WO1999027799A1

WO1999027799A1 - Method for producing and using lysine formiate

Info

Publication number: WO1999027799A1
Application number: PCT/EP1998/007437
Authority: WO
Inventors: Ulrich Eidelsburger; Linda VON DEM BUSSCHE-HÜNNEFELD; Joachim Meyer; Günter Gaus
Original assignee: Basf Aktiengesellschaft
Priority date: 1997-12-02
Filing date: 1998-11-19
Publication date: 1999-06-10
Also published as: AU1755899A; DE19753321A1

Abstract

The invention relates to the use of lysine formiate in foods and pharmaceutical and cosmetic preparations. The invention also relates to a method for producing said lysine formiate by eluting lysine from an ion exchanger.

Description

Process for the production and use of lysine formate

description

The invention relates to the use of lysine formate in foods, in pharmaceutical and cosmetic preparations and to a process for their preparation.

L-Lysine, as an essential amino acid, is widely used as an additive in food and feed. It is used particularly frequently for the manufacture of dietetic foods and animal feed. Lysine is also widely used in medicine, for example as a component of infusion solutions. L-lysine, for example, promotes bone growth and stimulates cell division and nucleoside synthesis.

Over 90% of the L-lysine synthesized worldwide is used for the production of animal feed, especially for the production of pig or poultry feed. The use of L-lysine for the production of feed serves to supply the animals with the essential amino acid present in the deficit in the protein sources used in animal nutrition. If lysine is fed, the limit is lifted and the animals' zoo performance can be increased significantly.

The most commonly used form of L-lysine is the L-lysine monohydrochloride (L-lysine-HCl) for reasons of stability and manufacturing. The use of lysine hydrochloride in poultry feed means that the animals absorb more water (US 4,919,945). This can very often lead to the undesirable so-called "wet litter" and thus to an increased risk of infection of the animals. Bird growth can also be adversely affected by increased chloride administration. In addition, basic amino acids in the form of their chloride salts have a very bitter taste, which also leads to less nutrient absorption by the animals.

For example, when using L-lysine hydrochloride in medicine, it can cause hyperchloraemic metabolic acidosis in infusion solutions. The use of such infusion solutions in patients suffering from renal insufficiency leads to an undesirable further stress on the already disturbed electrolyte balance of the patient by the chloride ions. The use of L-lysine in the form of the free Base is problematic in infusion solutions due to the basic pH.

The use of L-lysine in the form of the free base leads to acid-base problems. The high pH of the free base can lead to a Maillard reaction with sugars or isopeptide bonds in the food, particularly in the heat treatments customary for the production of feed, which leads to a reduction in the nutritional value of the feed.

By Edmonds et al. experiments on the effect of the amino acids methionine, phenylalanine, histidine, isoleucine, valine, leucine, tryptophan, threonine, arginine and lysine in animal nutrition in pigs and poultry (J. Nutr. Vol. 117, No. 5, 1987: 1396 - 1401 and J. Anim. Sci. Vol. 65, No. 3, 1987: 699 - 705) and the interaction between lysine and arginine (J. Anim. Sci. Vol. 65, 1987: 179 - 185) in the Amino acid uptake described by the animals. To avoid acid-base problems, L-lysine acetate was used in the experiments. A disadvantage of the use of lysine acetate is that lysine acetate has no or only an insufficient micro-static or micro-acidic effect.

WO 96/40618 describes concentrated aqueous lysine propionate solutions and their use in animal feed. Despite the use of very concentrated solutions, the addition of the lysopropionate brings a considerable amount of water into the feed. This results in wetter products that can spoil more easily and may need to be dried.

L-lysine propionate is claimed by US 3,0224,272. The claimed L-lysine propionate shows good fungicidal activity. It is therefore used, as can be seen from the description, for the preservation of baked goods or other foods intended for humans, which can be spoiled by the action of fungi. A disadvantage of the use of L-lysine propionate is that the propionic acid present in the salt has only a weak bacteriostatic or bacteriocidal effect and the spoilage of food can only be insufficiently prevented by the action of bacteria.

