SE461101B - USE OF STEMS FROM THE GROUP BACILLUS FOR THE EXTRACTION OF SUCCESS INHIBITORS AND WAY TO MANUFACTURE SUCCESS INHIBITORS - Google Patents

Description

461 101 can be used for the control and regulation of carbohydrate metabolism in humans and animals by inhibiting sucrose by means of these active substances.

To obtain suitable strains, the methods described below are used.

From soil samples, strains from the family Bacillaceae, in particular those from the group Bacillus, are isolated in a known manner. With inoculations from these strains, culture flasks are inoculated with nutrient solutions, which enable the growth of these strains. One can e.g. use a nutrient solution that contains S g peptone and 3 g meat extract per liter, but in principle many other types of nutrient solutions are usable, which contain suitable carbon and nitrogen sources and nutrients. The pH value of the nutrient solution can be varied within wide limits. Preferably, a starting pH in the nutrient solution is chosen between 6.0 and 8.0.

As a carbon source in the nutrient solution, the most diverse organic substances come into question. Carbohydrates, organic acids and alcohols are mentioned as examples.

As a source of nitrogen, yeast extracts, soy flour, peptones, meat extracts and many other organic substances can be used.

The concentration of carbon and nitrogen sources as well as the nutrients, of which FeSO4, CaCO3 and MQSO4 are specifically mentioned, can be varied within wide limits. The separate addition of nutrients can in many cases be omitted, as these often occur as additives in the complex nitrogen sources.

Since the formation of inhibitors often depends strongly on the composition of the industry, it is appropriate to grow the strains in different nutrient solutions to optimize production performance. Corresponding proposals are shown in the examples.

The nutrient solution is filled with e.g. 100-200 ml in a 1-liter Erlenmeyer flask, sterilize in a known manner, inoculate with the strain to be examined and grow the flask at 15-80 ° C, preferably at 24-40 ° C resp. about 70 ° C, for thermophilic bacilli 1 ekekmaekiner. if the culture shows growth, which can generally be seen after 1-10 days, usually after 1-5 days, a sample of e.g. 5 ml and separates the cells in this sample by filtration or centrifugation. 1-100 microliters of the culture solution are used in the tests described below, and the inhibitory capacity is calculated per ml.

The cells are extracted with 2 x 5 volumes (calculated on the cell volume) of acetone and then with 1 x 5 volumes of diethyl ether. The combined extracts are evaporated to dryness, taken up in water and lyophilized.

The lyophilisate is charged at a concentration of 10-1000 pg / ml in the tests described below.

Sucrase test A sucrase inhibitor unit (SIE) is defined as the amount of inhibitor that 50% inhibits two sucrase units. A sucrose unit (SE) is the amount of enzyme which, in 1 minute under specified test conditions, breaks down 1 pmol of sucrose into glucose and fructose. The number of pmoles of glucose formed is determined quantitatively by means of the glucose oxidase reaction under conditions in which a further sucrose cleavage by the sucrose does not take place. To perform the test, add 0,05 ml of a sucrose solution adjusted to 0,12 SE with 1-20 pg of inhibitor or 1-20 μl of the solution to be tested and make up to 0,1 M with sodium malinate buffer pH 6. 0 to 0.1 ml.

It is then equilibrated for 10 minutes at 35 ° C, and then 0.1 ml of a 0.05 M sucrose solution preheated to 35 ° C is added in 0.1 M sodium malinate buffer of 6.0. Then incubate for 20 minutes at 35 ° C, and the sucrose reaction is stopped by adding 1 ml of glucose oxidase reagent 2) and incubating for another 30 minutes at 3s ° C. Then 1 ml of 50% a2SO4 is added at a measurement at 545 nm against a corresponding blank. For evaluation, the percentage inhibition of the invested sucrose is calculated, and from the 50% inhibition point is calculated using a glucose calibration curve to SIE / g resp. SIE / 1. 461 101 1) Soluble sucrose from mucous membrane in pig intestine according to B. Borg-Ström, A. Dahlquist, Acta Chem. Scand. lg (1958), p. 1997.

Dilute with 0.1 m sodium malinate buffer pH 6.0 to the corresponding 2) SE content.

The glucose oxidase reagent is prepared by dissolving 2 mg of glucose oxidase (Boehringer, I.) in 100 ml of 0.565 m Tris-HCl buffer pH 7.0 and then adding 1 ml of surfactant (2 g Triton X 100 + 8 g 95% ethanol pa), 1 ml of dianisidine solution (260 mg o-Dianisidine. 2 HCl in 20 ml H 2 O) and 0.5 ml of 0.1% peroxidase solution in H 2 O. (Fa. Boehringer, Lyophilisate, Purity II).

According to the method described above, a number of strains from the family Bacillaceae were tested. In particular, strains from the group Bacillus had clear sucrose-inhibiting activities. The species B. subtilis, B. subtilis var were the most favorable with regard to the yield. niger, B. amyloliquefaciens, B. longisporus, B. polymyxa and B. coagulans.

The frequency with which strains were found according to the stated method, which in the residues turned out to be active inhibitors, was over 5%. Examples of particularly active strains are given in Table 1.

Table 1 Bacillus strains with sucrose inhibitory activity Art B.

B.

B.

B.

B.

B.

B.

B.

B. subtilis subtilis var. niger amyloliquefaciens coagulans longísporus polymyxa polymyxa polymyxa polymyxa polymyxa polymyxa polymyxa DSM DSM DSM DSM DSM DSM DSM DSM DSM DSM DSM DSM Stam nr. 704 675 (ATCC 9372) 7 (ATCC 23 350) 1 (ATCC 7050) 479 * 365 372 742 292 356 (ATCC 8523) 36 (ATCC 842) 740 5 461 101 ëšå Stam nr.

B -PO lymyxa osm 741 B. subtilis DSM 1050 B. subtilis DSM 1051 B. subtilis DSM 1052 B. subtilis DSM 1053 B. sultilis DSM 1054 B. subtilis DSM 1055 B. subtilis DSM 1055 B. subtilis DSM 1057 'I also inhibit amylases The strains listed are deposited under the DSM numbers indicated at the Deutsche Sammlung für Mikroorganismen (DSM), Göttingen, and can be obtained therefrom.

