DE19840489A1 - Use of specified enzymes, especially lysyl oxidase, as protein crosslinking agents for formulating compositions containing active ingredients - Google Patents

Abstract

An enzyme (I) selected from lipoxygenases, protein disulfide isomerases, phenol oxidases and peroxidases, lysyl oxidases, protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases is used to formulate compositions containing active ingredients. Independent claims are also included for the following: (1) a process for producing compositions in which one or more active ingredients are surrounded by at least one layer, where at least part of at least one layer comprises a protein that has been crosslinked with (I). (2) a composition in which at least part of at least one layer surrounding one or more active ingredients comprises a protein that has been crosslinked with (I); (3) an isolated protein comprising at least one of amino acid sequences (II)-(XIII): (a) G(S)(C)Q(C)KTNEKVNIEAPKPNI(C)DT(L)(S) (b) EYP(C)APDVVYNTK (c) GGTYSTVTQNPTLNR (d) DYNIMPGGGXVHR (e) ATGGTYSTVXAQN (f) APETENNAR (g) GPL(P)VNE(E)TTIEPLSFYNT (h) IYELSLQELIAEYGSDDPNNQHTFYSDI (i) DNVDDLSCTIIQR (j) VAPETENCAR (k) NVDVEYPCAPGVVYNTK (l) GYPNAEY(S)LDF/ER, in which lysine iota -amino groups are optionally oxidized to aldehyde groups; and (4) a food or animal feed containing the composition of (2).

Description

The present invention relates to active ingredient preparations which one or more active ingredients from at least one layer or parts of a layer surrounded by a protein that with an enzyme selected from the group of lipoxygenases, Protein disulfide isomerases, phenol oxidases, lysyl oxidases, Protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases was cross-linked.

The invention also relates to a method of production of active ingredient preparations in which one or more active ingredients are surrounded by at least one layer, marked thereby net that at least one of the surrounding the active ingredient (s) Layers or parts of these layers consist of a protein, that with an enzyme selected from the group of lipoxygenases, Protein disulfide isomerases, phenol oxidases, lysyl oxidases, Protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases has been cross-linked as well as the use of the said Enzymes for the formulation of active ingredients.

Furthermore, the invention relates to food, feed or pharmaceutical preparations containing the invention Active ingredient preparation and the enzyme lysyl oxidase. In particular The invention relates to dry powder containing vitamins, enzymes, Food and feed additives, such as B. Carotenoids contain. In addition, various Additives to be embedded.

Powdered active ingredient preparations such as vitamin and carotenoid Preparations are well known and are used in pharma chemical industry as well as in the food and feed widely used in industry. Be in literature many processes for the production of suitable preparations are described.

For the production of powdered preparations in which Oxidation-sensitive substances such as oil-soluble vitamins or Carotenoids should be protected against oxidative influences, different manufacturing processes, especially spray procedure described.

In the German patent 10 35 319 it is described how a dispersion of an oily vitamin in a large excess of powdered starch with a low moisture content (below 8%) is sprayed. The dry starch powder makes the water removed from the sprayed particles. This freezes them leaving a large amount of the starch on the surface of the particles sticks. In addition, the excess starch must be removed separates and then fed back into the process.

In the Swiss patent 488 455 it is shown that a mixture of inorganic substances as a powdering agent is made of water-repellent and water-absorbent Substances. This is to reduce the risk of explosion due to the dry, finely divided starch, avoided will.

From Swiss patent 389 505 it is known that the dispersion of the active ingredient in a cooled, gaseous Medium is sprayed in which the sprayed particles through Cooling solidify. For this process, heights of fall of up to required to 15 m, in addition, the temperatures must be clear be kept below room temperature.

Another method of solidifying the sprayed particles can also be made by collecting in a powder made from metal salts higher fatty acids. This procedure is used in the Swiss patent specification 431 252 described.

An alternative method for the production of active ingredient-containing, stable preparations is described in the European patent EP-A-0 618 001 described. This is where the spherical particles that embed the active ingredient in represent a mixture of different carrier substances, by first making beads through controlled division a primary oil-in-water emulsion that forms from the addition of active ingredients, oily substances, proteins and water for a water-immiscible solvent, and then separating the beads obtained. For the her A special mixing system is required to position the beads lich. The particles obtained in this way are then with an aldehyde, for example acetaldehyde, glutaraldehyde or Glyoxal post-treated, creating a chemical crosslinking that is also reflected in the water insolubility of the material obtained expresses, and thus an additional stabilization of the effect substance is reached.

In US Pat. No. 4,670,247, another Method for the production of crosslinked particles is described. For this purpose, first an emulsion, which essentially consists of a oil-soluble vitamin, a protective colloid such as. B. gelatin and a reducing sugar consists of a spray and dry tion process converted into powdery particles. These particles are then in a thermal process at temperatures treated between 105 and 180 ° C. In a Maillard reaction between the amino groups of the proteins and the oxo groups of the reducing sugar becomes insoluble in water Dry powder particles obtained by crosslinking the Matrix components is achieved.

In EP 782883 edible microcapsules are described which one The capsule wall is formed by salting out the protein with a edible salt and cross-linking of the capsule wall with transglutaminase getting produced. The disadvantage of this method is that they is not broadly applicable. So is the transglutaminase for example wise unsuitable for spray formulations as they sprayed and dried active ingredient formulations no longer properly transversely can crosslink and so the active ingredients already during storage are exposed to increased degradation. You wait until the cross crosslinking through the enzymatic activity is complete, so the formulations can no longer be sprayed.

If oxidation-sensitive compounds, in this case especially fat-soluble vitamins and carotenoids, with air come into contact, a reaction with oxygen takes place to a loss of active ingredient through conversion to undesired Ver ties leads. To prevent this oxidation, you can go to Example add certain additives to the preparations that by reacting with reactive groups of the proteins these in turn make it less permeable to oxygen and thereby the effect give fabrics a stabilizing protection. This can be done with for example done by converting proteins with reducing sugars in the sense of a Maillard reaction Solubility of the proteins in water is prevented. Farther can cross-link by reacting the proteins with aldehydes can be achieved, which also increases the carrier matrix Gives stability.

