DK172818B1 - Antibiotic polypeptide, method for its preparation and its use - Google Patents

Description

in DK 172818 B1

The invention relates to an antibiotic-acting polypeptide, hereinafter referred to as Epidermin, a process for its preparation by a novel strain-resistant strain of Staphylococcus epidermidis, the novel resistant strain, antibiotic formulations containing the active substance Epidermin and use of the active substance to control infectious diseases.

From European Patent Publication EP-A-0,027,710 it is known that a particular strain of Staphylococcus epidermidis, namely Staphylococcus epidermidis NCIB 11536 (deposited with the National Collection of Industrial Bacteria, Aberdeen) produces a low molecular weight, antibiotic active polypeptide with a broad effect 15 gram-positive germs, which peptide lyses bacterial cells.

Furthermore, it is known that the strain Staphylococcus epidermidis MF 205 (SM, Taylor et al., Int. J. Mass Spec. And Ion Physics 8, 161-164, 1983) also produces an oligopeptide having effect against 20 sensitive bacteria. It is not clear from this publication whether this strain is identical to the above strain NCIB 11536.

It has now been found that a resistant mutant of Staphylococcus epidermidis, deposited on October 26, 1984, with "Deutsche Sammlung von Microorganism" under number DSM 3095, and related to the above strain NCIB 11536, after a modified and work-up method produces the similar antibiotic, preferably on Gram-positive germ, acting polypeptide Epidermin, which, when compared with the products of Staphylococcus epidermidis NCIB 11536 and MF 205, exhibits, among other things, significant differences in amino acid composition:

Comparison of building components:

Y

2 DK 172818 B1

Epidermin Products from Staph.epidermidis _MF 205_NCIB 11536

Asn 1 - 5 Asx - 21

Glx - 31

Pro 1 11

Gly 2 22

Ala 2 2 1

Ile 2 2 1 10.

Phe 2 21

List 2 21

Lan 2 - E-Me Lan 1 -

Dhb 1 - 15 Tyr 1 1 S- (2-Aminovinyl) -D-cysteine 1 X - 1-2 20 Dhb corresponds to α, β-dehydroainiroic acid, Asx and Glx means Asn / Asp and Gln / Glu respectively.

After total hydrolysis of Epidermin with 6N hydrochloric acid (18 hours at 110 ° C), the quantitative amino acid analysis by ion exchange chromatography (standard program 25 with equipped Biotronik LC 6000 E) showed the following α-aroinoic acid composition:

Asp (1.00), Pro (0.95), Gly (2.09), Ala (2.03),

Ile (2.03), Tyr (0.30), Phe (2.02) and Lys (2.00) (cf.

In Fig. 1). By adding thioglycolic acid, the tyrosine content of the total hydrolyzate was increased to the far-reaching stoichiometric value of 0.93.

The configuration of the protein amino acids was found by gas chromatography of the pentafluoropropionyl amino acid n-propyl ester from the total hydrolyzate. Separation 35 occurred on the chiral stationary phase L-valentine-butylamide-polysiloxane (Chirasil-Val). By comparison with a test mixture of amino acids of known configuration, the above-mentioned structural components could be assigned to the L configuration (cf. Fig. 2).

In addition to these protein amino acids, there are the following non-protein amino acids: Lanthionine (2), β-methyllanthionine (1) and α, β-dehydroaminobutyric acid (1). Of these, lanthionine has meso configuration and β-methyllanethonine 2S, 3S, 6R configuration. In addition, Epidermin contains as component S- (2-aminovinyl) -D-cysteine.

The novel compound Epidermin can be characterized by the following indications: 1. Nature: Colorless powder.

2. Solubility: Very well soluble in mixtures of water / glacial acetic acid or methanol / glacial acetic acid, soluble in lower alcohols, insoluble in chloroform, acetone, diethyl ether, petroleum ether.

3. Color reaction on silica gel plates: Ninhydrin ^ chloro / TEM (TDM * 4,4'-bis- (dimethylamino) diphenylmethane), orcin / sulfuric acid, anisaldehyde / sulfuric acid. Destruction-free detection in UV light at 254 runs and at spraying 20 with water.

4. Thin-layer chromatography: Silica gel finished plates 60 F254 (Merck) were used.

System A: Chloroform / methanol / 17% ammonia (2/2/1) rf - 0.73

System B: Chloroform / methanol / 17% ammonia (70/35/10) RF * 0.30

System C: n-Butanol / ice blanket / water (4/1/1) RF * 0.05 5. HPLC: See Fig.7.

