biosynthesis of L-lysine.

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To maintain the concentration of the biomass at or at the optimal level for the yield of L-lysine the above the mentioned level not more than 5 g / l of the culture liquid the second fermenter takes you to the lying level periodically or continuously the amino acids necessary for the growth of the biomass and the stimulators of biosynthesis of L-lysine or those containing the substances mentioned Sources a. As sources what amino acids and stimulator s contained in the biosynthesis of L-lysine, for example Corn tract, molasses, yeast hydrolyzate or peanut flour hydrolyzate be used·

To increase the concentration of L-lysine in the culture liquid periodically or continuously, completely assimilable carbon is introduced into the second fermenter.

oclchtn

swell in amounts that the concentration of L-lysine in the culture liquid 1? up to 40 g / l, converted to L-lysine monochlorohydrate, achieved. Various sugars, for example sucrose, glucose or lactose, volatile Pett-

• ' or

acidic, for example acetic acid or butyric acid, ethanol can be used.

The culture liquid obtained by culturing

u
of the producer strain is obtained by both the periodic and the continuous process, is acidified with hydrochloric acid to a pH value of 4.0 to 5.5

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and the stabilizer, for example sodium hydrogen sulfite, iatriolithiosulfate or sulfamic acid, is added. The culture liquid is then evaporated in an evaporation plant to a dry substance content of Zö to 55 percent by weight to obtain the liquid feed concentrate, which contains 15 to 20 percent by weight of L-lysine (converted to L-lysine monochlorohydrate). In addition, the culture liquid can be evaporated and then dried until dry feed concentrate with an L-lysine content of 20 to 40 percent by weight (converted to the L-lysine monochlorohydrate) is obtained «

It is used to produce L-Lysine in crystalline form the biomass of the producer strains from the culture liquid, for example by filtering or centrifuging. From the obtained after the separation of the biomass L-lysine becomes native solution in a manner known per se separated by, for example, uie the native solution a column with synthetic resin ion exchanger passes through, then the L-lysine is eluted with aqueous iismonia solution, evaporate the eluate, acidify the concentrate obtained with hydrochloric acid, treat the concentrate with ethyl alcohol and that L-lysine crystallizes.

For the better . of the present invention

the following examples of the preparation

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dry and dry feed concentrates of L-lysine or crystalline L-lysine listed. In all of the examples, the quantitative parameters are based on L-lysine, namely the concentration of L-lysine in the culture liquid, the yield L-lysine, based on the weight of the sugar used, the content of L-lysine in the liquid or dry feed concentrate and the yield of crystalline L-lysine converted to the L-lysine monochlorohydrate indicated.

Example 1. The homoserine deficient producer strain of lysine Brevibacjterium sp. 22 (Depositnuinmer Sun; Museum of cultures of microorganisms of the August Kirchenstein- -Instituts of Microbiology, Academy of Sciences of the Latvian Soviet Socialist Republic), was heated in a thermostat to 2% meat peptone agar slant hydrogenated at a temperature of 29 to 3O ⁰ C. Increased for 24 hours. The culture was rinsed off the agar slant with sterile physiological solution (10 ml) and the suspension was transferred in an amount of 50 ml to laboratory-type inoculation tanks which contain 2 liters of pre-sterilized inoculation suturing medium of the following composition:

Beet molasses (the sugar content of molasses is 50 percent by weight, L-methionine 0.36 mg / g, L-threonine 2.6 mg / g, of biotin 0.05 / g / g, of thiamine 0.15 / g / g) 200 g Maize extract (the dry matter content of the maize extract is 50 percent by weight, on L-methionine 4 mg / g, on L-threonine 22 mg / g, on biotin 0.55 mg / g, on thiamine 7.5 g / g) 60 g

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Ammonium sulfate 40 g

Potassium dihydrogen phosphate 1 g

Potassium Hydrogen Phosphate 1 g

Water until 2 1

pH of the medium 7 * 0.

The sterilization of the Impfnährmediums was carried out by heating it to a temperature of 126 ⁰ C and then holding at a temperature of 120 ⁰ C for 1 hour.

The cultivation of the seed material was made by the periodic process at a temperature of ^ i to 30 ⁰ C, an aeration intensity of 60 mg Og / l.min "a stirring intensity of 300 U / min and a pH value of the medium from 7.0 to 7 | 1 (the pH value was maintained at the stated level with 25% aqueous ammonia solution). Sperm whale oil was used to destroy the foam. The cultivation of the inoculation material under the conditions described was carried out for 20 'hours until a concentration of the biomass in the culture liquid of 10.2 g / l was achieved, after which the inoculation material by compressed air into the inoculation tank with pre-sterilized inoculation medium (50 l) of the following composition was transferred:

Beet molasses (the molasses content was 50 percent by weight, L-methionine 0.36 mg / g, L-threonine 2.6 mg / g, biotin 0.05 Ag / g, Itaün 0.15 ye / e 409849 / 0677 _$

Corn extract (the content of the corn extract it to dry matter was 50 weight percent of L-methionine 4 mg / g, in L-threonine 22 mg / g, to biotin 0.55 / <g / g, thiamine 7 »5 M g / g) 1 | 5 kg

Ammonium sulfate I kg

Potassium dihydrogen phosphate 25 g

Potassium hydrogen phosphate 25 g

Water . up to 50 1

pH of the medium 7.0

The sterilization of the Impfnährmediums was carried out by heating it to a temperature of 126 ⁰ C and then maintaining the same at a Tempeatur of 122 ⁰ C for hour.