However, these methods show certain drawbacks to it appear desirable for improved stability to look for a method of reconciliation. So the application means one Maillard reaction between a protein and reducing Sugaring is in any case a thermal load and thus at least to a small extent a degradation of the active ingredient. In addition, the products tend to be brown in color. A Process for the formulation of active ingredients via the Maillard reaction can be found in patent application EP-A-0 547 422, for example to take.

Disadvantageous when using chemical agents such as Aldehydes or acids such as tannic acid are used as crosslinking agents, that for networking highly reactive and not unaffected by health Possible additives are used. The products that after These processes are produced, find with the consumer only conditional unrestricted acceptance.

It was therefore the object of the invention to be easy, cost cheap, broadly applicable process for the formulation of Develop active ingredients that have the disadvantages mentioned above does not have.

This object was achieved by the method according to the invention for Her provision of active ingredient preparations in which one or more Active ingredients are surrounded by at least one layer, thereby characterized in that at least one of the one or more active ingredients surrounding layers or parts of these layers from one Protein made with an enzyme selected from the group of lipoxygenases, protein disulfide isomerases, phenol oxidases and Peroxidases, lysyl oxidases, protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases was cross-linked, dissolved.

The chemical function of these enzymes in the metabolism or their chemical reaction is for example the writings of Green et al. (Biochem. J. 1983, 211, 481-493), Kagan et al. (Am. J. Respir. Cell Mol. Biol., 5, 1991, 206-210), Haywood et al. (Biochem. J., 1981, 199, 187-201) or Matheis et al. (J. Food Biochem. 11, 1987, 309-327).

Due to the advantageous enzymatic crosslinking in the fiction according to the method is both a thermal load of the Active ingredients as in the thermal crosslinking of the protein / Sugar layer and an associated tanning the active ingredient formulations about the Maillard reaction and its use reactive chemical substances such as aldehydes are avoided. the Maillard reaction in thermal crosslinking leads to a Linking of reducing sugars with the free amino groups of proteins or other free amino groups and thus to one he stabilization of the active ingredient formulations. When using of chemical compounds to form crosslinking proteins Bridges between the aldehydes are usually the dialdehydes and the amino groups of the proteins or other amino groups the end. In the method according to the invention, the proteins are direct for example above in the proteins or lipoproteins existing fatty acid residues, via cyctein-cysteine bridges (= cystine bridges), via Michael addition products or via the formation of Aldehydes from lysine with subsequent crosslinking via free Amino groups cross-linked.

This leads to the new active ingredient preparations according to the invention containing at least one one or more active ingredients vice versa layer or parts of a layer of a protein that with an enzyme selected from the group of lipoxygenases, Protein disulfide isomerases, phenol oxidases and peroxidases, Lysyl oxidases, protein disulfide reductases, tyrosine oxidases or Sulfhydryl oxidases was crosslinked. This invention Active ingredient preparations do not have any good stability brownish color and do not contain any chemical crosslinkers.

As active ingredients in the process according to the invention or in the Active ingredient preparations according to the invention are in principle all of them active in pharmacy, human or animal nutrition fabrics suitable. Preferred active ingredients are vitamins, enzymes, Food and feed additives.

Among enzymes in the process according to the invention or in the active Substance preparations are active ingredients such as enzymes. B. hydrolases such as amidases, proteases, lipases, esterases, phospholipases, β-glucosidases, amylases, nitrilases, mannanases, phytases or xylanases, transferases such as methyltransferases or amino transferases, oxidoreductases such as glucose oxidase or isomerases to be understood as an example.

In particular, hydrophobic active ingredients are preferred, particularly those which are easily oxidizable. Such are the vitamins of groups A, D, E and K and their mixtures, especially the mixtures with carotenoids and / or xanthophylls. In the context of the invention, they can be used in the form of vitamin solutions in oils, as provitamins and as pure vitamins of natural or synthetic origin. Preparations of vitamin D ₃ , vitamin K ₁ , vitamin A and its derivatives, in particular vitamin A acetate, vitamin A palmitate and vitamin A propionate, and mixtures thereof are of particular interest. The various vitamin E derivatives such as vitamin E acetate and vitamin E palmitate are also of interest. Preferred food and feed additives are carotenes, xanthophylls and carotenoids such as β-carotene and such. E.g .: astaxanthin, astacin, bixin, norbixin, capsorubin, violaxanthin, rubixanthin, phodoxanthin, apo-8'-carotenic acid ethyl ester, neurosporaxanthin, citranaxanthin, canthaxanthin, zeaxanthin, β-apo-4'-carotinal, β-apo-8'-carotinal carotinaL, β-apo-12'-carotinal, β-apo-8'-carotenic acid, lutein, capsanthin, lycopene and esters of hydroxy- and carboxy-containing representatives of this group, e.g. B. the lower alkyl esters such as methyl or ethyl esters or mixtures thereof.

The proportions of the active ingredients are in the inventive Active ingredient preparations generally about 1 to 75% by weight preferably 5 to 50% by weight, particularly preferably 10 to 35% by weight, based on the dry weight of the powder.

The proportions of vitamins or carotenoids are in all generally about 5 to 50% by weight, preferably 10 to 35% by weight, based on the dry weight of the powder.

Processes according to the invention are preferred in which an effective Substance-containing emulsion or dispersion in a water-repellent Silica-laden atmosphere which, if necessary, is rendered inert can be sprayed. The atmosphere can be beneficial also with another powdering agent such as an agent Based on a carbohydrate such as starch or modified starch be loaded for example corn starch.

In addition, methods are preferred in which after production the preparation of the active compound, for example by spraying it dry net is. It is preferred to have a residual moisture level below 10% by weight, preferably below 6% by weight, dried. Even lesser ones Residual moisture can be set.

The temperature of the process of the invention is essentially Lichen below 80 ° C, preferably below 60 ° C, kept.