The object was therefore to provide a solid or liquid L-lysine salt which does not have the disadvantages mentioned above and which has a high nutritional effect with a high and / or permanent biocidal and / or biostatic effect. The solid end product should also have good storage and Have processing properties. It should advantageously be able to be produced in a more solid form than in a liquid form.

It has been found that the desired properties can be achieved with lysine formates. The crystal structure of these lysine formates was m Acta Crystallographica by Suresh et al. (1995: 353-358).

The lysm formates produced from L-lysm formate, D-lysm formate or DL-lysine formate for the use according to the invention advantageously contain one mole of lysine per one mole of formic acid. However, Lysm formic acid salts can also be prepared which contain more or less than one mole of formic acid per mole of lysine. By increasing the .formic acid concentration over one mole per mole of lysine, the preservative effect of the lysine formates can be significantly increased. The lysine formates are preferably produced from L-Lysm. Liquid lysiformates can also formic acid in excess if necessary. m neutralized form or lysine in excess. In addition to lysine formate, the solid or liquid lysine formates can contain further salts of formic acid, such as alkali, alkaline earth, iron or ammonium salts.

Lysine sources for the production of lysine formates are, for example, proteins from which L-lysine can be obtained by hydrolysis with acids. D, L-Lysm and, through subsequent resolution, also L-Lysm and D-Lysm are accessible via chemical synthesis, for example from DL-α-.Amιno-ε-caprolactam. L-Lysm is usually obtained via a fermentative process with the aid of microorganisms essentially of the genera Corynebacteriu, Brevibacteπum, Arthorbacter, Microbacterium, Bacillus or Nocardia and a subsequent purification, advantageously via an ion exchanger. The fermentation broth can be added to the ion exchanger directly or after the biomass has been separated off. Microbiological processes for the production of L-Lysm are described, for example, in Trends of Biotechnology 1 (1983: 70-74). To produce the lys formates, the lysine bound to the ion exchanger is advantageously eluted from the cation exchanger with formic acid, concentrated and optionally dried. The fermentatively produced Lysm is usually bound to an anion exchanger and eluted with ammonia or another base. The counterion can then advantageously be wholly or partly, for example, by electodialysis or, in the case of NH ₃ as base, by stripping to produce the

Lysine formates are separated. The production of Lysi formates directly from the fermentation broth after separation of the Biomass, addition of formic acid and subsequent concentration is conceivable. The lysine formate solutions thus produced contain at least 0.1 molar equivalents of formic acid, preferably 0.5 to 5 molar equivalents of formic acid per molar equivalent of lysine. These lysine formate solutions particularly preferably contain lysine and .formic acid in stoichiometric amounts.

In addition to lysine formate, the lysine formate solutions can also contain further alkali metal or alkaline earth metal salts and / or ammonium salts of formic acid which arise, for example, from the neutralization of the base which has not been completely removed and which was used to elute the lysine from the ion exchanger. These solutions can also contain an excess of formic acid.

Solid lysine formates can be produced from the above-mentioned liquid lysine formate solutions by crystallization and / or by concentration and / or drying methods known to those skilled in the art, such as spray drying or fluidized-bed drying methods.

Lysine formates can also advantageously be prepared starting from aqueous lysine solutions or from solid lysine which contain lysine in the form of the free base by adding .formic acid. The solutions obtained are then advantageously concentrated in vacuo with heating, which produces either a concentrate or a solid product.

In addition to the nutritional effect of lysine, the formic acid component has a preservative effect, which protects the feed from microbial spoilage and, due to its bactericidal properties, can improve performance and health, particularly in young animals such as piglets. In contrast to free acid, the formic acid component in the lysine formates has no or only a very slight corrosive effect. Depending on the formic acid concentration used for the production, the lysine formates are odorless to slightly smelling, which means that a pungent formic acid odor cannot be detected. The product is therefore very easy to handle.