The cultivation of the seed material was made by the periodic process at a temperature of 29 to 50 ⁰ C, an aeration intensity of 60 mg 0 ^ / 1 »min, a stirring intensity of 500 U / min and a pH value of the medium from 7.0 to 7.1 for 20 hours. After the specified time, the concentration of the biomass in the culture liquid was g / l. The finished inoculation material was transferred by compressed air into the fermentation tank with pre-sterilized Mhermedium (10ü0 1) of the following composition:

Hüb'en molasses (the sugar content of the molasses was 50 percent by weight, L-methionine 0.36 mg / g, L-threonine 2.6 mg / g, biotin 0 ^ 05 ν g / g, thiamine 0, 15 u g / g) 100 kg

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50 kg • 20th kg 0.5 kg 0.5 kg 0.3 kg 1000 1 7.0

Maize extract (the dry substance content of the maize extract was 50% Oewiclit, L-methionine 4 mg / g, L-threonine 22 mg / g, Thi _a min '7.5 λ / g / g) and biotin 0.55 mkg /G

Ammonium sulfate zinc iodine hydrogen phosphate Hydrogen phosphate

Magnesium sulfate

Water up

pH of the medium

The cultivation of the producer stazome in the fermentation tank was carried out according to the periodic method. In the first 18 hours, the process at a temperature of 32 ⁰ C, an aeration intensity of 60 mg 0p / l "min, a pH of the culture liquid of 7.1 and a stirring intensity was of 300 U / min carried out. After the specified time, the concentration of the biomass in ß «r culture liquid reached 12.8 g / l, which was optimal for the yield of L-lysine. At that moment, the culture liquid contained no methionine and no threonine (the above * amino acids were used for the cultivation of the biomass), while the biotin and thiamine (the stimulators of the biosynthesis of L-lysine) in the culture liquid were used for the biosynthesis of L-lysine un-

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Sufficient quantities were available. The content of the culture liquid it of unused sugars was 2.2 percent by weight. from At that point in time (i.e. after the first 18 hours), the fermentation tank was fed continuously for 40 hours the following substances:

DL-threonine 5 g / pc

DL-methlonin 1.25 g / pc

Thiamine 1.25 mg / pc

Biotin 0.75 mg / pc

Sucrose 1.25 g / pc

The total amount of the fermenter during the whole The sugar added to the cultivation process was 10%, based on the weight of the nutrient medium.

Simultaneously with the addition ^ in the fermentation tank) of the above Substances, the ventilation intensity was reduced to 40 mg 0_ / l · min and the pH of the culture liquid was increased to 7 »8 · The temperature and the stirring conditions remained the same on the same level. Under the conditions mentioned, the cultivation process was continued for 25 hours, after which the pH of the culture liquid is lowered to 7.2 and the cultivation process was continued for another 25 hours. The total time of the cultivation process was 68 hours.

After finishing the cultivation process contained the culture fluid contains the following substances:

L-lysine 33.5 _g / i

Biomass 409 8 4 9/0677 14.8 g / l

Dry substances 8 | 7% by weight

Sugar 0.8% by weight

The yield of L-lysine was 35 % by weight, based on the weight of the sugars used. ..

The separation of L-lysine from the culture liquid in crystalline form was carried out as follows. The biomass of the producer strain was raised by the culture liquid a centrifuge (5000 rpm, 30 minutes) or a separator severed. The native solution obtained after separation of the biomass was passed through a column with synthetic resin cation exchanger directed. The sorbed L-lysine was eluted with aqueous ammonia solution and the eluate was evaporated by the ammonia was removed together with water vapor. The received The concentrate was acidified to pH value with hydrochloric acid

€ 5

from 3.3 to 4.0, treated with ethyl alcohol and precipitated the L-lysine in crystalline form.

Example 2. For the synthesis of L-lysine, the homoserine-deficient producer strain Brevibacterium sp. 22 J] (deposit number 192; Museum of Cultures of Microorganisms of the August Kirchenstein Institute for Microbiology of the Academy of Sciences of the Latvian Soviet Socialist Republic).