The invention also relates to active ingredient preparations obtainable according to a method according to the invention, and those that are considered to additional feature 0.025 to 4 times the weight fraction with regard to active ingredient on release agents or release agent mixtures contain, as well as food or feed containing a such active ingredient preparation.

Preferred release agents are hydrophobized silicas, corn starch, corn starch made hydrophobic by chemical treatment, Metal salts of higher fatty acids and other vegetable starches or mixtures of these release agents. Are particularly preferred Mixtures of at least one of these release agents with others Auxiliaries such as inorganic compounds that act as lubricants act, such as Neusilin® or Zeolex®.

In the case of hydrophobic silica, the proportion is relative to Active ingredient preferably in a range between 0.025 and 0.4, particularly preferably between 0.05 and 0.2. When corn starch lies the corresponding ratio preferably between 0.25 and 2, particularly preferably between 0.5 and 1.5.

The active ingredient preparations according to the invention are available by preparing a dispersion containing essentially these active ingredients, a protein and other carriers and fillers z. B. from the group of carbohydrates and / or natural or chemically modified starches. You can add other additives such as stabilizers or emulsifying agents. aside from that it contains an enzyme that is able to act on various Way to link protein molecules together. The result achieved crosslinking gives the protein and thus also the Matrix in which the active ingredients are embedded, a diminished Water solubility and thus increased stability.

As a protein in the method according to the invention, in principle all proteins are used. Preferred crosslinkable proteins are for economic reasons gelatine, casein, soy protein, Wheat proteins, corn protein and collagen.

Preferred crosslinkable proteins are all forms of vegetable or animal proteins such as gelatin, e.g. B. bone gelatin, Beef gelatin, fish gelatin, milk proteins, such as. B. Casein, Soy proteins, wheat proteins such as gluten, corn proteins and Collagens, particularly preferred proteins are gelatine, milk proteins and soy proteins. Among gelatin in the invention Process means all forms of gelatin, no matter whether it is natural gelatins or chemical modified derivatives.

For the preparation of the dispersion is in the invention Process advantageous gelatin of the A and B type in one wide Bloom area used. It is particularly advantageous to work with Gelatin with a Bloom of about 50 to about 250.

The gelatin is used according to the invention in amounts of 10 to 50% by weight, preferably 15 to 40% by weight, in particular 20 to 35% by weight, based on the dry mass of the active ingredient preparation.

The proteins are advantageous for mechanical stabilization Plasticizers such as sugar and / or sugar alcohols such as sucrose, Glucose, sorbitol, sorbose, mannitol or polyols such as glycerin are added puts.

The crosslinking enzymes according to the invention are selected from Group of lipoxygenases, protein disulfide isomerases (preferred E.C. class 5.3.4), phenol oxidases (preferably E.C. class 1.14.) and peroxidases (preferably E.C. class 1.11), lysyl oxidases (preferably E.C. class 1.4.3), protein disulfide reductases (before assigned to E.C. class 1.6.4), tyrosine oxidases (preferably E.C. class 1.14) or sulfhydryl oxidases (preferably E.C. class 1.8.). These can be of animal, vegetable or microbial origin be. They are preferably of microbial origin, that is to say from Eukaryotes such as fungi or yeasts or prokaryotes such as gram positive or gram-negative bacteria or archaebacteria. The various lysyl oxidases are preferred as the enzyme Used of origin. The lysyl oxidases are particularly preferred from yeasts such as from the genera Candida, Hansenula, Pichia, Sporobolomyces, Sporopachydermia, or Trigonopsis are used. Lysyl oxidases from the genera and are very particularly preferred Species Candida nagoyaensis, Candida nemodendra, Candida boidinii, Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Hansenula minuta, Hansenula polymorpha, Pichia pinus, Pichia pastoris, Sporobolomyces albo-rubescens, Sporo pachydermia cereana or Trigonopsis variabilis. Outstanding Lysyl oxidase is suitable for the method according to the invention from the genus and species Pichia pastoris.

The Pichia patoris lysyl oxidase according to the invention can be from the cell dimensions of Pichia pastoris cells via a cell digestion, which was carried out with conventional methods, one Ion exchange chromatography with a subsequent molecular drill chromatography and finally repeat ions Purify exchange chromatography. With this cleaning scheme could the lysyl oxidase with a purity of over 90% before added by more than 95%, particularly preferably shown by more than 99% will.

The purified lysyl oxidase was subjected to protein sequencing after Edman subjected. This was the N-terminal end as well various peptides obtained in a digestion with trypsin were determined (see Example 5).

The invention thus relates to an isolated protein which contains at least one of the following sequences:

an amino terminal sequence

G (S) (C) Q (C) KTNEKVNIEAPKPNI (C) DT (L) (S)

or partial sequences corresponding to peptides of the protein obtained after digestion with trypsin:

EYP (C) APGVVYNTK
GGTYSTVTQNPTLNR
DYNIMPGGGXVHR
ATGGTYSTVXAQN
APETENNAR
GPL (P) VNE (E) TTIEPLSFYNT
IYELSLQELIAEYGSDDPNNQHTFYSDI
DNVDDLSCTIIQR
VAPETENCAR
NVDVEYPCAPGVVYNTK
GYPNAEY (S) LDF / ER

and that the ε-amino groups of lysine especially in a protein can oxidize to aldehyde groups. The letters and characters in the above sequences have the following meaning: X is a unknown amino acid not identified at this position could be, the brackets do not mean clearly determinable Amino acids, being the specified amino acid that is likely correct amino acid is, the slash means amino acid alternatives, with the amino acid before the slash or the after the slash at the position can be correct. By the oxidation of the ε-amino groups finally comes to a cross cross-linking of proteins, with areas of the same protein can be linked to each other or different proteins.