6. Stability: Stable from pH = 2 to pH = 7, at higher 30 pH values, a strong decrease in activity occurs.

7. Molecular mass: In the region of 2160. Depending on isolation methods, as anions may be contained e.g. chloride, acetate or phosphate.

8. Ultraviolet absorption spectrum: In aqueous solution 35 long-wave maximum at 267 nro (Fig. 3).

v 4 DK 172818 B1 * 9. Infrared absorption spectrum; IR spectrum of a sample in a potassium bromide tablet (Fig. 4). in 10. Nuclear Magnetic Resonance Spectra: HH-NMR Spectrum: Fig Fig. 5, CC-NMR Spectrum: Fig.6.

Suitable fragments for sequencing the epidermin were obtained by tryptic cleavage. The reaction with trypsin over a short period of time led to loss of antibiotic activity. Macroscopically, by tryptic cleavage, a jelly-like white precipitate was observed that could be separated by centrifugation. The supernatant was lyophilized and gel chromatographed on a Spehadex G-25 (cover strain from Pharmacia company) in 1% acetic acid.

The chemically homogeneous fraction of ninhydrin, chlorine / TDM and water (hereinafter referred to as Pi) 2 was found to be the N-terminal fragment of the tryptic cleavage of Epidermin (see below).

The extremely hydrophobic, jelly-like precipitate consisted of several chemical components that arose in the tryptic cleavage from the C-terminal 20 fragment of the total molecule. A chemically homogeneous product (hereinafter referred to as P2) was obtained by dissolving the precipitate in dimethyl formamide and subsequent gel chromatography on Sephadex LH-20 (dextran produced by hydroxy propylation from Sephadex G-25, Pharmacia) with dimethylformamide as eluent. P2 could be detected with water, chlorine / TDM and under UV light. The ninhydrin reaction to P2 was negative.

The amino acid analysis of the fragment Pi showed the following composition: Pro (11, Lan (1), β-Me-Lan (1), Gly (1), 30 Ala (2), Ile (2), Phe (1), Ly3 (2) Since isoleucine, when dancing the total molecule, was determined as N-terminal amino acid and the fragment P2 does not contain isoleucine, the fragment pi must be the N-terminal cleavage product of the total molecule. The C-terminal amino acid of 35 fragment PI must be based on the tryptic cleavage] be lysln.

For further structural study of Pi, £ -: ¾

Ha DK 5 DK 172818 B1 the C-terminal amino acid lysine enzymatically removed by carboxypeptidase B (company Boehringer Mannheim),

The fragment P12 thus formed could be isolated in pure form by gel chromatography on Sephadex G-25 (1% vinegar ** 5 acid). P12 consists of the following amino acids: Pro (1), Lan (1), Ø-Me-Lan (1), Fly (1), Ala (2), Ile (2), Phe (1),

List (1).

The sulfur bridges in the thioether amino acids lanthionine and β-methyllanthionine prevent a sequence analysis 10 according to Edman. By reacting P12 with Raney-Ni W2, a sulfur-free dodecapeptide is obtained. Hereby, meso-Lan I D- and L-alanine, and β-methyllanthionine in D-amino-butyric acid and L-alanine are transferred. The dodecapeptide sequence was elucidated by Edman degradation and FAB spectrometry: 15 Ile1-Ala2-Ala3-Lys4-Phe5-Ile6-Ala7-Abu8-aro9-Gly10-Ala1-Ala12.

Various studies regarding the placement of the sulfur bridges in fragment P12 revealed the following structure: 20 I-S -1

Ile-Ala-Ala-Lys-Phe-Ile-Ala-Abu-Pro-Gly-Ala-Ala

! -s_I

The C-terminal fragment P2 obtained by tryptic cleavage can be characterized as follows: Asn (1), 25

Lan (1), Gly (1), Phe (1), Tyr (1), α-keto butyric acid and S- (2-aminovinyl) -D-cysteine.

The o-keto butyric acid is derived from the dehydroamino butyric acid, which in the sequence of the total molecule follows directly after lysine * 3. The tryptic cleavage releases the amino group 30 from the dehydroamino butyric acid. Since dehydroamino butyric acids with free amino groups are unstable, they are, for example, rearranged to σ-keto acids.

The tryptic fragment P2 is N-terminal with the α-keto butyric acid blocked (ninhydrin reaction to P2 advances negatively). In addition to the amino acids lanthionine, glycine, phenylalanine, tyrosine and aspartic acid identified by total hydrolysis and amino acid analysis, P2 contains an additional component which is destroyed by acidic total hydrolysis. This could be characterized by its signals in the C C-NMR spectra of Epidermin (Fig. 6) and j in the fragment P2 (Fig. 8).