The breeding conditions of the inoculum were those in Example 1 described analogously. The finished inoculation material was

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de transferred into the fermentation tank in an amount of 8%, based on the volume of the pre-sterilized liährmediums in this fermentation tank. The above mentioned Bahrmedium (pO mr) had the following composition:

Beet molasses (the sugar content of the molasses was 48 percent by weight, L-Lethionine 0.42 mg / g, L-Threonine 2.4 mg / g, Biotin 0.08 μg / g,

of thiamine 0.20 ^ g / g) 4500kg

Alae extract (the retention of the maize extract Dry substances was 50 percent by weight L-threonine 4 mg / g, L-threonine 22 mg / g, 3io-

"tin 0.55. g / g" of thiamine 7.5 g / g) 750 kg

iunmonium sulfate at '900 kg

Kai iumd ihydrogen phosphate 15 kg

Potassium hydrogen phosphate 15 kg

Magnesium sulfate. 9 kg

Water up to 30m ^

pH of the medium 7.0

The cultivation of the producer strain on the culture medium of the composition mentioned took place according to the periodic method under the following conditions: temperature 32 ° C .;

C; Ventilation intensity 50 mg 0p / l »iain; Kührint ens it ät Rpm; pH of the culture liquid 7.2; Duration of the process 70 hours. Reached after the first 16 hours

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the concentration of the biomass in the culture liquid 10.8 g / l, which was optimal for the yield of L-lysine. The culture liquid it contained no methionine at this point in time and no threonine, while the biotin and thiamine in the culture fluid are insufficient for the biosynthesis of L-lysine Quantities were included. "From that point on (i.e. after the first £ hour) the fermentation tank was run molasses as a source continuously for 40 hours

of L-methionine, L-threonine, biotin and thiamine with one Speed of 22.5 kg / h. In 1 g of the additional added molasses contained the substances mentioned in the following quantities:

L-methionine 0.42 mg; L-threonine 2.4 mg; Biotin 0.08 g; Thiamine 0.20 /..g.

Do the termination of the cultivation process (i.e. after 70 hours) contained the culture liquid the following substances:

L-lysine 29.7 g / l

Biomass 10.8 g / l

Dry substances 12.2% by weight

Sugar 0.5 wt

The yield of L-lysine was 35 weight percent based on the weight of the sugar used.

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The L-lysine in the culture liquid was stabilized by adding sodium hydrogen sulfite and the pH of the culture liquid was brought to A- 5 with hydrochloric acid. Then, the culture fluid in-a was concentrated to a i'rockensubstanzengehalt of 36 weight percent and to a spray dryer at a -Kestfeuchtigkeitsgehalt of 3 _t 5 percent by weight of dried plant Endampf vacuum. 3060 kg of dry feed concentrate of L-lysine was obtained, which contains the following substances:

L-lysine.

Sugar betaine total nitrogen protein Thiamine riboflavin

24, 3 wt.% Weight, 2 Weight 6th Weight, 16, .% .% .% 8 wt.% 9.3 /
ι /
118 " / g / g
, g / g

Example 3. Dea producer strain of L-Lysine Brevibacte-

riuin sp. 22y? lack of homoser, increased nan in one

¹ one

Thermostats on agar slants, 2% meat peptone

agars at a temperature of 29 to 30 ⁰ C for 24 hours. The culture was rinsed off the agar slant with sterile physiological solution (10 ml) and the suspension in each case in an amount of 1 ml in sterile flasks of 750 ml.

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like with pre-sterilized inoculation medium (50 ml) the following Transfer the set ζixng:

Beet molasses (the sugar content of molasses was 48 percent by weight of L-threonine 2.4 mg / g, . on L-methionine 0.42 mg / g; on biotin 0.06 -g / g; of thiamine 0.15 A g / g) 5 g

Corn extract (the content of the corn extract at Dry substances was 50 percent by weight; L-methionine 4 mg / g, L-threonine 22 mg / g; of biotin 0.55 g / g, of thiamine 7.5 g / g) 1.5 s

Ammonium sulfate 1.25 g

Calcium dihydrogen phosphate 0.025 g

Potassium hydrogen phosphate 0.025 g

Water, up to 50 ml

pH of the medium 7.0.

The sterilization of the Impfnährmediums was carried out by heating it to a temperature of 126 ⁰ C and then holding at a temperature of 120 ⁰ C for 1 hour.

The flask containing the Impfnährmedium and the cells of the producer strain was brought into a thermal cautery on a swing and led the growth of the culture at a temperature of 32 ⁰ C, a speed of rotation of the swing shaft 200 U / min, at a pH value of Aiediums of 7.0 to

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2 A 01 b

7.2 for 20 hours.

The further breeding of the producer strain took place according to the continuous process in an inoculation tank with a capacity of 5 nr. First of all, pre-sterilized inoculation medium (1 v?) Of the following composition (in percent by weight) was introduced into the above-mentioned vaccination tank:

Beef molasses (the sugar content of the molasses was 48 percent by weight, L-threonine 2.4 mg / g, of L-methionine 0.42 mg / g, of biotin 0.08 ^ g / g, of thiamine 0.20 / t / g / g) 10

Corn extract (the dry matter content of the corn extract was 50 percent by weight, of L-methionine 4 mg / g, of L-threonine 22 mg / g, of biothin 0.55 / ng / g, of thiamine 7.5 / «g / g) 5

Ammonium sulfate I.