The lysyl oxidase (= PROTEIN-LYSINE 6-OXIDASE, EC 1.4.3.13 or LYSYL OXIDASE) especially the lysyl oxidase from Pichia pastoris enables the enzymatic cross-linking of proteins, whereby gelatine is advantageously used as the protein. The ε-amino groups of the lysines are oxidized to aldehyde groups. These aldehydes can then react with the amino groups, other lysines or other free amino groups, for example amino sugars, to form Schiff bases. A particular technical advantage for the user is the time separation within the entire reaction sequence, that is, the formation of the aldehydes from the ε-amino groups of the lysines and the reaction of the amino groups with the aldehyde groups formed is separated in time. Compared to, for example, enzymatic crosslinking with the aid of transglutaminase, this offers a significant advance, since in this reaction the crosslinking always takes place immediately and therefore no temporal separation in the enzyme reaction and the crosslinking is possible. This temporal separation enables the advantageous use of the enzymatic reaction for processes such as, for example, the production of active ingredient preparations via spray drying or via the so-called micro-nization process (see EP-A-0 065193). Another advantage is the harmlessness of the molecules formed, as they are a natural component of the connective tissue ( Fig. 1 and 2). There they are produced by a tissue lysine oxidase.

Fig. 1

Formation of an aldol cross bridge from two lysine side chains

The enzymes mentioned can be in the form of the purified enzymes or in the form of raw extracts from natural sources such as from Eukaryotes such as fungi, yeast, animal or vegetable Cells or prokaryotes such as gram-positive, gram-negative Bacteria or archaebacteria can be used. Even whole Organisms or cells can be used for the method according to the invention be used as long as the enzymes enter the extracellular environment be secreted or the cells have become permeabilized. Before The method according to the invention with purified enzymes is added or, as long as there are no undesirable side activities, performed with crude extracts.

The amount of enzyme needed to cross-link the proteins or activities can be carried out in a conventional manner in the Determine a person skilled in the art by means of simple preliminary tests. Usually the enzymes are added in an amount that 20 to 100% of the crosslinkable groups, for example the Lysine residues in the protein are linked. Advantageously be 0.001 to 1000 units of enzyme per gram of protein, preferably 0.01 to 100 units of enzyme per gram of protein, particularly preferred 0.1 to 10 units per gram of protein added.

The active ingredient preparations can contain one or more active ingredients Contain different classes of active ingredients such as vitamins or carotenoids or several active ingredients of an active ingredient class for example several different carotenoids or vitamins or mixtures thereof can be contained in the preparations.

The active ingredients in the preparations can be of one or be surrounded by several layers. These layers can be made from one or more proteins crosslinked according to the invention layers or of one or more transversely according to the invention crosslinked protein layers and at least one chemically cross-linked protein layer and / or further layers consist of natural or synthetic polymers. The after Processes according to the invention cross-linked proteins can also Share at least one layer surrounding the active ingredient (s) be.

Among natural and / or artificial polymers are in principle all polymers conceivable for the formulation of active ingredients are suitable. As natural proteins, for example Polymers such as guar gum, alginates, carrageenan, pectins or Polymers based on mannose and / or galactose called. as artificial proteins are, for example, polymers based of acrylic acid, methacrylic acid, acrylates, methacrylates or Mixtures of these monomers such as, for example, Eudragit L30D-55 (methacrylic acid: ethyl acrylate, 1: 1) or Eudragit S (Methacrylic acid: methyl methacrylate, 1: 2). Other monomers too can be contained in the polymers, for example monomers, that have a positive charge like trimethylammonioethyl methacrylate, polylysine or polyallylamine. Depending on the used Polymer or polymer mixture can be with the help of the fiction produce appropriate layers that allow the advantageous or to formulate the active ingredients so that they are targeted at the site of action can be released. This is how layers can be created for example, are resistant to gastric juice that dissolves in the intestine sen, which dissolve in a targeted manner in the stomach, caused by the rumen leu do not dissolve or which when introduced into the body release the active ingredient after a while. In feed or Food, the active ingredient can be formulated so that it release only after the active ingredient preparation has been absorbed into the body is set.

The active ingredient or the active ingredients of one is advantageous or more crosslinked via the method according to the invention Surrounding protein layers.

The emulsions containing the active ingredient preparations are advantageously using hydrophobic release agents such as hydrophobic silica, natural starch, such as B. corn starch, hydrophobized starch derivatives, such as. B. hydrophobized corn starch, salts of long-chain fatty acids or mixtures sprayed on these substances. The release agent can also be used in the Spray chamber z. B. as hydrophobic silica particles in air or Inert gas (e.g. nitrogen) must be presented. Then takes place the drying of the sprayed particles, if necessary, afterwards Separation of the release agents, if necessary under easier Heating up to 80 ° C, preferably up to 60 ° C, particularly preferably at Room temperature, by treatment in an air or protective gas current.

In addition to the mandatory components, the Dispersion with advantage still others, in the manufacture of Compounds common to the active ingredient dry powders, added.

Fig. 2

Reaction of the lysine residues with allysine to form Schiff's base and further reaction to lysine non-leucine, desmosine and isodesmosine

Of particular importance for a use of the dry powder as Feed additive is for active ingredients that are sensitive to oxidation an addition of antioxidants, such as ethoxyquin, butylated Hydroxytoluene (BHT), butylated hydroxyanisole (BHA) or Tocopherol, as well as stabilizers such as ascorbyl palmitate, mono- and diglycerides, esters of monoglycerides with acetic acid, Citric acid, lactic acid, diacetyltartaric acid, polyglycerol fat acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, Stearoyl-2-lactylate, phosphoric acid or phytic acid and their Alkali or alkaline earth salts, or complexing agents, such as Ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid (NTA).

Often the emulsion but also humectants, such as Glycerine, sorbitol, polyvinylpyrrolidone or polyethylene glycols or additional emulsifiers such as lecithin are added.

In addition, additives such as carbohydrates such as sugar, Starch or starch derivatives, in particular corn starch or malto dextrin or thickeners, such as gum arabic, guar gum, Tragacanth, agar agar, carrageenan, locust bean gum, alginate, Pectins, xanthan gum, curdlan and certain degraded starches for Adjustment of the viscosity of the emulsion has been found useful.

For more details on the meaning, type and amount of such For additions, we refer to the relevant specialist literature, for example refer to the above-mentioned monograph "Fat-soluble Vitamins", Vol. 9, in particular pages 128 to 133.