5 An information on the chemical nature of this amino acid was obtained by reaction of P2 with Raney-Ni W2, in which two main products arose. In both products isolated by preparative HPLC, after total hydrolysis, an additional D-alanine residue was found in the amino acid chromatogram, which residue arose from this reaction.

The structure of this building part could be resolved by heterogeneous hydrogenation of the native antibiotic.

The four reaction products H1, H2, H3 and H4 were purified by semi-preparative HPLC, total hydrolyzed, and subjected to chiral stationary phase chromatography (Fig. 15).

Hereby, compared with the chromatogram of Fig. 2, at H1 and H3, an additional peak was identified which over its mass spectrum was identified as S- (2-aminoethyl) -D-cysteine. In Epidermin, this amino acid contained 20 as an acid-labile component S- (2-aminovinyl) -D-cysteine.

The two peptides P21 and P22 formed from the C-terminal fragment P2 by treatment with Raney nickel W2 could be characterized by amino acid analysis and gas chromatography on Chirasil-Val:

Reaction products P21_ P22_

Amino acid analysis and gas 2 D-Ala 1 D-Ala chromatography on Chirasil-Val 1 L-Ala meso-Lan 1 L-Phe 1 L-Phe 30 1 L-Tyr 1 L-Tyr 1 L-Asp 1 L-Asp 1 Gly 1 Gly

In addition, both products were blocked C-terminal with ethylamine. S- (2-Aminovinyl) -D-cysteine was cleaved in sulfurization under simultaneous hydrogenation in D-alanine and ethylamine.

light DK tm 7 DK 172818 B1

Sequencing of the bridged heptapeptide P21 (partial hydrolysis, 0.1N hydrochloric acid, 93 ° C, 16 hours) showed the following structure: 5 H3C-CH2-CO-CO-Gly-D-Ala-L-Phe-L-Asn-D -Ala-L-Tyr-L-Ala-NHEt

The placement of the mesolan-thionine bridge contained in P22 according to partial hydrolysis and various chemical studies on enzymatic fragments of P21 and P22 was only compatible with the following structure for the tryptic cleavage fragment P2:

I s I

i c H, C-CH - CO-CO-Gly-Ala-Phe-Asn ~ Ala-Tyr-Ala-NH-CH 15 3 2 I || .

\ -S-CH

The formal substitution of the 2-keto butyric acid which blocks the P2 N-terminus with the amino acid dehydrobutyrin present in the native antibody and a subsequent fragment condensation of the tryptic peptides PI and P2 directly led to the sequence below for the heterodetric tetracyclic peptide antibiotic dermin: DK 8 DK 172818 B1

Structure of the ribosomally synthesized heterocyclic cyclic docosapeptide antibiotic Epiderroin: meso-Lar.thionine CK, -S -CH,

I I

H-Ile-Ale-NK-CK-CO-Lys-? He-Ile-NK-CK-CO— \ (S) (R) CK, β-Methyllanchionine

10 I

(S) CH-S -: - CH,

Ί I

L NK-CK-CO-Pro-Gly-HN-CK-CO-Ala-Lys- (S) (R) 15 - CH-, meso-lanthionine in »CK CK, -S ---- CK, II I m I il »L-KN-C-CO-Gly-KV-CK-CO-Phe-Asr.-KN-CK-CO-Tyr-KK-CE-CO-'KK-CK 20 (I) (S) I (R) ||

CH2-S - CK

S- (2-Aminquinyl) -D-cysteine

The N-terminal adjacent halves of the 25 individual thioether amino acids are each D-configured (corresponding to the (S) configuration of the R, S nomenclature).

The invention thus relates to a homogeneous and thus purely considered product, referred to as Epidermin, and further to a process for its preparation, isolation and purification.

The cultivation of the producer Staphylococcus epider-midis DSM 3095 takes place aerobically at 37 ° C in a complex medium with the composition 2-4% meat extract, 1-3% malt extract and 0.25-1% CaCO 2 or 0.25-0.5% Ca (OH) 2 »35 (Unless otherwise indicated, the percentages refer to weight percentages here and below).

9 DK 172818 B1

The maximum for antibiotic activity is reached after 18-23 hours.

For example, attempts to enrich the antibiotic were made on the culture filtrate from 10 liter 5 culture vessels, the effectiveness of the individual steps being tested in the plate diffusion test. The active components could be enriched by extracting the culture filtrate liberated from cells and lime with n-butanol. However, the extraction with n-butanol only succeeded at the natural final pH of the culture filtrate of 8.0. A further purification could be obtained by separating lipidic companions by ether precipitation. To this, the butanol extract was evaporated, the residue was dissolved in methanol and stirred in a five times the amount of 15 cold diethyl ether. The activity thus remained completely in the precipitate (Scheme in Fig. 9).