After the use according to the invention, the lysine formates are advantageously used in foods, preferably in animal nutrition, particularly preferably as an additive in feed for pigs, piglets and poultry, such as, for example, laying hens, broilers or turkeys. You can use all common animal feed such as compound feed and / or individual feed such as feed - cereals such as wheat, oats, rye, barley and legumes, Corn or "corn cob mix" can be added. The lysine formates can be added directly to the feed or in the form of so-called "premixes" in which they have been mixed with other feed additives before use.

All auxiliaries customary in agriculture, such as flavorings, colorants, appetite stimulants, antibiotics, probiotics and / or enzymes, can be added to the lysine formates as auxiliary substances. The auxiliaries are advantageously added in an amount of 0.1% by weight to 50% by weight, based on the weight of the lysine formates, preferably in an amount of 0.1 to 10% by weight.

Depending on the composition of the feed, different application rates of the lysine formates are advantageous. As a rule, an addition of 0.1 - 10 kg per tonne of animal feed is sufficient for lysine supplementation and successful consumption. The lysine formates are preferably added in amounts of 0.5-5 kg per ton of animal feed.

The lysine formates can be used for the production of the feed or feed mixtures in a manner known per se. The feed can be added directly after harvesting or after storage. The solid and / or liquid lysine formates can be added to the feed directly or in the form of premixes, advantageously during pelleting or extrusion of the feed via, for example, a metering device. Spraying the feed or premixes with lysine formate solutions is also possible.

Also in pharmaceutical or cosmetic preparations, the Lysinformiate ve ^* can be rwendet. The lysine formates can be added to all customary pharmaceutical preparations which are suitable for oral or parenteral (subcutaneous, intravenous, intramuscular, intra-perotoneal) administration. Use in parenterals is preferred.

In principle, pharmaceutical or cosmetic preparations are to be understood to mean all common galenical application forms, whether solid or liquid, such as tablets, film-coated tablets, capsules, powders, granules, dragees, suppositories, solutions, ointments, creams or sprays. These are manufactured in the usual way. In addition, the pharmaceutical or cosmetic preparations can also contain other active ingredients, preservatives, loose agents, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents. Contain salts for changing the osmotic pressure, buffers, coating agents and / or anti-oxidants.

Examples

Example 1: Fermentative production of L-Lysm

Fermentation broth from a conventional lysine fermentation with Corynebacteπum glutamicum ATCC 21526, as described in US Pat. No. 3,708,395, was freed from the cell mass by centrifugation for 20 minutes at 20,000 × g and 4 ° C. The line-free solution was absorbed on an Amberlite 252H catalyst exchanger and washed with three column volumes of water. The column was then eluted with formic acid (85% in water). An eluate of the following composition was obtained:

11% Lys, 70% formic acid, 19% water.

Example 2: Preparation of lysmformate from fermentation broth

The solution produced according to Example 1 was brought to a final concentration of 50% Lysm and 23.6% formic acid by concentration in vacuo and then analyzed. After a long storage period or storage at 4 degrees Celsius, the solution produced in this way increased crystallization. If such a solution was evaporated to dryness in vacuo, the result was a constant. Formic acid value of 15.7 g. Formic acid. The NMR analysis of these crystals is shown in Table I.

Example 3: Preparation of lysm formate from solid or liquid lysm

Starting from commercially available Lysm x HC1 (Luta ^® -Lysm), an HCl-free Lysm-Base solution (50% m water) was generated from an aqueous solution of Lysm x HC1 by electrodialysis. In a second approach, commercial Lys base (Fluka) with water was made into a 50% solution. The absolute value of the Lysm content does not play a special role. The Lysm content can be determined very easily from the weight used, assuming solid Lysm, if solutions were concentrated, an experimental water determination was carried out to analyze the mathematical determination. A solution thus obtained with a defined lysine content was then mixed with at least one molar equivalent of formic acid and concentrated to constant weight in a vacuum with heating.

Under these conditions, an excess of formic acid used led to the same product which was obtained if exactly one equivalent of acid was used, since all excess acid (i.e. any amount greater than one equivalent) was removed in vacuo. If there was more formic acid in the product, it either had to be added afterwards or the product was not allowed to be completely concentrated.

The solid obtained after the concentration was the desired lysm formate, which no longer had to be purified.