Kaii and hydrogen phosphate 0.025

Calcium hydrogen phosphate 0.025

Water to 100

pH of the medium 7.0

Then said inoculation medium was inoculated with the culture in the permenter by transferring 12 liters of inoculum from the flask. After the inoculation, the incubation period took place for 6 hours, which was carried out at a temperature of 50 ^° C. and an aeration intensity of 60 mg O ₂ 4. ^ ₁₁ one

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Stirring intensity of 800 rpm and a pH of the culture liquid of 7.1 was carried out. After expiry of the said time is continuously introduced to the inoculum tank sterile Impfnährmedium the composition described above at a speed of 0.6 to MvsT · The cultivation of the producer strain according to the continuous process was conducted at a temperature of 32 ⁰ C ₁ a ventilation intensity of 60 mg O ^ l ^ min, a stirring intensity of 800 rpm and a pH of the culture liquid of 7.2 carried out. Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the inoculation tank: content of biomass 11 g / 1; Concentration of L-lysine 4.3 g / l; Concentration of sugars 2.2 percent by weight.

The cultivation of the producer strain was then carried out by the continuous method in three fermentation tanks connected to one another. The volume of the nutrient medium in each fermentation tank was 30 mr. The inoculum was continuously fed to the first fermentation tank at a rate of 0.6 mVSt and pre-sterilized nutrient medium at a rate of 3 nr / hour. The composition of the nutrient medium in the first Gärtaruc was as follows (in percent by weight):

Beet molasses (the molasses content in sugars was 48 percent by weight, L-methionine 0.42 mg / g, of L-threonine 2.4 mg / g; of biotin 0.08

; of thiamine 0.20 / µg / g) 17.2

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-'27- 240 Ί

Shark ext raid; (The dry matter content of the corn extract was 50 percent by weight, L-methionine 4 mg / g, L-threonine 22 mg / g, biotin 0.55 y "S / s", thiamine 7.5 yg / g) ²

Ammonium sulfate "2

Calcium hydrogen phosphate 0.075

Potassium hydrogen phosphate 0.075

Magnesium sulfate 0.035

Water to 100

pH of the medium 7.0

The cultivation of the Prοduzentenstammes in the first fermenter was carried out at a temperature of 32 ⁰ C, an aeration intensity of 60 mg Op / l min, a stirring intensity of U / min and at a pH value of the culture liquid of 7.1. Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the first fermentation tank: content of biomass IJ g / l; Concentration of L-Lyein 8.4 g / l; Concentration of sugars 5.2 percent by weight.

This concentration (13 g / l) of the biomass in the culture liquid was optimal for the yield of L-lysine.

The culture liquid emerged continuously from the first fermenter in the second fermenter (the dilution coefficient is 0.12 St ~ \). The culture liquid emerging from the first fermenter into the second fermenter did not contain any methionine " and ke.in threonine, while the biotin and thiamine in

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the cooling fluid in insufficient for the biosynthesis of L-lysine Quantities were included. Therefore, the second fermenter was run continuously at a speed of 0.6 nr / St Eait water diluted molasses as a source of amino acids and the stimulators of the biosynthesis of L-lysine. The sugar content of the molasses solution was 15 percent by weight, L-methionine 0.17 mg / g, L-threonine 0.95 mg / g; of biotin 0.032 g / g; of thiamine 0.08 Ag / g.

The cultivation of the producer strain in the second fermentor was carried out at a temperature of 3O ⁰ G, an aeration intensity of 50 mg 0 ₂ / * min l, a Sührintensität of 500 U / min, pH of the culture fluid of 7 »8 and a dilution coefficient of 0.14 St ". Under the cultivation conditions mentioned, the following state of equilibrium was established in the culture liquid of the second fermentation tank: content of biomass 13» 9 g / 1 »concentration of L-lysine 18.8 g / l; concentration of sugars 2 Weight percent.

The culture liquid passed continuously from the second fermenter into the third fermenter (the dilution coefficient was 0.14 St ' ¹ ). The cultivation of the producer strain in the third fermentor was carried out at a temperature of 30 ⁰ C, an aeration intensity of 50 mg 0p / l.min,

a stirring intensity of 400 rpm and a pH-value of the Culture fluid of 7.0. Under the mentioned cultivation conditions turned into the culture fluid of the third

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Fermentation tanks enter the following state of equilibrium: content of biomass 14 g / 1; Concentration-of L-lysine 27 g / l; concentration of sugars 0.7 percent by weight.

The yield of L-lysine was 32.2% based on the Weight of sugars used

The evaporation of the culture liquid and the subsequent drying to a residual moisture content of 2.9 percent by weight took place analogously to Example 2. The dry feed concentrate of L-lysine obtained contained the following substances: L-lysine 20.5 percent by weight; Sugar 51.3 percent by weight, betaine 2.2 percent by weight; Total nitrogen 5t & weight percent, protein 18 weight percent, thiamine 8.6 z / g / g riboflavin 92 // g / g.