A particularly suitable process for enrichment is also adsorption of the centrifuged culture filtrate on Amberlite XAD-8 or related types of this polymer 20 on acrylic ester basis (company Serva). The attachment of

Epidermin is thereby destroyed not only by simple adsorption, but by the cation exchange effect of free acrylic acid groups on the resin. This indicates that the active components could only be dissolved from the resin by elution with methanol / wife. HCl (99: 1). The highly acidic eluate must be neutralized with ammonia before evaporation in vacuo. After this reprocessing by adsorption on Amberlite XAD-8, a subsequent reaction of methanol / ether could be dispensed with.

An isolation of Epidermin by adsorption can and does occur directly in the culture fluid already during the cultivation of the microorganism.

Stability tests and chromatographic studies were performed on lyophilized butanol extract.

The incubation of the extract with aqueous solutions of different pHs resulted in a strong decrease in activity from pH 10, but in the range of pH 2-7 the antibiotic is stable.

10 DK 172818 B1 In the thin layer chromatogram of the evaporated butanol extract there were several, with different spray reagents, colorful compounds, the pair of which carried out bioautograms giving important information 5 on the nature of the novel active substance. In acidic and almost all neutral systems, the antibiotic by thin layer chromatography on silica gel 60 remains seated at start, while chromatography with alkaline eluent yields Rp values between 0.3 and 0.75. The combination of 10 bioautography with thin layer chromatography in alkaline systems showed a correlation with a compound that could be stained with ninhydrin.

As a result of these experiments, it could be maintained that the antibiticum is a highly basic peptide that avoided further purification by column chromatography on silica gel 60 as it could not be eluted from the column with acidic or neutral systems.

Chromatography of the Amberlite XAD eluate or the lipid-free butanol extract on Sephadex 20 LH-20 with methanol / acetic acid (95: 5) separated a large number of small peptides, amino acids and salts contained in the medium from the antibiotic (Scheme Fig 0.9).

In the subsequent multiplicative countercurrent distribution according to Craig, the experiences gained in extracting the antibiotic from the culture filtrate could be used. In a first liquid-liquid distribution with the system n-butanol / ethyl acetate / 0.1N acetic acid (3: 1: 3), the antibiotic remained seated at start. In the second Craig distribution with the neutral system 2-butanol / 0.05N 30 ammonium acetate (1: 1), the antibiotic was found in the center of the apparatus. Ammonium acetate could again be removed by lyophilization in high vacuum.

After freeze-drying, epidermin exists as a homogeneous powder in all thin-layer systems used.

While the purification of the process according to the European patent application 0.027.710 was carried out by freeze-thaw extraction, evaporation of the extraction agent, ultrafiltration, precipitation with ammonium sulphate,

Efl «11 DK 172818 B1 ion exchange chromatograph1 and gel filtration on Sephadex G-50 or G-25 or G-15 or on Biogel P2, the present purification leads to a homogeneously pure product by adsorption of the centrifuged culture filtrate on Amber-5 lite XAD -8, followed by a gel chromatography on Sephadex LH-20 and then twice countercurrent distribution according to Craig. Thus, the isolation and purification of the process according to the above-mentioned European patent application and according to the process according to the invention are completely different.

Further differences from the known process according to the above-mentioned EP-A-0.027.710 consist in the choice of the complex nutrient solution. While the known nutrient solution Brain-Heart Infusion (37 g BHI / liter) only resulted in very poor antibiotic yield, a nutrient solution used according to the invention, consisting of 2-3% meat extract, 1-3% sugar or sugar alcohols, such as malt extract, maltose, galactose, lactose, mannite, glucose or glycerol, and 0.25-1% calcium carbonate or 0.25-0.5% calcium hydroxide, excellent results. The best production was achieved with a nutrient solution of the following composition: 3% meat extract, 2% malt extract and 0.37% calcium hydroxide. Of all C sources, maltose after malt extract showed the best production. Glucose should only be used in conjunction with other C sources. The combination of lactose and maltose or of galactose and maltose also produced good results. All other usual C sources showed no or only poor production. Addition of all 20 amino acids as N-30 source at concentrations of 2 g / liter for each produced the same results as the meat extract. Casamic acid (company Difco) is suitable as an N source only by the addition of tryptophan (1-2mM) and vitamins. Suitable vitamins (preferred concentrations in parentheses): Biotin (0.006 35 mg / l), nicotinic acid (2.3 mg / 1), thiamine (1.0 mg / 1), pyrodoxine, HCl (12.0 mg / 1), calcium pantothenate (1.2 mg / l).