The strains DSM 704, 740, 741 and 742 are described in Table 2. The other strains are known from the literature and are found in "Cataloque of Strains 1974" for DSM.

For the recovery of sucrose inhibitors, the strains listed above are grown in the nutrient solutions described above. In doing so, it must be taken into account that practically every strain for optimal production needs a different qualitatively and quantitatively differently composed nutrient solution.

After cultivation 1 * 10 days at 15-80 ° C, preferably 24-40 ° C, resp. for thermophiles 50-70 ° C in shake flasks or in fermentation vessels of different sizes, the cells are separated from the culture solution, and as the inhibitors appear, the active ingredient is enriched from the culture solution and / or from the cells.

The inhibitors are recovered from the culture solution by lyophilization or precipitation with salts or water-soluble organic solvents (such as lower alcohols and ketones) or by adsorption of the active substances on ion exchangers. The inhibitors are recovered from the cells by extraction with organic solvents, such as e.g. alcohols, ketones, ethers, esters and sulfoxides.

For this purpose, the fermentation broth is centrifuged at 3000-20000 rpm, preferably 6-10000 rpm for 10-60 minutes or filtered, preferably under pressure and with the aid of filtration aids, and is thus separated into culture solution and cell residue.

Table 2 461 101 Strains properties Rods Length [u Width P Gram ~ rcaction Spores clipsqid / cylindrical round - - terminal / aubtnrminal ccntral / plraccntral Swollen spore oder cells Mobility Haxinnl growth cmprature Growth positive at ° C TM1 Growth negative at ° C 2-S DSH 732 3-7 DSN 70Hf 2 ~ k 0.6-0.8 0.6-o, e 0,7-o, s 0,6-0,8 Ö 6 04-014 '# 0 35 IQ' O- OOOIO H0 NS Ib O OOOIO 35 50 å O IOOIO S5 60 Catalase Anacrob growth Vogca-Proskaucr reaction pH 1 VP - Hediun Eigolb ~ roaktinn Growth of 5.7 NACI $ \ HQCI 7 \ HaCl 10 Lyaozyme (0.001 \) I 1 nlo 6 OIIIO IIIIO IOOQO Acid Formation from D ~ glucose L-arabinose D-xylol D-mannitase formation from glucose Degradation of Starkclsc Casein Galatia Tyrosine Hippurate O 0064- II§OQ Q OQO-O IIOÖÅP Ö 0600 llO-QO 0 # 049 (continued) The properties of the strains on gsm Es? DSH Ds ”vuo 1u1 1u2 voá Pectin, å * Use of Citrate _ _ _ 4 Propionate _ _ _ _ Desamincring of Penylalanine - _ _. aecuxrion of NO3 ': 111 xoz' +. . , Nitrate gas formation - - _ - Formation of crystalline dextrins - _ _ Indole '' - _ dihydroxyacetone + + _ Strains DSM 740, DSM 741, DSM 742 and DSM 704 are assigned to the Bacillus group due to spore formation and aerobic growth. The morphological and physiological characteristics obtained for strains DSM 740, DSM 741 and DSM 742 correspond to those of Bacillus polymyxa. while the strain DSM 704 should be subordinate to the species Bacillus subtilis.

The identification took place according to R.E. Gordon, W.C. Haynes, C. Hor-Nay Pong: The Genus Bacillus, Washington 1973.

The isolation of the inhibitor from the respective culture solution can take place in different ways: a) Evaporation of the culture solution under reduced pressure (10-50 torr) at bath temperatures of 20-100 ° C, preferably 40-80 ° C to about 1 / S-1/50 of the output volume. The evaporated extract is filtered or centrifuged, and the clear filtrate (the clear supernatant) is optionally lyophilized after the previous desalting. b) Precipitation of the inhibitors from the culture solution (or those according to a) evaporated culture solutions) by adding water-soluble organic solvents, such as e.g. alcohols or ketones, preferably methanol, ethanol, acetone, up to a content of 60-90%. Since inert concentrations of solvent inactive by-products precipitate, this precipitation process is particularly well suited for fractional precipitation for the separation of undesired by-products. c) Salting out of the inhibitors from the extracts (or the extracts according to a) evaporated), e.g. with ammonium sulphate, common salt, etc.

The precipitated precipitate is collected by centrifugation or filtration and washed either directly with acetone and ether and dried in vacuo or dialyzed after redissolution in water and lyophilized. d) Adsorption of the inhibitors on ion exchangers.

This method is suitable for the isolation of such inhibitors which, due to their chemical nature, carry charges. The desorption of the inhibitor takes place by changing the ionic strength or pH of the elution medium.

In addition to the inhibitor, unwanted by-products are often found in the culture solutions. The separation of these by-products can take place in different ways, e.g. by heat denaturation of the by-products of inhibitors which are heat-stable, or by dialysis through corresponding membranes of low molecular weight inhibitors, the undesired by-products being retained by the membrane, or by fractional precipitation (compare b)) or by absorption of the by-products on ion exchangers.

Extraction of inhibitors from the cells takes place by extraction several times of the cells with organic solvents, preferably two extractions with 3-5 volumes of acetone (calculated on moist cell volume) for 10-20 minutes and then once with ether for 5-10 minutes. The acetone and ether extracts are evaporated to dryness in vacuo, taken up in water and lyophilized.

The new substances dissolve well in water. One group among the inhibitors is heat stable at neutral pH values, acid stable (pH 2), alkali stable (pH 12) and dialyzable. These inhibitors are not inactivated by trypsin and pepsin, on the other hand they do not inhibit the mentioned enzymes. They cannot be stained with ordinary protein dyes. Judging by the gel filtration, the molecular weight of these inhibitors is above 100 but below 2000. The best inhibitors in this group are characterized by an extremely high inhibitory activity against sucrase, which activity exceeds all hitherto known sackazase inhibitors.