17.0 g of formic acid (0.369 mol) were added to 100.1 g of a 50% strength lysine solution (Fluka, 0342 mol) with stirring. The lysine / carboxylic acid ratio was 1: 1.08. The initially clear solution soon formed a crystalline solid. The entire mixture was concentrated in vacuo and the resulting solid was subjected to further drying. The constant weight was reached at 65 g. The product was a 1: 1 mixture (= salt) of lysine and formic acid, which did not require any further purification.

Accordingly, a solution of any concentration was first prepared from solid lysine, to which the acid was then added. Since excess water has to be removed again, an acid solution close to the saturation limit is recommended. Table I shows different approaches.

Table I. Manufacture of lysm formate

Example 4: Preparation of lysmformate by elution with base

A Lys solution produced according to Example 1 was subjected to a conventional workup. The fermentation broth was adsorbed on Dowex50 ion exchangers, washed with water and eluted with NH ₃ solution. The proportion of NH3 and additional water were removed from the eluate by means of vacuum and heating. A 38.4% Lys base solution in water was then obtained. This was mixed with stochiometric amounts of .2imeιsensaure, concentrated to 50% lysm content and analyzed. A lysformate composition was determined which corresponded to the lys formates mentioned in Example 1 (Tables I, I and II).

Example 5: Feeding trial with piglets

The experiment was carried out with 48 piglets (Deutsche Landrasse x Pietram). The animals were 30 days old at the start of the experiment and had an average live weight of 7.6 kg. The animals came from 9 litters, with 5 animals selected from the litter, which were randomly distributed to the 5 treatment groups according to the principle of block formation. Groups I, II and IV were assigned an additional animal from a litter. The effectiveness of the two lysing preparations used (L-Lysm x HC1 and Lysmformiat) was examined according to the treatment scheme shown in Table II. Table II: experimental groups and lysine supplement

Group I as a negative control received the basic feed without any lysine supplement. In group II, and III to the base feed were 0.12 and 0.24% lysine equivalents in the form of the commercial product Luta-lysine ^®: added (L-lysine HCI x,> 78% L-Lysine, sales BASF AG, Ludwigsha-fen) . In groups IV and V, 0.12 and 0.24% lysine equivalents were also supplemented with the test substance L-lysine formate. The lysine formate prepared according to Example 1 or 2 contained 75% lysine activity and a formate content of 25%. The composition of the basic feed used is shown in Table III. By using components that are customary in practice but low in lysine, the aim was to achieve a low base content of lysine, which should only cover about 60% of the lysine requirement of the piglets. This should create good conditions for measuring the effectiveness of the lysine supplements. The basic feed was supplemented with DL-methionine and L-threonine in order to ensure a supply of these amino acids that was appropriate to the needs and thus lysine had a clearly first-limiting effect. With the exception of lysine, the feed used with 17.7% crude protein, 13.1 MJ ME / kg (= convertible energy), 0.9% calcium and 0.7% phosphorus met the requirements for piglet rearing feed II according to the standard type (in accordance with the Feed Act. Appendix II, compound feed No. 2.3). The analyzed .amino acid pattern of the basic feed (Table IV) showed a lysine concentration of 0.77% and thus a significant undersupply compared to the requirement of 1.1 - 1.2% lysine in the feed. All other amino acids were included as needed. The feed mixtures were pelleted.

The piglets were kept in a fully air-conditioned test house in individual pens in a cage system. The room temperature was 26 ° C at the start of the test and was continuously reduced to 22 ° C by the end of the test. The relative humidity was between 50-60%. The feed was presented to the animals for ad libitum intake. The feed troughs were replenished daily, feed not consumed was weighed out on the 3rd and 7th day of each week. The daily consumption of food was determined from the feed template and the back weight. The animals were Position of the live mass weighed. The feed conversion was determined from feed consumption and live mass gain (g feed per g gain).

The experimental data obtained was subjected to an analysis of variance, checked for significance (F-test) and the mean values of the individual groups compared with each other using the Student-Newman-Keuls test. The result table contains the mean values and the standard deviation of the individual values. Significant differences between the mean values (P <0.05) were marked with different capital letters. In addition, the slope ratio technique was used to determine the relative biological effectiveness of the two forms of lysine used using linear regressions.

