DD210069A1 - METHOD FOR PRODUCING LYSINE-ENRICHED FODDER WHITE CONCENTRATE - Google Patents

Abstract

The invention relates to a process for the production of lysine-enriched microbial feed protein and represents an improvement of the process according to DD-WP 160357, in particular with regard to the extension of the field of application. The object of the invention is the production of lysine-enriched feed protein concentrate based on an extended energy and carbon supply without any additional need for active ingredients and to the exclusion of the formation of hard-to-apply products. It is achieved by the coupling of an unsterile microbiological process for the production of nourishment based on n-alkane hydrocarbons of lysine on the basis of carbohydrates, alcohols and carboxylic acids as an energy and carbon source, the culture fluid from the non-sterile process has a content of at least 1000 ng amino acids / l and a biotin content of at least 10 my / l.

Description

Title of the invention

Process for the preparation of lysine enriched feed protein concentrate

IPK: C12N

Field of application of the invention

The invention relates to a method for producing lysine-enriched microbial feed protein and represents an improvement of the method according to DD '. O 6O 3S7- in particular with regard to the extension of the field of application «

Characteristic of the known technical solution ε η

From the main pat ent WS ^ f 6 O 3 S " is the production of lysine enriched feed protein concentrate by coupling an unsterile microbiological process for obtaining feed protein based on n-alkane petroleum products with a microbial sterile process for obtaining liquid based lysine η-paraffins known.

It has been found that the growth stemming symbiotic formed by the microbial accompanying flora in the non-sterile process stimulate the growth of lysine-producing microorganisms on n-alkanes. At the same time, the amino acids present in the culture liquid obtained after the separation of the microorganisms in the non-sterile process are used for the cultivation of the lysin-producing cultures. In this way, the active substance and amino acid additives otherwise required for the lysin-producing cultures can be dispensed with in this process the cleaning, d. H. Removal of the undesired hydrocarbons in the end product, the protein broth from the non-sterile process associated with the lysine-enriched product. However, this process was only applied to the use of hydrocarbons as the carbon source in both partial processes.

Object of the invention

The aim of the invention is the production of lysine-enriched feed protein concentrate on the basis of an extended supply of energy and carbon without an additional requirement for active ingredients and to the exclusion of the formation of products which are difficult to apply.

Presentation: the essence of the invention

In accordance with the present invention, the objective is achieved by providing a sterile microbial process for lysing, alcohol, carboxylic acid and carbohydrate based lysing with an unsterile process of biomass production with sole vial growth.

when the aqueous culture medium from the non-sterile process, which was separated from this after heating the microorganism suspension ₃ immediately after separation as the amino acid and active ingredient j, in particular as a source of biotin and thiamin, for the cultivation of the lysinproduzierenden Culture is used "When heated above 50 ^° C, microorganisms release amino acids, vitamins and other biogenic substances into the surrounding water"

The bacteria suitable for lysing are mutants that are characterized by a number of properties. "In addition to the need for vitamins, especially biotin, these properties include blocking the pathway for the formation of related amino acids, such as L-Hoπιοserin, L-methionine, L -Threonin, L-leucine, or isoleucine "Bas is expressed in the need for said amino acids or as sensitivity to L-threonine or L-methionine." Another feature is the variation in the control of feedback by related amino acids and manifests itself in resistance towards L- * lysine or L-threonine or their analogues.

The addition of the culture liquid from non-sterile biomass production into the lysine fermentation suspension eliminates the need for adding biotin. Surprisingly, it was found that a greater lysine yield was achieved on addition of this culture fluid which had at least an amino acid content of 1000 mg / l and a biotin content of 10 ng is than the case with the sole addition of the corresponding amino acids and biotin is the case. This ect could not be found so clearly in the technical solution according to WP "5" 7.