These additives make the active ingredient preparations advantageous stabilized and adapted to the special administration conditions fits.

To carry out the process according to the invention, for example, the procedure is such that, to prepare the dispersion containing the active ingredients, gelatin is dissolved in hot water (from 50 to 70 ° C.), and the sugars, the amino compounds, the vitamins and the solution are added to this solution / or carotenoids, stabilizers and the other customary additives and, if necessary, additional water are added and the whole is dispersed by vigorous stirring at elevated temperature. For the enzymatic crosslinking of the powder in the last processing step, the finished dispersion should be in a pH range of 4 to 10, which can be achieved by adding bases such as NaOH, KOH, Ca (OH) ₂ , MgO, soda or NH ₄ OH or can be adjusted by adding basic salts such as sodium acetate or Na ₂ HPO _4. The dispersion prepared in this way can be converted into a dry powder by customary methods, for example by spray drying, spray granulation or other customary drying methods.

Further embodiments of the method according to the invention are for example the use of enzymatic cross-linking with the different micronization processes.

In EP-B-0 065 193, for example, a method for Production of very finely divided free-flowing powders the carotenoids described, the carotenoid in one volatile, water-miscible organic solvent at temperatures between 50 ° C and 200 ° C, optionally below increased pressure, within a time of less than 10 seconds is dissolved and then the molecularly disperse obtained in this way Solution immediately by rapidly mixing with an aqueous solution a swellable colloid at temperatures between 0 ° C and 50 ° C the active ingredient precipitates in colloidal form. The dispersion is then removed from the solvent and the dispersing medium in usual way exempted. Proteins are used as swellable colloids used. This can be done by adding the enzymes mentioned above are crosslinked in the process according to the invention. Advantage The enzymes of the aqueous micronisate solution are liable to be present added to the drying step so that there are enough reactive groups for crosslinking to be made before drying he follows. Further information on the process can be found in EP-B-0 065 193 remove.

EP-B-0 410 236 describes another method of manufacture a colloid-dispersed carotenoid preparation, with a suspension sion of a carotenoid in a high-boiling oil during a Duration of maximum 30 seconds with superheated steam is brought into contact. Instead of the oil for loosening the Carotenoids can also contain organic solvents such as chloroform, Methylene chloride, carbon tetrachloride or trichlorethylene ver (see DE / OS 12 11 911). Then he will hold mixture with an aqueous solution of a protective colloid emulsified and the emulsion then sprayed and dried. Here, too, proteins are used as protective colloids, which are im Process according to the invention via the addition of the enzymes mentioned can be crosslinked prior to mixing and spraying. Further information on the process can be found in EP-B-0 410 236.

EP-B-0 498 824 describes an additional advantageous ver drive for the production of microdisperse active ingredient preparations. A carotenoid in the presence of a carotenoid is used as an active ingredient Protective colloid of a protein such as gelatin, for example ground and the suspension formed is then dried.

By adding those used in the process according to the invention Enzymes can be used to crosslink the proteins and the Stabilize active ingredient preparation. Details of the manufacturer procedures are to be found in the patent specification mentioned and WO 94/19411 remove.

The subsequent processing of the dispersion to form the Powders according to the invention can be according to all known from the literature Procedure.

Because of the desired particle size distribution of the powder (0.1 to 0.6 mm Diameter) those methods are preferred in which Vor Precautions are taken that the gelled droplets of the Dispersion remain separate from each other until their shape changes has stabilized.

For example, the method according to EP-B1-74050 may be mentioned which the dispersion in hydrophobic silica or a metal salt a higher fatty acid is sprayed or the process according to EP-B1 285 682, in which the dispersion in starch powder is sprayed. It has been shown that in particular Process used for preparations with gelatin contents below 35 wt .-% are used, the spray with hydrophobized Silicas can be carried out particularly advantageously as powdering agents is.

Have the powders produced by the methods described after drying a water content of less than 10%, usually less than 6%. The powdery Preparations consist of particles with a well-developed surface. They quickly dissolve into one in warm water of approx. 40 ° C milky dispersion.

The active compound preparations according to the invention are advantageously suitable liable for the production of pharmaceutical preparations, as well as for Manufacture of food and feed.

Examples example 1 Production of lysyl oxidase with Pichia pastoris - Lu 583 -

The strain was cultivated on YM agar:

3 g / l Malt Extract Difco 5 g / l Peptone Difco 3 g / l Yeast Extract Difco 20 g / l Agar Difco

The plates were incubated at 28 ° C. for 48 h and are thereafter Stable for at least 4 weeks at 45 ° C.

Preculture and fermentation medium

10 g / l glucose (autoclave for 30 min at 121 ° C)
prepare separately and autoclave for 30 min at 121 ° C:

0.2 g / l magnesium sulfate × 7 H ₂ O
3.0 g / l potassium dihydrogen phosphate
0.2 ml / l saline solution according to Vishniac & Santer *

prepare separately and filter sterile:

10 ml / l vitamin solution acc. To Lodder **

The medium components are combined after sterilization.

* Formulation of the saline solution. According to Vishniac & Santer

50 g / l Titriplex III
22 g / L zinc sulfate x 7 H ₂ O
5.54 g / l calcium chloride
5.06 g / L manganese chloride x 4 H ₂ O
4.99 g / L iron sulfate x 7 H ₂ O
1.1 g / l ammonium heptamolybdate × 4 H ₂ O
1.57 g / l copper sulfate × 5 H ₂ O
1.61 g / L cobalt chloride x 6 H ₂ O

Adjust to pH 6.0 with KOH

** Formula of the vitamin solution according to Lodder

20 µg / l biotin
20,000 µg / l calcium pantothenate
2 µg / l folic acid
10,000 µg / L inositol
200 µg / l p-aminobenzoic acid
400 µg / l niacin (nicotinic acid)
400 µg / L pyridoxine hydrochloride
200 µg / L riboflavin
400 µg / l thiamine hydrochloride

Preculture

For a 10 l fermenter, 2 × 250 ml preculture (= VK) were im 1 l Erlenmeyer flask (= EMK) with 3-4 Lu 583 platinum eyelets each inoculated and incubated for 24 h at 28 ° C and 200 rpm.