When fermenting the strain DSM 7 in a nutrient solution with composition A (see example 4) after fermentation 4 days over 400,000 SIE / 1 is recovered, during a cultivation of the strain in nutrient solution S3 (see example 3) after fermentation 6 days over 300,000 SIE / 1. By adsorption on strongly acidic cation exchangers in the form and subsequent desorption with aqueous NH3 solution as well as evaporation and lyophilization of the descrbate, a crude inhibitor of about 40,000 SIE / g is obtained. Extraction of the crude inhibitor with methanol, evaporation of the extract to dryness, redissolution in water and chromatography of the aqueous solution on weakly acidic ion exchangers based on dextran or cellulose, in particular carboxymethylcellulose, desorption with dilute mineral acids, preferential fractions of these and lyophilic fractions fractions give a 10 N hydrochloric acid, evaporation of the sucrose-inhibitory-enriched crude product with about 250,000 SIE / gu Chromatics of the enriched LD 20) in methanol, evaporation of the sucrose-inhibitively active fractions and the crude product on modified dextran ( Sephadex addition of concentrated mineral acids, preferably concentrated hydrochloric acid to a pH of 1-3, gives a crystalline product with 540,000 SIE / g. This substance is chromatographically pure, as a sum formula C6H13O4N and C6H14O4NCl were obtained for the hydrochloride. parameters (IR, NMR, UV spectra, melting point, specific angle of rotation) and chemical properties (per iodate oxidation, elemental analysis) is the identical with a compound described by S. Inoye et al, (Tetrahedron go, 2125 (1968)) with the sum formula C6H13O4N and C6H14O4NCl for the hydrochloride, respectively, which the authors have assigned to the structural formula R f] m ! no 5 í H H ß s "l 11 461 101 proposed by the authors the name" 1-deoxinojirimycin ".

This compound was obtained chemically by the authors by hydrating the antibiotic nojirimycin. The starting product nojirimycin is obtained according to T. Niida et al. (J. Antibiotics, Ser. A. 20, 62 (1967)) by fermentation of organisms from the group Streptomyces.

The present invention makes it possible for the first time to produce deoxinojirimycin with a procedure by direct microbiological synthesis with good yields without detour over the relatively unstable and thereby difficult-to-handle nojirimycin.

It is extremely surprising and could not be predicted that these compounds are produced by organisms from the Bacillus group, as these microorganisms are generally less suitable as producers of secondary substances and at most mainly form peptide-like secondary substances.

In addition, it is formed by individual strains, e.g. DSM 372, sucrase inhibitors, which in admixture with deoxinojirimycin and / or nojirymycin contain other other sucrose-inhibitory active components which can be clearly separated by thin layer chromatograms.

Other strains, e.g. DSM 741, forms inhibitors which, in thin layer chromatograms, do not show deoxinojirimycin or nojirimycin, and which thus have a different chemical structure.

It is known that in animals and humans, hyperglycaemia occurs after ingestion of carbohydrate-containing foods and beverages (eg cereal and potato starch, fruit, juice, beer, chocolate), which is caused by a rapid breakdown of carbohydrates by glycoside hydrolases (eg salivary and pancreatic amylases, maltases, sucrose) according to the following scheme: Amylase Malta Starch resp. glycogen -------- - Maltose -------- - Glucose Sucrose Sucrose --------- - Glucose + Fructose 12 461 101 These hYPef91Ychemicals are especially strong in diabetics and ihål- liät UtPfä9lade. In obese people, due to food, camouflage hyperglycaemia usually causes a particularly strong secretion of insulin, which in turn leads to increased fat build-up and reduced fat breakdown. In connection with such hyperglycaemia, hypoglycaemia often occurs in metabolically healthy and obese people due to insulin secretion. It is known that both hypoglycemias and chymus remaining in the stomach promote gastric juice production, which in turn triggers or promotes the formation of a Qastrit, a ventricular ulcer or duodenum.

It is further known that carbohydrates, especially sucrose, are broken down in the oral cavity by microorganisms and thereby promote caries formation.

Diseased absorption of carbohydrates, e.g. due to intestinal saccharase deficiency causes a diarrhea. Appropriate doses of a glucosidase inhibitor cause artificial pathological absorption and are therefore suitable for counteracting constipation.

The inhibitors of the invention are therefore suitable as therapeutics for the following indications.

Obesity, hyperlipoproteinemia, atherosclerosis, diabetes, prediabetes, gastritis, constipation, caries.

For broadening the spectrum of action, it may be appropriate to combine inhibitors of glycoside hydrolases which mutually complement their effects, whether they are combinations of the inhibitors of the invention with each other or combinations of the inhibitors of the invention with those already known. Then it can e.g. it may be appropriate to combine the sucrase inhibitors of the invention with already known amylase inhibitors.

Also advantageous in many cases are combinations of inhibitors of the inhibitors according to the invention with known, oral antidiabetic 05-cytotropic sulfonylurea derivatives and / or blood sugar-acting biguanides) and with blood lipid-lowering active substances, such as clofibrate, nicotinic acid, cholestyramine and others. 13 461 101 The compounds can be administered without dilution, e.g. as a powder or in a gelatin casing or in combination with a carrier or in a pharmaceutical composition. Pharmaceutical preparations may contain a greater or lesser amount of the inhibitor, e.g. 0.1-99.5% in combination with a pharmaceutically acceptable non-toxic inert carrier, wherein the carrier may contain one or more solid, semi-solid or liquid diluents, fillers and / or non-toxic, inert and pharmaceutically acceptable formulation aids. Such pharmaceutical preparations are preferably in the form of dosage units, i.e., physically discrete units, containing a certain amount of the inhibitor corresponding to a fraction or several times the dose required for the desired inhibitory effect. Dosage units may contain 1, 2, 3, 4 or more single doses or 1/2, 1/3 or 1/4 of a single dose. A single dose preferably contains a sufficient amount of active substance to achieve the desired inhibitory effect when administered according to a predetermined dosage schedule for one or more dosage units, with a whole, a half or a third or a quarter of the daily dose usually administered 1, 2, 3 or 4 times a day. Other therapeutic agents may also be taken. Although the dosage and dosing schedule in each case must be carefully weighed using thorough professional judgment and taking into account the patient's age, weight and condition, the nature and severity of the disease, the dosage is usually in the range of about 1-1 x 104 SIE / kg body weight per day. In many cases a sufficient therapeutic effect is achieved with a lower dose, while in other cases a larger dose is required.