12 DK 172818 B1

Cultivation takes place under good aeration at temperatures between 34 and 37 ° C. The best production process is obtained when the pH before cultivation is at 6.0-7.0. In the absence of carbonates or hydroxides of 5 divalent cations, such as calcium carbonate or calcium? hydroxide, no production takes place. After adding, for example, calcium carbonate, the pH shows a characteristic course of decrease into the acidic region, and thus no production took place. With subsequent increase of the pH into the slightly alkaline range, production started. Instead of calcium carbonate, magnesium carbonate can also be used, and calcium hydroxide produced better results than calcium carbonate. With 50 mM calcium hydroxide, the production compared to 25 mM calcium carbonate could be increased somewhat. By utilizing the C sources (sugar) of the strain, organic acids are formed, which are formed by bivalent cations, thereby simultaneously buffering the medium.

20 Fig. 10 shows the results regarding the course of the number of live germs and antibiotic production, expressed in ramification ring against Micrococcus luteus ATCC 9341, for the media in question compared with Brain-Heart-Infusion medium (company Difco) according to above-25 said European patent.

By means of an adjustment line, the plate diffusion test found the activity increases listed below at the time of production maximum.

Activity in the Nutrient Solution Brain-Heart-30 Infusion was set to 100%.

a) Brain-Heart Infusion Agar 100% I b) 3% Meat Extract, 2% Malt Extract, 25 mM Calcium Carbonate 200% 35 c) 3% Meat Extract, 2% Malt Extract, 50 mM Calcium Hydroxide 320% J.I.iflf 13 DK 172818 B1

These indications show no absolute production values. However, a comparison of the values obtained by the plate diffusion test clearly showed that compared with the known working method 5, a significant increase in the yields is obtained using the method according to the invention.

The strain used to prepare Epidermin is characterized by Schleifer & Kloos (Int.J.Syst.Bact.25, 50-61 (1975)) as follows: 10

Gram staining: positive

Cell size j diameter 0.5-0.8 µm

Colony size: approx. 1 mm diameter

Colony appearance: smooth, shiny, in the middle ^ slightly elevated

Colony color: greyish-white

Cells in culture: often single cells or two together, rarely larger heap 2Q Hemolysis: stratified cultures showed strong hemolysis on platelets

Anaerobic growth: the strain also grows under anaerobic conditions 25 Lysozyme sensitivity: cells are up to 2 mg / fol resistant to far lysozyme

Lysostaphin sensitivity j cells are already sensitive to lysostaphin 2Q at 20 µg / ml Epidermin Resistance: detected up to 1 mg / ml

Other resistances: in liquid culture, strain shows but a marked resistance to Streptomycin (up to 1 mg / ml) and Spectinanycin (0.5 mg / 35 ml)

Elevated NaCl content: the strain still grows well at 15 wt% sodium chloride.

14 DK 172818 B1

Colonies or extinct cultures are very easy to reach.

The utilization of various C sources during acid formation and other enzyme reactions was determined by aide 5 of the Api-Staph system (company Biomerieux, NOrtingen).

This system is based on a combination of 20 biochemical reactions that can be traced back to the classification According to KLOOS ft SCHLEIFER (J. Clin. Microbiol.

1, 82-88 (1975)).

10

Table I

C source utilization and other enzyme reactions C source or enzyme Pro-Staph. epid. Staph.epid.

____ dueer.t KioosfcSchleifer Aoi - Staph 15 Control - D-Glucose + k.A. + D-Fructose + + + D-Mannose {+) (+) +

Maltose +++

Lactose (+) (+) + D-Trehalose - D-Mannitol - xylitol - D-Melibiosis - k.A.

Raffinose - k.A.

D-xylose -

Sucrose + + + "Ί Q- Methyleluceside - k.A.

N-Acetylglucosarin - k.A. +

Nitrate Reduction + ♦ * I ^ 3q Phosphatase +++

Formation of acetylr.ethyl- + k.A. + carfcinol (Voces- ros - .. kauerraaction)

Areinindehydroiase - k.A. +

Urease + K.A. + 35 _ rn 11 ..... m

' "'" WiliiiS

15 DK 172818 B1 + positive reaction negative reaction (+) unambiguous positive reaction occurs later somewhat + variable property 5 k. A. no information

The strain Staphylococcus epidermidis DSM 3095 was seeded onto small incised tubes with a medium of the following composition:

Peptone 10 g

Disodium hydrophosphate 2 g Meat extract 8 g

Glucose 10 g (separate autoclaved)

Sodium chloride 3 g pH 7.2 g then incubated overnight at 37 ° C and finally frozen at -20 ° C. For new experiments, a fresh tube was always thawed as, with prolonged storage at 4 ° C, 20 activity losses were observed.