Example 4. The homoserine deficient producer strain of L-lysine Brevibacterium sp.22 Ji was grown under conditions analogous to those described in Example 1 for the production of inoculum material. The finished inoculum was transferred to the fermentation tank in an amount of 5%, based on the pre-sterilized nutrient medium in this fermentation tank. The above-mentioned drilling medium (30 rar) had the following composition:

Beet molasses (the sugar content in molasses was 50 percent by weight: L-methionine 0.36 mg / g; L-threonine 2.6 mg / g; biotin 0.05 M g / g; thiamine 0.15 A. g / g) 5100 kg

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Maiseirfcrakt (the content of the maize extract Dry substances was 50 percent by weight; an! .- methionine 4 mg / g; of threonine 22 mg / g;

on biotin 0.55 / "g / g; on thiamine 7t5 ^ s / s) ⁶⁰ O ^k S

Ammonium sulfate 900 kg

Kai iumd ihydrogen phosphate 15 kg

Calcium hydrogen phosphate 15 kg

Magnesium sulphate 10 kg of water up to 30 ^m

pH of the medium 7.0

The cultivation of the producer strain took place in the fermentation tank according to the periodic method. In the first 18 hours, the process at a temperature of 30 ⁰ C was a vent sintensität of 60 mg 0 ^ / 1-min, a stirring intensity of 500 U / min and carried out a pH of from 7.1 Kulturflussigkeit. After the specified time, the concentration of the biomass in the culture liquid reached 10.8 g / l. The concentration mentioned was optimal for the yield of L-lysine. At this point in time, the culture fluid contained no methionine and no threonine, while the biotin and thiamine in the culture fluid were contained in insufficient amounts for the biosynthesis of L-lysine. From this point on (ie after the first 18 hours) the fermentation tank was periodically fed corn extract as a source of L-ethionine, L-threonine, biotin and 'i'hiamin. In 1 g ilais extract

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were the substances mentioned in the following quantities: L-liethionine 4 mg / g; L-threonine 22 mg / g, biotin 0.55 / 'g / g, Thiainine 7.5 / ^ g. The corn extract was in a fermentation tank Serving of 7.5 kg every '' hour for 40

Hours fed.

After the first 18 hours of carrying out the cultivation process in the fermentation tank, the aeration was lowered intensity to 40 mg Op / l-min and increased the pH of the Culture fluid to 8.0. The temperature and the stirring intensity remained unchanged. Under the stated conditions the cultivation process was continued for 20 hours, after which the pH value of the culture liquid was lowered to 7.2 and the cultivation process continued for a further 22 hours was carried out. The total duration of the cultivation process is 60 hours.

After finishing the cultivation process contained the culture fluid contains the following substances: L-lysine 26.2 g / l; Biomass 14.4 g / l; irockensübstanzen 10.8 percent by weight; Sugar 0.8 percent by weight.

The yield of L-lysine was 33.5% based on the Weight of the sugars used.

The L-lysine was added to the culture fluid stabilized by sodium hydrogen sulfite and the pH value brought the culture liquid to 4.5 with hydrochloric acid. then

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the culture liquid was evaporated in a vacuum evaporation plant to a dry matter content of 35 percent by weight by mixing liquid putter concentrate of L-lysine in a. Received amount of 5400 kg. The said feed concentrate from L-lysine contained the following substances; L-lysine 8.5 percent by weight; Sugar 2.6 percent by weight; Betaine 1.2 weight percent; Total nitrogen 2.7 weight percent; Protein 7.8 percent by weight; Thiamine 8.9 α g / g; Riboflavin 125 / "g / g» dry substances 35 weight percent.

Example 5. The on. Homoserine deficient producer strain of L-lysine Brevibacteriunx sp. 22 was grown analogous conditions described for the preparation of inoculum under ^ um in Example. 1 The finished inoculum was transferred to the fermentation tank in an amount of 6% based on the pre-sterilized nutrient medium in this fermentation tank. The mentioned nutrient medium (10 1) had the following composition:

Glucose 250 g

Yeast hydrolyzate (the content of the yeast hydrolyzate an 'i'ro c kens excerpts was 20 percent by weight; of L-methionine 1.2 mg / g; at L-threonine 2.2 mg / g; of biotin 0.6 g / g; of thiamine 0.15 / * g / g) 500 g

Ammonium chloride 80 g

Potassium dihydrogen phosphate 5 g

Potassium hydrogen phosphate 5 g

Magnesium sulfate 3 g

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V / ater up to 10 1

pH value of the medium 7 * 0

The yeast hydrolyzate was prepared as follows. Dry yeast in an amount of 400 g was suspended in 1600 ml of 3% hydrochloric acid. The hydrolysis was carried out in an autoclave at a temperature of 127 ° C. for 2 hours. Then, the Hyldrolysat was cooled to room temperature, diluted with 25% strength aqueous ammonia solution ^it to a pH value of 7.3 is neutralized and filtered.

Culturing of the producer strain in the fermentor was carried out after the periodic process under the following conditions: temperature 28 ⁰ C, 45 mg Belui'tungaintensität 0p / l "min, stirring intensity 800'U / min, pH of the culture fluid 7. * 2 After 16 hours of cultivation, the concentration of the biomass in the culture liquid reached 10.2 g / l. The concentration mentioned was optimal for the yield of L-lysine. At this point in time, the culture fluid contained no methionine and no threonine, while biotin and thiamine (as stimulators of the biosynthesis of L-lysine) were contained in insufficient amounts for the biosynthesis of L-lysine. The unused glucose content of the culture liquid was 0.35 percent by weight. From this point on (ie after the first 16 hours) the fermentation tank was continuously fed with yeast hydrolysis as a source for 40 hours

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of amino acids and simulators of the biosynthesis of L-lysine concentrated at a rate of 3 g / hour Acetic acid fed as a completely assimilable carbon source at a rate of 18 g / h. It pointed the yeast hydrolyzate has the above composition. The pH of the culture liquid was until the end of the Process to 7.0 to 7.1 by adding 25% aqueous Ammonia solution held.