Oral administration can be performed using solid and liquid dosage units, such as e.g. powders, tablets, dragees, capsules, granules, suspensions, solutions and the like.

Powders are prepared by decomposing the substance into a suitable size and mixing with an even degraded pharmaceutical carrier. Although an edible carbohydrate, such as e.g. starch, lactose, sucrose or glucose can usually be used for this purpose. and also used here, it is desirable to use a non-metabolizable carbohydrate, such as e.g. a cellulose derivative. 461 101 '4 Sweeteners, condiments, preservatives, dispersants and colorants may also be used.

The capsules can be prepared by preparing the powder mixture described above and by filling the gelatin casings already prepared. The powder mixture can be pre-filled with lubricant, such as e.g. silica gel, talc, magnesium stearate, calcium stearate or solid polyethylene glycol. The mixture can also be added with a disintegrator or a solvent such as e.g. agar-agar, calcium carbonate or sodium carbonate, to improve the availability of the inhibitor when taking the capsule.

The production of tablets takes place e.g. by preparing a powder mixture, coarse or fine-grained, and adding a lubricant and its integrator. From this mixture tablets are formed.

A powder mixture is prepared by mixing the substance, which is suitably decomposed and supplemented with an excipient or other carrier substance, as described above. If necessary, a binder is added, e.g. carboxymethylcellulose, alginate, gelatin or polyvinylpyrrolidone, a solution retardant, such as e.g. paraffin, a resorption accelerator, e.g. a quaternary salt and / or an absorbent, such as e.g. bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated together with a binder, such as e.g. syrups, starch paste, acacia slime or solutions of cellulose or polymeric material. The product is then pressed through a large sieve. Alternatively, the powder mixture can be passed through a tableting machine, and the resulting irregularly shaped pieces are broken down into grain size. In order for the obtained grains not to get stuck in the tablet-forming nozzles, they can be added with a lubricant, such as e.g. stearic acid, stearate salt, talc or mineral oil. This mixture, which has been made slidable, is compressed into a tablet form. The active substances can also be combined with free-flowing, inert carriers and transferred directly into tablet form, omitting the granulation or decomposition step. The product can be provided with a clear or opaque protective cover, e.g. a coating of shellac, a coating of sugar or polymeric substances and a polished wax coating. Dyes can be added to these coatings so that a distinction can be made between the different dosage units. The dosage forms to be administered orally, such as e.g. solutions, syrups and elixirs, can be prepared in dosage units so that a certain amount of preparation contains a certain amount of active substance. Syrup can be prepared by dissolving the active substance in an aqueous solution, which contains suitable flavoring substances. Elixir is obtained by the use of non-toxic alcoholic carriers. Suspensions may be prepared by dispersing the compound in a non-toxic carrier material. Solvents and emulsifiers, such as e.g. ethoxylated isostearyl alcohols and polyoxyethylene sorbiters, preservatives, flavor enhancers such as e.g. peppermint oil or saccharin and the like, can also be added.

The dosing instructions can be stated on the capsule. In addition, the dosage can be ensured so that the active substance is released delayed, e.g. by introducing the active substance into polymeric substances, waxes or the like.

In addition to the above-mentioned pharmaceutical compositions, e.g. foods are also prepared which contain these active substances, e.g. sugar, bread, potato products, fruit juices. beer, chocolate and other confectionery and preserves, such as e.g. marmalades, these products being provided with a therapeutically effective amount of at least one of the inhibitors of the invention.

The inhibitors prepared according to the invention also have the property that in animals they greatly affect the ratio between the proportion of undesirable fat and the proportion of desirable low-fat meat (lean meat) in favor of lean meat. This is of special importance for the breeding and keeping of useful animals in agriculture, e.g. in pig breeding but also of great importance for the breeding and keeping of other useful animals and pets. The use of the inhibitors can further lead to a considerable rationalization of the feeding of the animals both in terms of time, quantity and quality. Since they cause a certain delay in digestion, the residence time of the nutrients in the digestive tract is extended, whereby an ad libitum feeding is made possible at a lower cost. Furthermore, when using the inhibitors according to the invention, in many cases a considerable saving of valuable protein feed is obtained. 461 101 16 The active substances can thus be used in practically all areas in the animal industry as a means of reducing fat build-up and for saving feed egg whites.

The effect of the active substances here depends to a large extent on the species and sex of the animals. The active substances are especially valuable for animal species, which at all or during certain life intervals have a strong tendency to fat storage.

Examples of animals for which the inhibitors can be used to reduce fat storage and / or save egg feed whites are mentioned e.g. the following livestock and pets: warm-blooded animals such as cattle, pigs, horses, sheep, goats, cats, dogs, rabbits, fur animals, e.g. nertz, chincilla, other pets, e.g. guinea pigs and hamsters, working and zoo animals, e.g. rats, mice, monkeys, etc., birds, e.g. broilers, chickens, geese, ducks, turkey roosters, pigeons, parrots and canaries and cold-blooded animals such as fish, e.g. carp and reptiles, e.g. snakes.

The amount of active substance administered to the animals to achieve the desired effect can be extensively varied due to the beneficial properties of the active substance. It is preferably between about 0.5 mg - 2.5 g, in particular 10-100 mg / kg of feed. The administration time can be a few hours or days up to several years. The appropriate amount of active substance and the appropriate duration of administration are closely related to the challenge goal. It depends in particular on the animal's species, age, sex, state of health and the way of caring for the animals and is easy to determine for each professional.

The active substances are administered to the animals by the usual methods. The mode of administration depends in particular on the species, behavior and general condition of the animal. Thus, the administration can take place orally once or several times a day at regular or irregular time intervals. For reasons of convenience, an oral administration is used in these cases, especially in the rhythm of the animals' nutritional and / or beverage intake. .

The active substances can be administered as pure substances or in formulated form, by formulated form is meant both in the form of premix, i.e. in mixture with non-toxic, inert carriers of 90dtYCkli9t kind, and also as part of a total ration in the form of an additional feed or as a mixing component in a single mixed feed. This also includes the administration of suitable preparations via the drinking water.