In the following, the preparation of the antibiotic drug Epidermin is discussed in more detail.

The culture can be carried out in suitable shake flasks, but for the production of larger amounts of substance 25 a culture container of 200 liters or more can be used.

For flask experiments, 500 ml Erlen flask with side inserts were used. The flasks were filled with 100 ml of nutrient solution and autoclaved for 20 minutes 30 at 121 ° C. As seed material, 1% of a 4 hour old pre-culture served. The incubation was performed at 37 ° C on a rotary shaker at 140 rpra.

For 10-liter culture, 10-liter culture vessels (Model MF-14 New 35 Brunswick, Scientific Co., New Brunswick, USA) were filled with 9.9 liters of nutrient solution and autoclaved for 30 minutes at 134 ° C. After cooling, 100 ml of a 4 hour old preculture was seeded with 100 ml of 16 culture and grown at 37 ° C, 0.6 in wm and 240 rpm for the blade stirrer. The relatively strong foaming was counteracted by repeated addition of sterile polyol.

. 5 For 25-liter culture, a 25-liter culture vessel (type b 25, Braun / Melsungen with sorting system) was provided with 25 liters of nutrient solution with the addition of 3 ml of polyol and sterilized at 121 ° C for 30 minutes. As seed material, 300 ml of 10 served a 4-hour old culture. The cultivation took place at 37 ° C, 0.6 wm and 1000 rpm.

The progress of a 10 liter scale cultivation in a nutrient solution containing 30 g of meat extract, 20 g of malt extract and 5 g of calcium carbonate in 1 liter is shown in Fig. 11. Intensive growth associated with utilization of the C sources offered during acidification can be seen by the decrease in pH. At the start of the alkalization, the antibiotic can be detected in the culture filtrate. Production reaches its maximum after 12-18 hours.

20 To control the course of cultivation, samples were taken sterile at various times during cultivation. The samples were evaluated for the following: a) pH-Value Measurement with a laboratory pH meter (Knick pH-mV meter).

25 b) Growth course: Growth could be followed by the increase in the number of 1 live germs. To this, 0.5 ml of sterile culture culture was diluted in brine, of which 0.1 ml was plated on plates (medium; peptone 10 g, meat extract 8 g, sodium chloride 3 g, disodium hydrophosphate 2 g, glucose 10 g, to 1 liter). After 18 hours of incubation at 37 ° C, the single colonies could be counted; c) Antibiotic concentration:

The samples were centrifuged in an Eppendorf centrifuge 3200 for 2 minutes, and 20 µl of the supernatant was tested by the plate diffusion test.

In parallel, an adjustment curve of known concentrations was produced.

7s' ΪΪ 17 DK 172818 B1

After reaching the production maxima, the culture liquid was centrifuged by continuous centrifugation (centrifuge: Type LA 71b-4, Loher & Sohne, Ruhstorf /

Rat) at 1380 rpm. For optimum cell separation, the passage rate should be kept very low.

An initial enrichment of the active components was obtained by the above-mentioned adsorption on Amberlite XAD-8, according to Example 1 below.

Example 1 80 liters Culture filtrate was poured over approx. 8 liter Amberlite XAD-8. No activity could be detected during the passage. On subsequent washing with water, no activity was also eluted. Then 15 were added with 13 liters of methanol. No activity was detected in the wash water. The elution was carried out with methanol / hydrochloric acid 99: 1 to give the following fractions: 2q "Fraction 1: 0.8 liters active

Fraction 2: 4.5 liters Main activity

Fraction 3: 2 liters active

Fraction 4: 2 liters no activity

Fraction 5: 1 liter no activity 25

The fractions were tested thin-layer chromatographically for their content of Epidermin, then the active fractions were pooled and evaporated to dryness on a rotary evaporator (81.2 g), the residue was taken up in methanol / acetic acid (95/5) (400 ml), insoluble matter 30 rial was centrifuged and portions of each 50 ml were gel chromatographed on Sephadex LH-20 (column 100x5 cm, eluent methanol / acetic acid 95: 5). Positive fractions were collected and evaporated (14.8 g).