Table V summarizes the results of weight development, feed intake and feed conversion. The animals in group I showed significantly worse weight gain, feed intake and feed utilization than the animals in the groups supplemented with lysine. Accordingly, there was a significant undersupply of lysine in the basic feed. The addition of 0.12% and 0.24% lysine in the form of L-lysine x HCI significantly increased the daily gain from 214 g to 308 and 354 g, respectively by 44% and 63% compared to control group I. The corresponding addition of the product lysmformate also significantly improved the daily gain to 330 g and 367 g, respectively by 54% and 72% compared to the negative control. The lysine formate thus showed a greater increase in growth than the addition of L-lysine x HCI.

The feed consumption of the piglets was also significantly influenced by the lysine supply. When 0.12% and 0.24% lysine in the form of lysine x HCI was added, the piglets ingested 21% and 30% more feed than the animals that received the basic feed (group I). In comparison, the feed intake when using the lysine formate was 30% and 33% above the level of the base group. The somewhat higher feed consumption in the groups with the addition of the lysine formate suggests good acceptance of the feed supplemented with this lysine source.

In order to quantify the biological effectiveness of lysine formate in comparison to the commercial lysine x HCI, linear regressions between the growth rate and the intake of supplemented lysine were estimated. The relative effectiveness of the lysine formate and its significance can be read from the quotient of the regression coefficients of lysine x HCI and lysine formate and its 95% confidence interval. This statistical ver The slope ratio technique (Finney 1978) is the slope of two regression coefficients, where the confidence interval is calculated using the Fieller formula. Supplemented lysine intake, ie lysine consumption, which was achieved solely through supplementation, was chosen as the influencing factor. In the model used, the negative control represents the additive constant of the regression equation, since its lysine uptake is entered with 0. The variable supplemented lysine intake enables a comparison of the substances based on the same lysine intake. The influence of different feed intake is eliminated. The corresponding regression equation and the course of the regression are shown in FIG. If the biological effectiveness of lysine x HCI = 100 is set, an effectiveness of 105% is shown for the lysine formate.

Table III: Composition of the base feed used

Table IV: Analyzed amino acid contents of the base feed used (% in 88% DM)

¹ • Methionine and cystine were determined by oxidation with performic acid

^{2 ■} nb - not scanned

Table V: Influence of L-Lys x HCl and lysine formate on live weights, growth rate, feed consumption and feed conversion of piglets

5

0

5

Claims

Claims:

1. Use of lysine formates in foods, in pharmaceutical or cosmetic preparations.

2. Use according to claim 1 in food

3. Use according to claim 1 or 2 in animal feed.

4. Use according to. Claims 1 to 3 in feed for monogastric animals.

5. Use according to .Claims 1 to 4 in pig or poultry feed.

6. Use according to claims 1 to 5 in amounts of 0.01 to 1.0 wt .-% based on the total amount of food.

7. Cosmetic or pharmaceutical preparations containing lysine formates.

8. Foods containing lysine formates.

9. Feed containing lysine formates.

10. A process for the production of liquid lysine formates, characterized in that lysine is eluted with .formic acid from an ion exchanger.

11. A process for the preparation of liquid lysine formates, characterized in that lysine is eluted from the ion exchanger with a base, the base is subsequently removed in whole or in part and the solution thus obtained is mixed with .formic acid.

12. The method according to claim 11, characterized in that NH _{3 is} used as the base.

13. The method according to claim 11 or 12, characterized in that the solution is completely or partially neutralized.

14. The method according to any one of claims 11 to 13, characterized in that the solution based on the lysine contained is mixed with at least 0.1 molar equivalents of formic acid.

15. The method according to any one of claims 11 to 14, characterized in that the solution based on the lysine contained in stoichiometric amounts (1: 1) is mixed with formic acid.

16. A process for the production of solid lysine formates from the liquid lysine formates as claimed in claims 11 to 15, characterized in that the salts are isolated from the solutions and then dried.