Main culture

Preparation in the 10 l Infors fermenter with the following conditions:

Temperature: 28 ° C
Ventilation: 5 l / min. via ring gassing
Speed: 400 rpm.
Fermenter equipment: 3 × standard stirring blades and built-in breakwaters

Before the start of the fermentation was by the addition of 50% (V / V) n-butylamine solution in water, the pH is adjusted to about 7.0. The fermenter was then precultured with 2 × 250 ml inoculates. The fermentation time was about 28-30 hours. The time point of the end of fermentation was determined by determining the OD at 600 nm set. The OD 600 of the fermenter samples was thereby measured against air and should be 0.9-1.0. At higher OD rapidly decreases the activity of the enzyme.

Work-up

The fermenter contents were in a Hereaus Cryofuge 8000 at 45 ° C and 5000 rpm (corresponds to approx. 9500 × g) centrifuged for 20 min. the The entire wet biomass was dissolved in 250 ml of 50 mM potassium phosphate buffer washed and centrifuged again. The cell mass can be stored at -15 ° C be stored in the freezer.

Cell disruption and measurement of lysyl oxidase

The wet biomass (100 ml) was mixed with 100 ml glass beads (diameter knife 0.5 mm) in the ball mill with ice cooling for 30 minutes crushed at 5000 revolutions per minute. The homogenate was filtered through cheesecloth and then for 10 minutes at 10 000 rpm and Centrifuged 4 ° C. The activity of the supernatant was determined by an HPLC test. The lysyl oxidase converts benzyl amine in benzaldehyde (detection at 250 nm). The amount of Benzaldehyde formed was determined using a calibration curve.

Running conditions

Buffer A: water, 0.1% TFA
Buffer B: acetonitrile, 0.1% TFA

0 minutes 40% B
6 minutes 70% B
6.1 minutes 100% B
6.5 minutes 100% B
Flow 1 ml / min
6.5-9 min back to 40% B, flow 1.5 ml / min

The lysyl oxidase containing supernatants from 7 x 10 l fermenters were collected and pooled. An activity of 125.5 U / l in 1.8 l.

Example 2 Marking the gelatin with fluorescent dyes and cross cross-linking of the gelatine by the lysyl oxidase

Gelatin (10 g) was dissolved in 50 mM sodium borate buffer pH 9.0. The pH was checked and adjusted _{with 0.5 M NaHCO 3.} The fluorescent dyes Cy-5 or Bodipy 630/650-X, SE) were dissolved in 5 ml of DMSO (= dimethyl sulfoxide) and added to the proteins in a weight ratio of 1: 100. The succinimidyl esters of the dyes reacted with the free amino groups of the proteins for 16 hours at room temperature. The reaction batches were then dialyzed 5 times against large volumes of 20 mM sodium phosphate buffer with 0.01% azide and stored as a 1% protein solution at -20 degrees Celsius.

In a 4 l stirred flask were in 1800 ml of a solution, consisting from pooled supernatants from lysyl oxidase fermentation solutions (ZH 29303/5; 125.5 U / l) at 40 ° C 454 g gelatine A 100 Bloom brought into solution by stirring. This was done with Bodipy doped gelatin (see above) given. The whole approach was made Stirred for 48 h at 40 ° C, whereby the flask was not closed, about permanent air access and thus the access of Sauer fabric to enable.

300 g samples were taken from the solution at the specified time intervals:

Sample 1: immediately after the start of the experiment
Sample 2: after 2 hours
Sample 3: after 5 hours
Sample 4: after 24 hours
Sample 5: after 29 hours
Sample 6: after 48 hours

The samples were processed as follows:

• a) 200 g were poured directly into a cloud with a single-fluid nozzle from hydrophobized silica (Sipernat D 17) to particles sprayed about 200 microns in diameter. The obtained moist Product was shared; one half was at room temperature and the other at 80 ° C each on a suction filter in the air electricity dried.
• b) After adding vitamin A, Isosweet and corn starch, after emulsifying the oil phase, the remaining 100 g were sprayed in the same way with an Ultraturrax device and converted into a dry powder by drying the spray product at room temperature. These products had roughly the following composition:
  25% vitamin A acetate stab. with ethoxyquin and BHT
  30% gelatin A 100 Bloom
  25% corn starch
  15% isosweet
  Remainder: water + Sipernat D 17 + residues from fermentation residue

Example 3 Measurement of cross-linking by fluorescence correlation Spectroscopy (= FCS)

The FCS is a method based on number fluctuation spectroscopy. With the FCS, diffusion coefficients and number concentrations of fluorescent molecules can be measured in highly dilute solutions. Figs. 1 and 2 show the structure of an FCS apparatus and its operating principle. A laser beam is focused into a very small volume of a liquid sample using a microscope objective and the fluorescence excited in it is measured.

Fig. 1 shows an FCS apparatus. The focused laser beam and the confocal diaphragms define a very small observation volume (the confocal volume) in the sample. The time fluctuations of the fluorescence from this volume are analyzed with a correlator.

Fig. 2: (= number fluctuations in the observation volume lead to fluctuations in the FCS signal I (t). The mean residence time τ (= mean time for the diffusion of a particle through the observation volume ) and the number of molecules N in the measuring volume can be read off. τ is directly proportional to the size of the fluorescent molecules.

The volume is so small that on average there are only a few particles in it. The number of particles in the volume fluctuates constantly around a mean value N due to diffusion. This means that the fluorescence intensity I (t) also fluctuates around a mean value Im. The diffusion time τ (proportional to the molecule size) and the number concentration of the fluorescent Molecules calculated (see Fig. 1).