For multiplicative distribution, according to Craig, appliance from the company Labortec (Basel) is used. An initial separation was performed with a 50 ml * s apparatus in the system n-butanol / ethyl acetate / Ο, ΙΜ acetic acid (3/1/3).

The sample (5 ml) dissolved in the lower phase (5 g) was subjected to the following separation conditions in 18 DK 172818 B1; In Trintal: 160 5 Shaking movements / steps: 70 '5 Shaking intensity: 45 • Separation time: 20 min.

For the final purification, the crude product obtained (4.2 g) in portions of each 2 g was dissolved in the lower phase of the system 2-butanol / 0.05N ammonium acetate (50 ml) 10 and purified in a 10 ml apparatus with 440 elements .

Trine number: 440

Shaking movements / steps: 50

Shaking intensity: 50

Separation time: 10 min.

The Epidermin-containing elements were collected, evaporated, taken up in water and lyophilized several times in high vacuum. The antibiotic (2.6 g) was found to be homogeneous in all studies performed.

20

During the manufacture and isolation of the Epidermi net, the plate diffusion test was used for the biological characterization, namely both for recording the spectrum of effect and for controlling production, processing and enrichment. The test, as described by Zåhner and Haas, 25 Biology of antibiotics (Springer Verlag, Berlin-Heidelberg-New York, 1972), was conducted in Petri dishes from Greiner, Nurtingen, with 6 mm diameter filter rounds (Machetey & Nail, Diiren). The filter rounds were wetted with 20 µl of the test liquid in question, dried at room temperature on a glass plate and placed on the test plates. The test plates were incubated at 37 ° C and after ca. At 16 hours, growth inhibition was assessed.

As a routine test germ, Streptococcus pyogenes 35 ATCC 8668 and, because of its problem-free handling, served Micrococcus luteus ATCC 9341.

IH SKI

! Ti r Ti 19 DK 172818 B1

The test plates with Streptococcus pyogenes ATCC 8668: Before preparation of the test plates, the test germ had to be germinated fresh on platelets with mucin (medium: 500 ml agar pH 7.4, 160 ml mucin solution (10 wt), 3.5 5 ml glucose solution (50 wt), 70 ml bed blood).

After 15-18 hours of incubation at 37 ° C, a saline suspension (E ^g 0.5) was prepared and pipetted into 100 ml of nutrient substrate (medium: peptone 10g, meat extract 8g, 10 sodium chloride 3). g, Na 2 HPO 4 2 g, glucose 10 g to 1 liter, pH 7.2). The poured plates of 10 ml portions were durable for some days at 4 ° C.

Test plates with Micrococcus luteus ATCC 9341:

An overnight culture (medium: peptone 10 g, 15 meat extract 8 g, sodium chloride 3 g, Na 2 HPO 4 2 g, glucose 10 g to 1 liter, pH 7.2) was diluted to an extinction of 1.0 at 578 nm. With 0.25 ml of this suspension, 100 ml of agar was seeded and 10 ml * s plates were poured. The test plates could be stored for 20 days at 4 ° C.

Test plates for the spectrum of effect:

Bacteria test plates: a) Aerobically live bacteria:

Overnight cultures were introduced into the respective 25 test media and the plates prepared as described for Micrococcus luteus ATCC 9341.

8) Anaerobic live bacteria:

The precultures of Clostridium pasteurianum ATCC 6013 and Propionibacterium aenes DSM 1897 were introduced into 30 test tubes filled with nutrient solution up to the cotton plug to displace the oxygen.

With 3 ml of pre-culture, 100 ml of agar were seeded and 10 ml of plates were poured.

The incubation took place in Anaerobier-Topf (BBL-Gas Pak 35 100, Becton, Dickinson GmbH, Heidelberg) at the optimum temperature in each case.

20 DK 172818 B1

Test plates with yeast-growing fungi: In shake culture, cells were cultured in the test medium in question for 18-20 hours. After counting in

Thoma counting chamber, the test plates were seeded at a density of 106 organisms / ml of agar.

Test plates with fungi and Streptomyceter:

The test organisms were cultured on small swab (medium: yeast extract 4 g, malt extract 10 g, glucose 4 g to 1 liter) at the relevant temperatures until 10 sporulation. The spores were rinsed with 3 ml of saline-Tween 80 (1 drop of Tween 80 to 100 ml of saline) and poured into test plates.