The total duration of the cultivation process in the fermentation tank was 66 hours. After the completion of cultivation During the process, the culture liquid contained the following substances: L-lysine 23 * 4 sA »biomass 13.3 SA» dry substances 3.4 Weight percent; Acetic acid 0.4 percent by weight; Sugar - was missing.

The yield of L-lysine was 30% based on that Weight of acetic acid used. L-lysine was separated from the culture liquid in crystalline form analogous to example 1

Example 6. Whoever is deficient in homoserine producer strain of L-lysine Breνibacterium sp. 22 was grown for the production of inoculum under ^ m. "In the analogous conditions described in Example 3. The finished inoculum was in the fermentation tank in an amount of β ^ _y based on the

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Volume of the pre-sterilized in this fermentation tank Nutrient medium, transferred. The mentioned nutrient medium (IO liter) had the following composition:

Sucrose 250 g

Peanut flour hydrolyzate (the dry matter content of peanut flour hydrolyzate 50 percent by weight; of L-methionine 0.7 mg / g; of L-threonine 4.5 mg / g; of biotin 0.2 A g / g; of thiamine 4.9 ^ g / g) 600 g Urea 80 g

"Potassium dihydrogen phosphate 5 S

Potassium hydrogen phosphate 5 g

Magnesium chloride. 2g

Water until 10

pH value of the medium 7 »0

The peanut flour hydrolyzate was prepared as follows. The peanut flour in an amount of 600 g was suspended in ml of 3% hydrochloric acid. The hydrolysis was carried out in an autoclave carried out at a temperature of 127 ° C for 2 hours. Then the hydrolyzate was cooled with 25% aqueous ammonia solution to a pH value of 7 | 0 and filtered.

The cultivation of the producer stanimes in the fermentation tank was carried out according to the periodic procedure under the following conditions

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conditions carried out: temperature 28 ⁰ C; Ventilation intensity -45 mg Op / 1 «min; Stirring intensity - 800 rpm; pH of the culture liquid 7.2. After 18 hours of cultivation, the concentration of the biomass in the culture liquid reached 11 g / l. This concentration was optimal for the yield of L-Ly-

£ l, iutiiionin una Ice in> sin. The culture liquid did not contain O at this point Threonine, while the biotin and thiamine are in the culture fluid in insufficient for the biosynthesis of L-lysine Quantities were included. The content of the unused Sucrose in the culture liquid was 0.54 percent by weight. From this point on (i.e. after the first 18 hours) the fermentation tank was kept for 40 hours peanut flour hydrolyzate as a source of L-methionine, L-threonine, Biotin and thiamine at a rate of 10 g / h and concentrated butyric acid as fully assimilable carbon swell continuously at a rate of 10 g / h to. The peanut flour hydrolyzate had the composition given above. The pH of the culture fluid was up to the end of the process in a range from 7.0 to 7 * 2 by adding the 25% aqueous Ammonia solution held. The total time of the cultivation process in the fermentation tank was 70 hours. After the termination of the cultivation process removed the culture liquid the following substances: L-lysine 17.5 g / l; Biomass 16 g / l; Dry substances 3.2 percent by weight; Butyric acid 0.55

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Weight percent; Sugar - absent.

The yield of L-lysine was 50%, based on the weight of the butyric acid used. The separation of L-lysine from the culture liquid in crystalline form was carried out analogously to Example 1

Example 7 “ The homoserine deficient producer strain of L-lysine Brevibacterium sp. 22 was grown under conditions analogous to those described in Example 3 for the production of inoculum material. The finished inoculum was transferred to the fermentation tank in an amount of 8%, based on the volume of the pre-sterilized nutrient medium in this fermentation tank. The nutrient medium mentioned (10 liters) had the following composition:

• sucrose 250 g DL-methionine ■ 2000 mg DL-threonine 8000 mg Biotin 250y, & Thiamine 500 ^ " urea 80 g Potassium dihydrogen phosphate 5 g Kai i um hydrogen phosphate 5 g Magnesium chloride 2 g water up to 10 1 pH value of the medium 7.0

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Culturing the Prod uz ent s enst strains in the fermentor was carried out after the periodic process under the following conditions: temperature 52 ⁰ C; Aeration intensity 50 mg Og / min / stirring intensity 600 O / min; pH of the culture liquid 7.2. After 16 hours of cultivation, the concentration of the biomass in the culture liquid reached 11.2 g / l. This concentration was optimal for the yield of L-lysine. At this point in time, the culture fluid contained no DL-methionine and no DL-threonine, while the biotin and thiamine in the culture fluid were contained in insufficient amounts for the biosynthesis of L-lysine. The content of the unused sucrose in the culture liquid was 0.66 percent by weight. From this point on (ie after the first 16 hours) the following substances were continuously added to the fermentation tank for 40 hours: Di-methionine 12.5 mg / h; DL-threonine 50 mg / h; Thiamine 12.5 vi / g / st; Biotin 6.25 / i / g / st; Sucrose 25 g / pc.