Determination of the molecular weights by gel permeation chromatography graphics (GPC)

The molecular weight distribution (MGV) was determined by means of GPC. The most important test parameters:

Column: TSK Gel, 4000 SWXL, 250.4 mm
Mobile phase: 0.01 M NaH ₂ PO ₄ , 0.1 M Na ₂ SO ₄ , 1% SDS
pH: 5.3
Temperature: 30 ° C
Flow: 0.5 ml / min
Detection: UV spectrometer, 220 nm

To prepare the sample, approx. 0.5 g of substance in 100 ml of mobile phase were stirred at T = 60 ° C. for 15 min. The so he The remaining cloudy solution was filtered through a 0.2 µm filter and then injected into the sample loop of the GPC device (approx. 20 µl). Water-insoluble gelatin ingredients could cannot be detected.

With this method, the intensi was obtained as a direct measured variable ity signal of the detector (measure of the concentration) as a function the elution time. At GPC, the molecules were named after their hydrodynamic radius rH separated. Molecules with a large rH are eluted faster than molecules with a small rH.

By means of a calibration with standard polymers (here: Pullul standards) it was possible to calculate MGV from the elution diagrams [see MD Lechner, K. Gehrke and EH Nordmeier, in: "Makromolekulare Chemie", Chapter 4.3.6.1., Birkhäuser Verlag (1996 )]. As a first approximation, the following proportionality applies between the elution time t _E and the molar mass M of the Pullul standards:

t _E ∞ log (M) (1)

However, when plotting t _E vs. log (M), the data can be described more precisely with a third-degree polynomial. Gelatin molecular weights can be calculated from the gelatin elution times from the inverse of this polynomial. The molecular weights obtained in this way are therefore not absolute gelatin molecular weights (as can be obtained, for example, from the LS), but only relative molecular weights (based on the standards used here). It should also be noted that, due to the pullul standards available, the elugrams can only be reasonably converted into the corresponding molecular weights ^{in the range of 12 min <t <25 min (10 3} g / mol <MW <10 ^{7 g / mol)} . With a semi-logarithmic plot (t _E vs. log (M)), the area under the curve is approximately proportional to the amount of substance examined. The elution diagrams were normalized to the same total areas, so that the areas under the curves - and thus the respective substance quantities - could be compared directly with one another.

The following observations were made: With increasing duration of the incubation of the labeled gelatin with the unlabeled gelatin and the lysyl oxidase, the value τ (proportional to the molecule size) became smaller and smaller during measurements by the FCS and at the same time the intensity (I, kcps) of the fluorescence decreased (Table 1, Fig. 3).

Furthermore, a decrease in the high molecular weight gelatin content was observed in the GPC with increasing incubation time (Table 2, FIG. 4).

By incubating the gelatin with lysyl oxidase, the Gelatin cross-linked. These crosslinked parts could be in water can no longer be completely resolved, but rather lay as ge swollen gel particles. For FCS and GPC sample preparation these particles were removed by centrifugation or filtration separates and could no longer be detected.

With increasing incubation time the gelatin disappeared before adds the high molecular weight gelatine components from the Elugram. this means that just the high molecular weight gelatin shares over are proportionally affected by the networking. From statistical This behavior makes sense for reasons. Furthermore but could also have a molecular weight-dependent distribution of the Lysine groups may be responsible for this finding.

The decrease in τ by FCS measurements is called the decrease in the mole to interpret the molecular weight and was able to use gel permeation Chromatography can be shown directly. The decrease in intensity is due to the decrease in the number of fluorescent particles in excess was due to the redissolved preparations. Through cross servers wetting, large gelatin molecules were more likely affected as small molecules. In addition, small moles cool the network created by the action of the lysyl oxidase leave better than uncrosslinked, large gelatin molecules that are in the insoluble part remain.

It could thus be shown that by the lysyl oxidase a for a cross-linking sufficient number of allysines and later Schiff bases are formed.

Example 4 Proof of cross-linking by means of a fast, parallelizing test in microtiter plates

Gelatin was biotin activated with 1 mM / l N-hydroxysuccinimide labeled at pH 8.4 in a borate buffer and then against dialyzed several times a 20 mM phosphate buffer pH 7.4. The capable the modified gelatin, avidin or streptavidin bind, could be shown in preliminary tests. 10 and 100 mg / ml After biotin modification, gelatins bind to those with avidin modes fied surface of a Biacore sensor chip. Not modified Gelatin was not bound to this surface.

Test principle and implementation

Gelatin was adsorbed on the surface of microtiter plates beer. After that, biotin-labeled gelatin and the cross-server wetting substance / enzyme added. Adsorbed gelatin was cross-linked with biotinylated gelatin. After an intense Washing step, the biotinylation was carried out by a streptavidin Peroxidase complex detected.

0.4 ml of a 1% gelatin solution in 0.1 M NaHCO ₃ , pH 9.2 was adsorbed on NUNC MaxiSorp Elisa plates for 3 hours at room temperature. It was then washed three times with PBS, 0.05% Tween 20 solution.

After that it was biotinylated gelatin and in different Concentrations of crosslinkers added. This was in one PBS buffer with 5 mM DTT and 0.1% Tween 20 performed. the Microtiter plates were then placed for various times incubated at different temperatures. After that, the Plate washed 6 times to remove unreacted material distant.

Streptavidin peroxidase from the Boehringer company was used for detection 1: 10,000 in PBS, 0.1% Tween 20 diluted and 1 ml added per well give. It was again 30 minutes at room temperature (approx. 23 ° C) incubated. After a further washing step, 0.015 ml of reac tion mixture (0.1 ml 42 mM tetramethylbenzidine in DMSO, 10 ml 0.1 M sodium acetate, pH 4.9 adjusted with citric acid) added give. A blue color solution formed over the course of the reac tion. The reaction was stopped after 5 minutes with 0.1 ml of 2 M sulfur acid stopped. The blue color turned yellow. Afterward the absorption was measured at 450 nm.

Example 5 Purification of lysyl oxidase Homogenization

The cells of Pichia pastoris (approx. 18 ml) were after storage thawed at -20 degrees C and treated with buffer A (20 mM sodium phosphate, 1 mM ethanolamine, pH 7.0 diluted to 50 ml. There were 50 ml glass beads, diameter 0.5 mm, are added and 30 minutes homogenized at 5000 rpm while cooling with ice. The homo genat was filtered through cheesecloth. The filtrate was at 10 minutes Centrifuged 8000 rpm and 4 degrees C.