If the fermentation was carried out with auxotrophic mutants, the addition of the substrate also resulted in a greater lysine production than when the required amino acids and biotin were added »

The heating process of the biomass suspension from the unsterile process should therefore be carried out so that at least 1000 mg / l of free amino acids and 5 mg / l of biotin are present in the culture medium. Heating separated culture liquid is not first returned to the fermentation process of the unsterile process and then removed, but immediately supplied to the Iysin culture, as in the return to the fermentation process, the content of amino acids and active ingredients, especially biotin and thiamine, is lowered by accessory Utilisation «The lysine-enriched suspension from the sterile process, the biomass suspension is then fed from the non-sterile process and worked up together thermally dry biomass, which is purified by extraction in a further process stage for the removal of undesirable hydrocarbons. In certain cases, in which the lysine-enriched biomass suspension should be added in such amounts of the remaining dry biomass, that the originating from the aqueous culture medium of the unsterile process small amounts of dissolved hydrocarbons do not interfere with the quality of the final product, the lysine-enriched suspension can also after extractive cleaning in the desired end product is introduced.

applications

In a 500 liter stirred fermentor, the yeast becomes Lodderomyces elongisporus: under known, nonsterile conditions with a n-alkane petroleum distillate as the carbon source

continuously cultivated «The microbe-containing suspension is continuously removed from the per- mentor and returned to the tower

EADERSHIP subjected by decantation of separated V / ater a heat separation at 80 ⁰ C using an ethylene oxide-propylene oxide surfactant. Subsequently, by further separation of water by means of a jet separator, a further Xonzentrierung the yeast phase "A part of the separated by the separation process water is sterilized and serves as the basic medium (GM) for ( '' the Lysinfermentation

Using GM, 4 experiments were carried out, Example 1

A 13 / sin-forming strain of Corynebacterium glutamicum is aerobically cultured in a seed medium of the following composition at pH 7jO.

D-glucose KH ₂ PO ₄

40.0 g / l 0.4 g / l 1.4 g / l 3.0 g / l 0.4 g / l 25.0 g / l 1000 ml

Urea MgSO ₄ χ 7 peptone GM

3 media of the following composition are prepared.

LM-C-1 LM-C-2 LM-C -3

Glucose MgSO ₄

KH ₂ PO ₄

Urea biotin aqua dest.

GM

In each case 5 1 of the corresponding Lysinfermentationsmediums were inoculated with 500 ml of preculture. The experiment was carried out over a period of 96 h. The following amounts of lysine were determined in the individual batches:

40 0 G 40 O S 40.0 S ο, 3 G O, 3 G 0.3 G ο, 7 G O, 7 G 0.7 G 3, 0 G 3, 0 G 3.0 G 50 00 u m ml : 500 ml 1000 ml 500 ml

LM-C-1 6 g / l LM-C-2 42 g / l LM-C-3 19 S / 1 Example 2

An L-lysine-producing strain of Brevibacterium flavum is cultured in the following medium for 120 hours under aerobic conditions.

_; LM-B-1 LM-B-2

B-glucose (NHj ₀ SO

MgSO ₄ χ 7H FeSO ₄ MnSO ₁

KH ₂ PO ₄

FeSO. χ 7 H

4 thiamine biotin yeast extract

150 g / l 150 g / l 40 g / l 40 g / i 0 , 8 g / l O, 8 g / l 0 , 4 g / l 4 g / l 0 , 01 g / l Ö, 01 g / l 5 mg / 1 5 mg / 200 / «/ I - 200 / Ig / 1 - 2 g / l -

CaCO " 5 g / i DL-methionine 100 mg / 1 Distilled water 1000 ml GM mm

g / i

1000 ml

There were 38 g / 1) LM-B-1) and 51 g / 1 (LM-P-2) L-lysine detected in the culture broth

Example 3

A lysine-forming St on τη of Protaminobacter thiaminophagus is cultured in a methanol-containing medium of the following composition (pH 7j2) ·