The active substances may optionally be administered in formulated form also together with other nutritional and active substances, e.g. min / eral salts, trace elements, vitamins, egg whites, energy carriers (eg starch, sugar, fats), dyes Qchgeller flavors or other feed additives, e.g. growth promoters in suitable form. The active substances can be given to the animals during or after nutrient uptake.

It is convenient to carry out the oral administration together with the feed and / or the drinking water, the active substances being added to the total amount or only a part of the feed and / or the drinking water as required.

The active substances can be added to the feed and / or drinking water by simple mixing as pure substances, preferably in finely divided form or in formulated form in mixture with edible, non-toxic carriers, optionally also in the form of a premix or a feed concentrate.

The feed and / or drinking water can e.g. contain the active substances according to the invention in a concentration of about 0.001-5.0, in particular 0.02-2.0% by weight. The optimum concentration of the active substance in the feed and / or drinking water depends in particular on the amount of feed and / or drinking water taken up by the animal and can be easily determined by any person skilled in the art.

The type of feed and its composition are irrelevant in this respect. You can use all common, commercially available or special feed compositions, which preferably contain the usual balance of energy and egg white substances, including vitamins and minerals, necessary for a balanced diet. The feed can e.g. 461 101 18 composed of plant substances, e.g. oilcake waste, cereal waste, cereal by-products but also of hay, silage, turnips and other fodder plants, of animal substances, e.g. meat and fish products, bone meal, fats, vitamins, e.g. A-.D-, E-, K- and B-complexes and special. _ protein sources, e.g. yeasts, as well as certain amino acids and minerals and trace elements, such as e.g. phosphorus and iron, zinc, manganese, copper, cobalt, iodine, etc.

Premixtures may preferably contain about 0.1-50, in particular 0.5-5.0% by weight of the active substance of the formula I in addition to any edible carriers and / or mineral salts, e.g. carbonated lime and is prepared by the usual mixing methods.

Mixed feed preferably contains 0.001-5.0, in particular 0.02-2.0% by weight of the active substances of formula I in addition to the usual raw material components in a mixed feed, e.g. seed waste or by-products, oilcake waste, animal egg whites, minerals, trace elements and vitamins. They can be prepared by the usual mixing methods.

Preferably in premixes and compound feedingstuffs, the active substances can optionally also be protected against air, light and / or moisture by covering their surface with a suitable agent, e.g. with non-toxic waxes or gelatin.

Examples of the composition of a finished compound feed for birds, which contains an active substance according to the invention: 200 g wheat, 340 g maize, 360.3 g soy waste, 60 g nut algae, 15 g dicalcium phosphate, 10 g calcium carbonate, 4 g iodinated common salt , 4.5 g of vitamin-mineral mixture and 3.2 g of premix of active substance give, after thorough mixing, 1 kg of feed.

The vitamin-mineral mixture consists of: 6000 I.E. vitamin A, 1000 I.E. vitamin D3, 10 mg vitamin E, 1 mg vitamin K3, 3 mg riboflavin, 2 mg pyridoxine, 20 mg vitamin B12, 5 mg calcium pantothenate, 30 mg nicotinic acid, 200 mg choline chloride and 200 mg MnSO4 x H20, 140 mg ZnSO4 x 7H2O, 100 mg FeSO 4 x 7 H 2 O and 1 20 mg CuSO 4 x SH 2 O. The premix with active subatance contains “g 461 101 e.g. 1-deoxinojirimycin in the desired amount, e.g. 1600 mg and in addition 1 g of DL-methionine and so much soybean flour that 3.2 g of premix are obtained.

Examples of the composition of a pigmeal feed containing an active substance of the formula I: 630 g of feed waste (composed of 200 g of maize, 150 g of barley, 150 g of oat and 130 g of wheat waste), 80 g of fishmeal, 60 g of soy waste 58.8 g tapioca flour, 38 g brewer's yeast, 50 g vitamin-mineral mixture for pigs (composition eg as for chicken feed), 30 g flaxseed cakes, 30 g corn gluten flour, 30 g soy flour, 10 g sugar cane molasses and 2 g pre- mixture of active substance (composition eg as for chicken feed) gives after thorough mixing 1 kg of feed.

The specified feed mixtures are preferably adapted for rearing and fattening chickens and pigs, but they can nevertheless be used for rearing and fattening other animals with the same or similar composition.

As already mentioned, the inhibitors can be used individually or even in arbitrary mixtures with each other, whereby both the pure active substances and also the crude active substances obtained in the preparation are optionally charged after a coarse purification.

Example 1 If inoculating a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 2.0% corn starch 1.0% glucose 0.5% casein hydrolyzate 1.0% yeast extract pH is adjusted with Na2CO3 to 7.2 + 0, 4% CaCO3 sterilization 30 minutes at 121 ° C. with a spore suspension of the strain DSM 704 and grows the flask at 28 ° C on a round shaker, the culture loading after 5 days has an activity of 70 SIE / ml. 20 461 101 Exemgel 2 About a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 7.5% malt extract 0.3% casein hydrolyzate 0.7% yeast extract 0.31% 'caco3' 0.3% KZHPO4 tap water , sterilize for 30 minutes at 121 ° C The pH is adjusted after sterilization to 6.6-6.8 with K2CO3 inoculated with a spore suspension of strain DSM 704 and the flask is grown at 28 ° C. On a round shaker, after 5 days the culture solution has an activity of 197 SIE / ml.

Example gel 3 - Nutrient solution S3 If a 140 liter yeast tank, containing 100 liters of nutrient solution with the composition 7.5% malt extract 0.3% casein hydrolyzate 0.7% yeast extract 0.3% CaCO3 0.3% KZHPO4 tap water, sterilization 30 minutes at 121 ° C The pH is adjusted after sterilization to 6.6-6.8 with KZCO3 inoculated with 1.2 l of preculture, obtained by culturing 10 1-liter Erlenmeyer shake flasks with 120 ml each of nutrient solution of the same composition, inoculated with the DSM strain 704, and if grown under stirring and aeration for 5 days at 28 ° C, a culture solution containing 260 SIE / ml is obtained.