The total time of the cultivation process in the fermentation tank was 70 hours. After the end of the cultivation process, the culture liquid contained the following substances: L-lysine 38 »5 g / 1» biomass 12.4 g / l; Dry substances 2.5 Weight percent; Sucrose 1 percent by weight.

The yield of L-lysine was 35% based on weight the sucrose used. The separation of L-lysine

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from the culture liquid in crystalline form was carried out analogously to example 1.

Example 8. The lack of homoserine producer strain of L-lysine Brevibacterium sp. 22 was used for the production of inoculum under Zm in Example 1 described analog

Bred under conditions

The cultivation of the producer strain was carried out according to the continuous process in three interconnected fermentation tanks. The volume of the nutrient medium in each fermentation tank was ^ O rs? Speed of 3 he / h continuously increases. The composition of the nutrient medium in the first fermentation tank was as follows (in percent by weight):

Beet molasses (the sugar content of molasses was 48 percent by weight; L-methionine 0.42 mg / g; of L-threonine 2.4 mg / g; of biotin 0.08 / & g / g; at Thhlamin 0.2 / g / g) 5

Corn extract (the content of the corn extract at Dry substances was 50 percent by weight; of L-methionine 4 mg / g; of L-threonine 22 mg / g, of biotin 0.55 y * g / g »of thiamine 7.5 / <g / g) 2

Ammonium sulfate - 2.5

Potassium dihydrogen phosphate 0.075

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Magnesium sulfate. 0.035

Water to 100

pH of the medium 7.0

Culturing of the producer strain in the first fermentor was carried out at a temperature of 32 ⁰ C, an aeration intensity of 60 mg 0p / l "min, a stirring intensity of 400 V / min, a pH of the culture fluid of 7 | O. Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the first fermentation tank: content of biomass 11 g / l; Concentration of L-lysine 2.2 g / l; Concentration of sugars 2.1 percent by weight.

This concentration (11 g / l) of the biomass in the culture liquid was optimal for the yield of L-lysine.

From the first fermentation tank, the culture liquid continuously passed into the second fermentation tank (the dilution coefficient was 0.12 St "*). There was no culture liquid in the culture liquid that passed from the first fermentation tank into the second fermentation tank Methionine and no threonine contain, while the biotin and thiamine are inadequate for the biosynthesis of L-lysine Quantities were included. The content of the culture liquid of sugars after the first fermentation tank was also insufficient and amounted to 2.1 percent by weight. Therefore, the second was carried out Fermentation tank corn extract of the above composition

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as a source of amino acids and stimulators of biosyn-

■ z

synthesis of L-lysine in an amount of 0.1 mr / h and 50% aqueous SaocharoselÖsung as fully assimilable carbon source in an amount of 0.5 nr / St periodically ·

The cultivation of the producer strain in the second fermentor was carried out at a temperature of 30 ⁰ C, an aeration intensity of 50 mg Op / l-min, a stirring intensity of 500 u / min, a pH of the culture liquid of 7.8 and a dilution coefficient of 0.14 St ". Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the second fermentation tank: content of biomass 11.2 g / l; concentration of L-lysine 22.5 g / 1» concentration of sugars 3, 8 percent by weight.

The culture liquid entered the third fermentation tank continuously from the second fermentation tank (the dilution ^{coefficient was 0.14 St "1} ).

Temperat "ur ^ 0 ⁰ C, a central strain in the third fermentation tank was ₂ A 'min, a Hührintensität of 400 U / min and a pH of the culture fluid of 7.0, an L / aeration intensity of 50 mg O. Under the above culturing conditions the following equilibrium was established in the culture liquid of the third fermentation tank: content of biomass 11.6 g / l; concentration of L-lysine 35.7 g / l; concentration of sugars

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0.5 percent by weight.

The yield of L-lysine was 36% based on weight the sugar used "

The evaporation of the culture liquid to a dry substance content of 55 percent by weight was carried out analogously to Example 4 - The resulting liquid feed concentrate of L-lysine contained the following substances: L-lysine 20 percent by weight; Sugar 2.8 percent by weight; Betaine 0.6 percent by weight; Total nitrogen 1.6 weight percent; Protein 5.2 percent by weight; Thiamine 6.2 weight percent; Riboflavin 92 yc / g / g; Dry substances ^ percent by weight.

Example 9 » The homoserine deficient producer strain of L-lysine Brevibacterium sp. 22 Jl was described in Example 1 for the production of inoculum under τ

grown under analogous conditions.

The cultivation of the producer strain was carried out according to the continuous method in three connected fermentation tanks. The volume of the mixing medium in each fermentation tank was 20 m. The inoculum was continuously fed to the first fermentation tank at a rate of 0.6 nr / h and pre-sterilized nutrient medium at a rate of 3 Br / h. The composition of the sewing medlurna in the first fermentation tank was as follows (in percent by weight):

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Beet molasses (the molasses content was 48 percent by weight; L-methionine 0.42 mg / g; L-threonine 2.4 mg / g; biotin 0.08 / 1 g / g; thiamine 0.2 ^ i g / g) 5

Maize extract (the dry matter content of the maize compromise was 50 percent by weight, L-methionine 4 mg / g; L-threonine 22 mg / g; biotin 0.55 ^ 6/6 % thiamine? _T 5y & / B) 2

Ammonium sulfate 2.5

Potassium dihydrogen phosphate 0.075

Potassium hydrogen phosphate 0.075

Magnesium sulfate 0.035

Water up

pH of the medium 7.0.