___ LM-P-1 LM-P-2 - LM-P-3

Methanol 20 ml 20 ml

(3TH ₄ ) 2 SO ₄ 6 g 6 g

KH ₂ PO ₄ 2g 2g

K ₂ HPO ₄ 7g 7g

MgSO ₄ χ 7 H ₂ O 0.6 g 0.6 g

FeSO ₂₁ χ 7 H ₃ O 8 mg 8 mg

MnSO ₄ χ 4 H ₂ O 8 mg 8 mg

   _. 'Thiamine 1 mg 1 mg

Biotin 1 ng 1

Yeast extract 0.05 g

Distilled water 1000 ml -

GM - 1000 ml 1000 ml

Cultivation was carried out over a period of 120 hours. "After 48, 72 and 96 hours, 10 ml of methanol were added in each case. After 120 hours, 6.9 g / l were added to the culture fluid. 1 (LM-P-1), 12.1 g / l (LM-P-2), 12.0 g / l (LM-P-3) L-. Lysine found.

20 ml 6 G OJ G 7 G 0.6 G 8th mg 8th mg

Example 4

A 1-lysine-producing strain of Corynebacterium glutamicum is cultured in a seed medium of the following composition (pH 7.0) _#

Glucose 50 g / l

Peptin 15 g / l

Yeast extract. 15 g / l

NaCl 2.5 g / l

Urea 2.5 g / l

Biotin 50 μg / l

With 1 1 of the resulting bacterial suspension 10 1 of a F'ermentationsmediums be inoculated. The fermentation medium has the following composition,

LM-E- * 1 LM-S-2

Ammonium acetate 5.0 g / l 5.0 g

2.0 g

MgS0 ₄ x 7 H ₂ O 0.5 g 0.5 g

0 δ / 1 2, 0 G ο, 5 S ο, 1 G  ο, 02 G 30 / Ug 70 mg

₄ χ 7 H ₂ O 0.1 g 0.1

MnSQ ₄ χ 7 H ₂ O 0,02g 0,02g

Biotin thiamine

Distilled water 1000 ml GM - 1000 ml

The cultivation is carried out over a period of 48 h at a temperature of 30 ⁰ C. For 5 h, 7.5 l of a mixture containing 7 % ammonium acetate and 35 % acetic acid are added continuously over a period of 40 h. The pH of the medium is adjusted to 6.9. After culture has ended, the culture broth is added Use of LM-SI 27.0 g lysine / 1, while with the use of LM-E-2 a lysine content of 37.1 g / l was found.

The further work-up is the same for all 4 examples. The lysine suspensions were each concentrated to such an extent that the lysine concentrations were 90-100 g / l.

For the further work-up, from each example the suspension was used which was obtained using the GM, ie the inventive solution (Example 1: LM-G-2 / Example 2: LM-3-2 / Example 3: LM-P -3 / Example 4: LM-E-2). The lysine suspension is mixed with the yeast suspension obtained as described initially after the hot separation in the ratio of 1: 5 (1 part lysine suspension + 5 parts yeast suspension) and processed in a spray dryer to a fine-grained dry matter having a water content of 8 10 % 5-stage extraction, this powder is cleaned with a mixture of ethanol and gasoline (1/1) ·

The following amounts of lysine were determined in dry yeast per 100 g of protein:

Example 1 22 g

Example 2 21.5g

Example 3 19.8 g

Example 4 21.7g

The further purified fodder, which was won in contrast to the 'inventive technical solution based on n-alkali nen, contains only about · 7 g of lysine per 100 g protein ·

Claims (1)

ErfindungsansOruch Process for the preparation of lysine-enriched feed protein concentrate according to WP Ί6Ο 3 $ "7 , characterized in that an unsterile microbiological process for obtaining n-alkane-based carbohydrate feedstocks with a sterile microbial process for obtaining lysine based on carbohydrates, alcohols and carboxylic acids coupled as a carbon source
is carried out by carrying out the lysine fermentation using the culture liquid from the non-sterile process, said culture liquid having a content of at least 1000 mg amino acids / 1 and a biotin content of at least 10 ug / 1, and processing both microbial products into a common end product.