Example 4 - Nutrient solution A If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 3.0% soybean meal 3.0% glycerin 0.2% CaCO3 tap water sterilization for 30 minutes at 121 ° C is inoculated with a spore suspension of strain DSM 7 and the flask is grown at 21 ° C. 28 C on a round shaker, the culture solution after 4 days has an activity of 437 SIE / ml.

Example 5 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 7 and the flask is grown at 28 ° C on a shaker, then the culture solution has an activity of 162 SIE / ml.

Example gel 6 About a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 1.0% glucose 1.0% starch solution 0.5% casein hydrolyzate 0.75% meat extract 0.75% peptones 0.5% yeast extract 0 .1% KZHPO 4 0.3% NaCl 0.1% MgSO 4. 7H2O tap water pH is adjusted with Na2CO3 to 7.2 sterilization 30 minutes at 121 ° C inoculated with a spore suspension of strain DSM 7, and the flask is grown at 28 ° C on a round shaker, then the culture solution after 5 days has an activity of 36.4 SIE / ml.

Example 2 If a 1 liter Erlenmeyer flask containing 200 ml of a nutrient solution according to Example 3 is inoculated with a spore suspension of strain DSM 7 and the flask is grown at 28 ° C on a round shaker, the culture solution after 6 days has a activity of 224 SIE / ml.

Example 8 If a 140 liter yeast tank containing 100 liters of the nutrient solution of Example 4 is inoculated with 1.2 liters of preculture, obtained by culturing 10 1-liter Erlenmeyer shake flasks each containing 120 ml of nutrient solution of the same composition, inoculated with a strain DSM 7, and if grown under stirring and aeration for 5 days at 28 ° C, a culture solution containing 286 SIE / ml is obtained.

Example 9 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 1, and the flask is grown at 28 ° C on a round shaker, then the culture solution after 6 days has a activity of 28.4 SIE / ml.

Example 10 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 365, and the flask is grown at 28 ° C on a round shaker, then the culture solution after 4 days has a activity of 15.6 SIE / ml.

Example 11 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of the DSM 365 strain, and if the flask is grown at 28 ° C on a round shaker, the culture solution has an activity after 4 days of 25.8 SIE / ml.

Example 12 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with a spore suspension of strain DSM 741, and if the flask is grown at 28 ° C on a round shaker, the culture solution has an activity after 5 days. of 3.2 SIE / ml. Example 13 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution of the composition of Example 1 is inoculated with a spore suspension of strain DSM 741, and if the flask is grown at 28 ° C on a shaker, the culture solution after 5 days an activity of 26.4 SIE / ml.

Example 14 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution of the composition of Example 2 is inoculated with a spore suspension of strain DSM 741 and the flask is grown at 28 ° C on a round shaker, the culture solution after 3 days an activity of 24.0 SIE / ml.

Example 15 About a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 2.0% corn starch 0.5% glucose 0.3% casein hydrolyzate pH adjusted with Na 2 CO 3 to 7.2 + 0.4% CaCO 3 sterilization minutes at 121 ° C are inoculated with a spore suspension of the strain DSM 741, and the flask is grown at 28 ° C on a round shaker, then the culture solution after 2 days has an activity of 81.7 SIE / ml and after 3 days an activity of 121 SIE / ml.

Example 16 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with a spore suspension of strain DSM 675 and the flask is grown at 28 ° C on a round shaker, then the culture solution after 5 days has a activity of 8.6 SIE / ml.

Example 17 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 675, and if the flask is grown at 28 ° C on a round shaker, the culture solution has an activity after 5 days. of 53.0 SIE / ml.

Example 18 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 675 and the flask is grown at 28 ° C on a round shaker, then the culture solution has a activity of 17.8 SIE / ml.

Example 19 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 4 is inoculated with a spore suspension of strain 24 461 101 DSM 372, and the flask is grown at 28 ° C on a round shaker, then the culture solution after 5 days an activity of 5.0 SIE / ml.

Example 20 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 372 and the flask is grown at 28 ° C on a shaker, then the culture solution after 4 days has a activity of 21.6 SIE / ml.

Example 21 If a 1 liter Erlenmeyer flask containing 200 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 372 and the flask is grown at 28 ° C on a round shaker, then the culture solution after 3 days has a activity of 26.8 SIE / ml.

Example gel 22 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 2.0% corn starch 1.0% glucose 0.5% casein hydrolyzate 0.5% yeast extract pH adjusted with Na2CO3 to 7.2 + 0, 4% CaCO3 sterilization for 30 minutes at 121 ° C is inoculated with a spore suspension of strain DSM 372, and the flask is grown at 28 ° C on a single-shaker, then the culture solution has an activity of 26.5 SIE / ml after 3 days.

Example 23 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 22 is inoculated with a spore suspension of strain DSM 479 and the flask is grown at 28 ° C on a round shaker, the culture solution after 3 days has a activity of 7.9 SIE [ml.

Example 24 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 6 is grown with a spore suspension of the strain 25 461 ml DSM 36, and if the flask is grown at 28 ° C on a round shaker, then the culture solution after 3 days an activity of 5.4 SIE / ml.

Example 25 If a 1 liter Erlenmeyer flask, containing 120 ml of a nutrient solution similar to GXêmPel 2, is inoculated with a spore suspension of strain DSM 36, and if the flask is grown at 28 ° C on a shaker, the culture reading after 3 days has an activity of 5 , 4 SIE / ml.

Example 26 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 356 and the flask is grown at 28 ° C on a shaker, then the culture solution after 3 days has a activity of 8.8 SIE / ml.

Example 27 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 292 and the flask is grown at 28 ° C on a round shaker, then the culture solution after 3 days has a activity of 9.0 SIE / ml.

Example 28 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 292, and if the flask is grown at 28 ° C on a round shaker, the culture solution after 3 days has an activity of 9 .2 SIE / ml.

Example 29 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 2 is inoculated with a spore suspension of strain DSM 742, and if the flask is grown at 28 ° C on a round shaker, the culture solution has an activity after 3 days. of 9.8 SIE / ml.