Ion exchange chromatography

The supernatant was readjusted to pH 7.0 with NaOH and applied to a Q-Sepharose (Q-Sepharose Fast Flow, Pharmacia, diameter 5 cm, height 13 cm, volume 250 ml). To the application (conductivity 0.7 mS / cm, 540 ml) was with 600 ml Buffer A washed. The column was created with a linear gradient of 1 l buffer A and 1 l buffer B (like buffer A with 1 M NaCl) eluted. The active fractions were collected.

Molecular sieve chromatography

The desired fraction was concentrated over a 10 kDa Omega filter on a preparative Superdex column (Pharmacia, Diameter 2.6 cm, length 60 cm, volume 320 ml) in 20 mM sodium phosphate buffer, 150 mM NaCl, 1 mM ethanolamine, pH 7.5 separately. Flow 3 ml / minute. The active fractions were collected.

Ion exchange chromatography

The fraction of value of the molecular sieve chromatography was again purified by chromatography on a Mono-Q column (Pharmacia, HR5 / 5). The gradient was generated from buffer A and buffer B. Flow rate 1 ml / minute, 100 fractions of 1 ml. The Lysyl oxidase eluted as the major active protein. The protein had a molecular weight in the SDS gel under reducing conditions of approx. 121,000 Da.

The following sequences were obtained:

Amino terminal sequence

G (S) (C) Q (C) KTNEKVNIEAPKPNI (C) DT (L) (S)

Sequences corresponding to peptides of the protein obtained after digestion with trypsin:
EYP (C) APGVVYNTK
GGTYSTVTQNPTLNR
DYNIMPGGGXVHR
ATGGTYSTVXAQN
APETENNAR
GPL (P) VNE (E) TTIEPLSFYNT
IYELSLQELIAEYGSDDPNNQHTFYSDI
DNVDDLSCTIIQR
VAPETENCAR
NVDVEYPCAPGVVYNTK
GYPNAEY (S) LDF / ER

Table 1

Table 2

Claims (20)

1. A process for the production of active ingredient preparations in which one or more active ingredients are surrounded by at least one layer, characterized in that at least one of the layers surrounding the active ingredient (s) or parts of these layers consist of a protein which is selected from the with an enzyme Group of lipoxygenases, protein disulfide isomerases, phenol oxidases and peroxidases, lysyl oxidases, protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases. 2. Process for the production of active ingredient preparations according to Claim 1, characterized in that the one or more active fabrics or one or more with at least one layer surrounding active ingredients in solution with crosslinkable protein and Enzyme for coating is mixed, taking the weight ratio between active ingredient and protein between 1 to 100 and 5 to 1. 3. The method according to claim 1 or 2, characterized in that as an active ingredient vitamins, enzymes, food additives or Feed additives are used. 4. The method according to claims 1 to 3, characterized net that the active ingredient is hydrophobic. 5. The method according to claims 1 to 4, characterized net that a crosslinkable protein selected from the group Gelatin, casein, soy protein, wheat protein, corn protein or collagen is used. 6. The method according to claims 1 to 5, characterized net that an enzyme obtained from microorganisms is used will. 7. The method according to claims 1 to 6, characterized net that the cross-linking with the enzyme lysyl oxidase by to be led. 8. The method according to claims 1 to 7, characterized net that you can at least one active ingredient with an aqueous Solution of crosslinkable protein and enzyme mixed and sprayed. 9. The method according to claim 8, characterized in that in one with hydrophobic silica, corn starch or hydro phobic cornstarch-laden atmosphere is sprayed. 10. The method according to claims 1 to 9, characterized net that the active ingredient preparation to a residual moisture is dried below 10 wt .-%. 11. The method according to claims 1 to 10, characterized net that the temperature in the manufacture of the active ingredient preparation is kept essentially below 80 ° C. 12. Active ingredient preparation obtainable by a method according to claims 1 to 11. 13. Active ingredient preparation containing at least one or layer or parts of a layer surrounding several active substances from a protein made with an enzyme selected from the Group of lipoxygenases, protein disulfide isomerases, phenol oxidases and peroxidases, lysyl oxidases, protein disulfide reductases, tyrosine oxidases or sulfhydryl oxidases t.s. has been networked. 14. Active ingredient preparation according to claim 13, wherein the active ingredients are selected from the group:
Carotenoids
Xanthophylls
Vitamin A
Vitamin E.
Vitamin D ₃
Vitamin K ₁ 15. Active ingredient preparation according to claim 13 or 14, which the 0.025-fold to 4-fold weight proportion with regard to active contains substance of release agent or release agent mixtures. 16. Active ingredient preparation according to claims 13 to 15, wherein the active ingredient content in the active ingredient preparation between 1 to 75% by weight. 17. An isolated protein that contains at least one of the following amino acid sequences
G (S) (C) Q (C) KTNEKVNIEAPKPNI (C) DT (L) (S)
EYP (C) APGVVYNTK
GGTYSTVTQNPTLNR
DYNIMPGGGXVHR
ATGGTYSTVXAQN
APETENNAR
GPL (P) VNE (E) TTIEPLSFYNT
IYELSLQELIAEYGSDDPNNQHTFYSDI
DNVDDLSCTIIQR
VAPETENCAR
NVDVEYPCAPGVVYNTK
GYPNAEY (S) LDF / ER
and which can oxidize the ε-amino groups of the lysine to aldehyde groups. 18. Use of an enzyme selected from the group of Lipoxygenases, protein disulfide isomerases, phenol oxidases and peroxidases, lysyl oxidases, protein disulfide reductases, Tyrosine oxidases or sulfhydryl oxidases for formulation of active ingredient preparations. 19. Food or feed containing an active ingredient Preparation according to claims 13 to 16. 20. Pharmaceutical preparation containing an active ingredient Preparation according to claims 13 to 16.