The "cultivation of the producer strain in the first fermentor was carried out at a temperature of 32 ⁰ C, an aeration intensity of 60 mg 0p / l? Min, a stirring intensity of 400 V / min, a pH of the culture fluid of 7» 0th Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the first fermentation tank: content of biomass 10.8 g / l; Concentration of L-lysine 8.5 g / l; Concentration of sugars weight percent.

This concentration (10.8 g / l) of the biomass in the culture liquid was optimal for the yield of L-lysine.

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The culture liquid came into contact from the first fermentation tank.

Whether he

continuously into the second fermentation tank (the dilution coefficient re? 0.12 pieces). The culture liquid that entered the second fermentation tank from the first fermentation tank did not contain any Methionine and no threonine, while the biotin and thiamine were contained in the culture liquid in insufficient amounts for the biosynthesis of L-lysine. The content of the culture liquid of sugars after the first fermentation tank was also insufficient and was 2 percent by weight. That's why one led the second fermentation tank of corn extract of the above composition as a source of amino acids and the One-speed stimulators of the biosynthesis of L-lysine from 0.1 mvfit and 52% aqueous acetic acid solution as a fully assimilable carbon source with a Speed of 0.5 nr / h continuously increases.

The cultivation of the producer strain in the second fermentor was carried out at a temperature of 30 ⁰ C, an aeration intensity of 50 mg Op / l "min, a stirring intensity of 500 V / min, a pH of the culture liquid of 7.8 and a dilution coefficient of 0.14 pc.

Set under the cultivation conditions mentioned The following state of equilibrium is established in the culture liquid of the second fermentation tank: content of biomass 11.4 g / l; Concentration of L-lysine 29 g / l; Concentration of sugars 0;

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"T ■ i

2407519

Acetic acid 1.2 percent by weight.

The culture liquid emerged from the second fermentation tank

continuously, into the third fermentation tank (the dilution coefficient was 0.14 St). The cultivation of the producer strain in the third fermentor was carried out at a temperature of 3O ⁰ C, an aeration intensity of 50 mg 0 ₂ / liter min, a stirring intensity of 400 U / min and at a pH value of the culture liquid of 7.0. Under the cultivation conditions mentioned, the following equilibrium was established in the culture liquid of the third fermentation tank: content of biomass 11.9 g / 1 »concentration of L-lysine 23.2 g / 1; Concentration of sugars 0; Acetic acid 0.1 percent by weight.

Evaporation of the culture liquid to a content on dry substances of 45.4 percent by weight was carried out analogously to Example 4. -What obtained liquid feed concentrate of L-lysine contained the following substances: L-lysine 17.2 percent by weight; Sugar 0; Betaine 0.4 percent by weight; Total nitrogen 1.6 weight percent; Protein 5 »4 percent by weight; Thiamine 4.2 / g / g; ßiboflavin 105 // g / 6 »dry substances 45.4 Weight percent

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Claims (4)

L / Br PATENT CLAIMS: 1.Microbiological process for the production of liquid and dry feed concentrate of L-lysine or crystalline L-lysine by cultivating the producer strains Brevibacterium sp Growth of the biomass contains necessary amino acids and stimulators of the biosynthesis of L-lysine, at a temperature of 28 to 35 ⁰ C with aeration and stirring of the culture liquid by
and subsequent evaporation of the culture liquid on a Dry matter content from 35 to 55 percent by weight to achieve liquid feed concentrate of L-lysine or subsequent evaporation of the culture liquid and drying until the dry feed concentrate is obtained of L-lysine or subsequent separation of L-lysine from the Culture fluid in crystalline form, characterized in that that after reaching the optimal concentration of the biomass for the yield of L-lysine the culture liquid periodically or continuously the Amonic acids necessary for the growth of biomass and Stimulators of the biosynthesis of L-lysine or sources of the named amino acids and stimulators of the biosynthesis of _ such L-lysine in quantities adds that the concentration of the biomass / ►0 9849/0677 is kept at a level in the culture liquid »that for the yield of L-lysine is optimal, or this level does not exceed by more than 5 g / l culture liquid 2. Microbiological method according to claim 1, characterized in that one additionally zaznführ ¹ as the KuI-turfluigkqit. Sources of the amino acids necessary for the growth of the biomass and the stimulators of the biosynthesis of L-lysine maize extract, molasses or yeast hydrolyzate are used. 2 · Microbiological method according to claim!, Characterized characterized in that after reaching the optimum concentration for the yield of L-lysine the biomass of the culture liquid periodically or continuously completely assimilable carbon sources adds in amounts: aaü uie concentration of L-lysine in the Culture liquid 17 to 40 g / l, converted to L-lysine monochlorohydrate, achieved. 4. Microbiological method according to claim 3, d adurch characterized in that sugar or acetic acid are used as completely assimilable carbon sources used. 409849/0677