Example 30 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution according to Example 1 is inoculated with a spore suspension of strain DSM 740 and the flask is grown at 28 ° C on a round shaker, the culture solution after 4 days has an activity of 5.7 SIE / ml. 26 461 101 Example 31 The culture solution from a 100 liter yeast tank according to example 3 is adjusted with semi-concentrated HCl to pH 3.0-3.5, and the bacterial mass is centrifuged off after flocculation. 70 liters of dark brown culture solution with a SIE content of 220,000 SIE / liter were obtained. This solution was applied at a flow rate of 20 liters / hour on a column packed with 12 kg of Lewatit SC 104 in H + form with a diameter of 30 cm.

The run-through liquid had virtually no sucrose inhibitory activity and was discarded. The column was washed with 50 liters of distilled water (wash water I), 20 liters of 0.01 N HCl and finally with another 20 liters of distilled water (wash water II).

For desorption of the activity, 2.5% ammonia was connected. In parallel with increasing the conductivity of the eluate and increasing browning, the sucrose inhibitory activity was eluted. The first 20 liters up to the increase in conductivity were discarded, and the activity-containing fraction (35 liters) was evaporated in a roller evaporator and redissolved in some water (concentrate 3 liters). The concentrate was lyophilized. 288 g of dark brown crude product I were obtained with 38,000 SIE / g.

Example 32 The culture solution from a 100 liter culture tank according to Example 8 was adjusted with semi-concentrated HCl to pH 3.0-3.5, and the bacterial mass was centrifuged off after flocculation. 60 liters of dark brown culture solution with a SIE content of 240,000 SIE / liter were obtained. This solution was charged at a flow rate of 20 liters / hour on a column packed with 12 kg Dowex 50 W x 4 in H + form with a diameter of 30 cm. The liquid passed had virtually no sucrose inhibitory effect and was discarded. The column was then washed with 50 liters of distilled water (wash water I), 20 liters of 0.01 N HCl and then further with 20 liters of distilled water (wash water II). For desorption of the activity, 2.5% ammonia was then added. In parallel with the increase in conductivity in the eluate and increased browning, the sucrose inhibitory activity was eluted.

The first 20 liters up to increase the conductivity were discarded. The fractions which had activity (35 liters) were evaporated in a roller evaporator and redissolved in some water (concentrate 3 liters).

The concentrate was lyophilized. 360 g of dark brown crude product I with 24,000 SIE / g were obtained. 27 461 101 Example 33 50 9 crude product I from Example 31 was comminuted in mortar and then extracted with 3 x 300 ml of technical methanol. Thereafter, the batch was first homogenized for 2 minutes in an ultraturrax homogenizer and then stirred for 20 minutes in a water bath at a temperature of 40-50 ° C. After each extraction, it was filtered through a pleated filter. The residue remaining in the pleated filter after the third extraction was dried in vacuo (28.4 g). Because tests showed only low specific activity in this residue, it was discarded.

The methanolic extracts were combined and evaporated in vacuo to dryness. The active residue remaining in the flask was dissolved in 750 ml of H form (CM cellulose from the company Whatman, type C 52). It was then washed with 5 liters of water, after which 20 liters of 0.002 N HCl were connected for elution.

The first liquid, wash water and eluate were taken up in fractions of about 0.5 liters. The sucrose inhibitory effect was determined, and all fractions containing more than 30,000 SIE / liter were combined: in the first liquid obtained the dark brown colored fractions 2-4 and in the light yellow eluate fractions 16-35. The combined fractions were evaporated and lyophilized. 18.2 g of fractions 2-4 were obtained in the first liquid obtained with a specific activity of 4000 SIE / g (discarded) and 3.1 g of the so-called crude product II with 250,000 SIE / g (about 50-60% purity). The crude product II is highly hygroscopic and dissolves in air in a short time to a sticky mass.

Example 34 2.5 g of the crude product II from Example 33 were dissolved in 15 ml of methanol and chromatographed on a 5 x 90 cm column filled with Sephadex R LH 20 in methanol to separate the major amount of the accompanying peptides.

At a flow rate of 100 ml / hour and a temperature of 4-5 ° C, 10 ml fractions were collected. 10 μl of these fractions were applied to a silica gel plate (Merck) and examined by thin layer chromatography in the system ethanol / 25% NH 3 / H 2 O = 80/10/10. The fractions containing inhibitors according to the thin layer chromatography were combined, evaporated to 5 -10 ml and chromatographed on the same column.

The fractions were analyzed by thin layer chromatography and the inhibitor-containing fractions were combined to give only a very narrow range. Then evaporated to dryness. The inhibitor purified on LH 20 is about 90% pure. To separate the last remaining peptide impurities, the residue obtained after roller evaporation is added with 2-3 ml of methanol and the yellow methanolic solution is added with 50 μl of concentrated hydrochloric acid. After a short time, possibly even only after several hours, the inhibitor crystallizes in the form of slightly yellow cubes or squares. The peptides remain in the mother liquor. The crystals are centrifuged off, washed once with ice-cold methanol and the washed precipitate is then dissolved while heating in 2 ml of methanol. 4 ml of butanol are added and the mixture is allowed to stand overnight at 4 ° C. The colorless crystals which precipitate until the next morning are centrifuged off, washed once with ice-cold methanol, once with acetone and once with ether and then dried in vacuo. Yield 460 mg of the inhibitor in the form of hydrochloride with 540,000 SIE / g.

Example 35 To detect sucrose inhibitory components in culture solution or crude product, the following thin layer chromatographic procedure was used with subsequent enzyme reaction on plate, wherein inhibitory components are detected directly on the plate. In this thin layer chromatography, 1-5 μl of the culture solution or 1-5 μg of the preparation is placed on prepared silica gel plates (Merck company, type KG 60 F 254) and developed with ethanol / NH 3 / H 2 O = 8/1/1 (I ) or ethyl acetate / methanol / water = 10/6/4 (II). To directly make the sucrose inhibitory components visible, spray the developed and well-dried plate with enzyme gel (20 ml / 20 x 20 cm plate) and allow the gel to solidify. It is then pre-incubated for 5 minutes in a humid chamber at room temperature, after which it is sprayed to saturation with substrate gel. After these two gel layers solidify, the plate is introduced into a humid chamber and incubated at 40 ° C. Inhibition staining (light spots, reddish-brown background) develops after 60-90 minutes. At the time when the color development is optimal, the incubation is interrupted, and the plate with the agar layers present thereon is dried with a hot air fan.

Preparation of the gels Enzyme gel: 1.5 g of agarose (from the Biologique Francais industry) is suspended in 100 ml of 0.2 M Na-maleinate buffer pH 6.0 and then dissolved by boiling. The clear agarose solution is cooled to 5 ° C and shaken with 250 ml Triton X-100 solution (2 g Triton X-100 H 29 461 101 + 8 g ethanol pa) and 0.5 ml dianisidine solution (20 mg Dianisidine / 1 ml acetone). Immediately before using the gel, add 1 ml of GOD / POD reagent (12.5 mg glucose oxidase, purity grade I, Böhringer, order no. 15323 and 2.5 mg peroxidase, purity II, Böhringer, order no. 15302, dissolved in 5 ml of maleinate buffer) and 4-5 units are sucked from pig intestine (enzyme preparation in the same way as page 4, note 1).

The gel must be kept at SOOC until sprayed, otherwise it solidifies during spraying into the nozzle.

Substrate gel: 0.5 g of agarose is suspended in 100 ml of Na-maleinate buffer pH 6.0 and dissolved with boiling. Then cool to 50 ° C and 100¿pl Triton (2 g Triton X-100 + 8 g methanol p.a.) is added and 19 sucrose (Serva no. 35579). After dissolving the sucrose, the gel can be used.

Examination of the culture solutions for the strains by this method yielded sucrose inhibitory components with the following Rf values in system I: Strains Rf-Yärq? DSM 7 0.25 DSM 741 _ 0.25; 0.41; 0.50; 0.59; 0.71 DSM 704 0.25 DSM 372 0.25; 0.51; 0.60; 0.71 and the following Rf values in system II: Strains Rff value DSM 704 0.05 DSM 479 (0.05); 0.22-0.35; 0.62-0.67; 0.98 DSM 741 (0.05); 0.23-0.35; 0.62-0.67 DSM 372 (0.05): 0.20-0f22 Exemgel 36 About a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 1.0% starch 0.1% glucose 0 .5% casein hydrolyzate 1.0% yeast extract pH adjusted with Na2CO3 to 7.2 461 101 3 ”4 'sterilization 30 minutes at 121 ° C inoculated with a spore suspension of strain DSM 479, and the flask is grown at 2800 on a round shaker. the culture solution after 2 days an activity of 20.2 SIE / ml. gkemgel 37 About a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution with the composition 2.0% corn starch 1.0% glucose 0.5% casein hydrolyzate 1.0% yeast extract 0.1% K HPO 2 4 pH adjusted with Na 2 CO 3 to 7.2 + 0.4% CaCO 3 sterilization 30 minutes at 121 ° C is inoculated with a spore suspension of the strain DSM 372, and the flask is grown at 28 ° C on a round shaker, then the culture solution has an activity of 2.5 days. 50.3 SIE / ml.

Example 38 If a 1 liter Erlenmeyer flask containing 120 ml of a nutrient solution of the composition of Example 1, 2, 4 or 6 is inoculated with spore suspensions from the strains indicated and if the flask is grown at 28 ° C on a round shaker, the culture solution after 4 days the following activities: Nutritional solution The activity of the culture solution Strain according to example after 4 days in SIE / ml KA-63 DSM 1060 31 "6.6 25 7.9 50 4.3 3.8 2.7 8.4 3 , 6 3.1 un IAM-1523 DSM 1061 uuu CUT 8108 DSM 1062 NJpNm-JhNm-l-ë- II f 31 461 101 Nutritional solution Culture solution activity Strain according to example after 4 days in SIE / ml CUT 8110 DSM 1063 28 "71 69 104 38 10.6 14.4 54 2.7 28 1.5 10.3 17 3.8 4.1 n S 202 DSM 1Û64 nn S 204 DSM 1065 n S 219 DSM1066 u S 242 DSM TOG7 u IUI fi- * IU -Iøb-JoàIQ-JIÄNJON-lb

Claims (3)

461 101 12 Patent claims in Use of strains of the group Bacillus with the following names and deposit designations B.amyloliquefaciens DSM 7, B.coagulans DSM 1, B.longisporus DSM 479, B.polymyxa DSM 365, DSM 372, DSM 742, DSM 292, DSM 356 (ATCC 8523), DSM 36 (ATCC 842), DSM 740, DSM 741, B.subtilis DSM 704, B.subtilis var.niger DSM 675, B.subtilis DSM 1060, DSM 1061, DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066, DSM 1067, of which B.subtilis DSM 704, B.polymyxa DSM 740, DSM 741 and DSM 742 are new, for the recovery of sucrose inhibitors by culturing these strains by commonly known methods. Use of strains B.amyloliquefaciens DSM 7, B.subtilis DSM 704, DSM 675 and B.polymyxa DSM 372 for the recovery of the sucrose inhibitor 1-deoxinojirimycin. Method of preparing sucrose inhibitors according to claim 1, characterized in that strains from the group Bacillus with the following names and deposit designations B.amyloliquefaciens DSM 7, B.coagulans DSM 1, B.longisporus DSM 479, B.polymyxa DSM 365, DSM 372 , DSM 742, DSM 292, DSM 356 (ATCC 8523), DSM 36 (ATCC 842), DSM 740, DSM 741, B.subtilis DSM 704, B.subtilis var.niger DSM 675, B.subtilis DSM 1060, DSM 1061 , DSM 1062, DSM 1063, DSM 1064, DSM 1065, DSM 1066, DSM 1067, are grown in ordinary nutrient solutions at temperatures between about 15 and 80 ° for about 1-8 days during aeration in ordinary culture vessels, the cells are removed and the inhibitors isolated from the culture solution or cell extracts by standard purification procedures.