The drug Epidermin has an excellent antibacterial activity. It works particularly well against a wide range of gram-positive bacteria. The antibacterial effect of Epidermin was examined by comparison with the action of Nlsin (Nisin, cf., among others, German Ausleglegrift DE-A-2,000,818 or British Patent GB-B-1,182,156). For some important, clinically relevant 20 germs, fusidic acid was also used for comparison. The activity was found by the plate diffusion test and by determining the minimum inhibition concentrations.

a) Plate diffusion test: 25 In the plate diffusion test, the sensitivity of various microorganisms to Epidermin and Nisin was tested. Table II shows that both antibiotics work almost exclusively on gram-positive bacteria. Nisin with an activity of 4000 units was used.

, 30

Table II

Sensitivity to Epidermin and Nisin. Indications in mm inhibition ring, concentrations 1 mg / ml and 0.5 mg / ml.

”i · 21 DK 172818 B1

Microorganisms Culture conditions Epidermin Nisin

Temp. Medium 1 0.5 1 0.5

Bacteria ^ Eubacteriales, gram positive

Arthrobacter aureseens 27®C 5 8 Sp 9 8

Arthrobacter crystallopoietes 27®C 5 16 15 16 15

Arthrobacter globiformis 27®C 5 14 12 14 13

Arthrobacter oxydens 27eC 5 12 11 11 10 10 Arthrobacter pascens 27eC 5 13 12 14 13

Bacillus cereus 37®C 3 8 Sp

Bacillus pumilus 37eC 4 14 12 12 10

Bacillus subtilis ATCC 6051 37®C 4 12 11 9 8

Bacillus subtilis ATCC 6051 37®C 6 17 16 14 12 15 Bacillus subtilis ATCC 6633 37®C 4 14 11 9 8

Bacillus subtilis F 24-2 37 ° C 4 13 11 8 Sp

Bacillus subtilis A 14 37®C 4 12 10 9 Sp

Brevibacterium flavum 37®C 4 11 10 8 7

Clostridium pasteurianum 30 ° C 7 17 15 14 13

Clostridium sporogenes 37®C 8 10 '9 8 Sp

Corynebacterium spec. 27C 4 15 13 9 8

Corynebacterium insidiosum 27eC 4 19 17 10 8

Corynebacterium rathayi 27®C 4 14 12 10 9

Micrococcus luteus ATCC 381 27eC 5 10 9

Micrococcus luteus ATCC 9341 27®C 2 21 20 17 16 25 Propionibacterium aenes 37 "C 8 22 19 21 18

Sarcir.a lutea 37®C 5 15 14 15 14

Staphylococcus aureus Tu 202 37eC 4 13 10 10 9

Staphylococcus aureus DSM 683 37®C 4 11 9 Sp -

Staphylococcus aureus Pen.res 37®C 4 12 11 9 8

Staphylococcus cohnii 37®C 2 14 12 9 -

Streptococcus pyogenes 37®C 2 22 20 18 17

Eubacteriales, gram negative:

Proteus mirabilis 37 * C 4 11 9 9 7 35 Proteus vulgaris 37®C 5 9 8 8 Sp 22 DK 172818 B1

Actinomycetales:

Streptomyces glaucescens 27 * C 3 10 8 10 9 j Streptomyces violaceorubes 37 * C 3 9 8-- 5 Streptomyces vicido- 37 * C 3 11 10 98 cbromogenes (") means that no effect Sp" track 10 b) Minimum inhibitory concentration:

The minimum inhibitory concentration for clinically relevant germs, expressed in µg / ml, was tested in microtiter plates in the following medium: 25 ml of Na-Lactate, 5 g of Na 2 SO 4 0.5 g KI

MgCl₂, 5 g NH4 Cl, 10 g glucose, 50 g calcium pantothenate, 50 g thiamine, 0.25 g folic acid, 50 g niacin, 25 g p-aminobenzoic acid, 50 g pyridoxine hydrochloride, 25 g riboflavin in 1000 ml distilled water Table 1 shows the minimum inhibitory concentrations (µg / ml) of Epidermin and for comparison Nisin and fusidic acid. The Nisin used had an activity of & 2500 units.

Table III

Minimum residue concentration in µg / ml:

Epidermin Fusidic Acid Nisin

St. eoidermidls WG 99_ 2_ 4_128 30 Sc. pvooenes ATCC 8668_0.125__1_4

Sc. pneumoniae ATCC 6302_2 16 32

Me. luteus ATCC 159S7_0.25_0j_5_16

Hc. luteus ATCC 9341 _- <0.06 _0_l5_8

Cb. xerosis NCTC 9755_0,125__0,125_16 35 E. ~ coli ATCC 11775_128 __> 128_> 128 E. coll ATCC 9637_128_> 128_> 128

Propionib. aenes PC 904_0.06. 0,5_32

Propionib. aenes ATCC 25746 0.25 1 64 • i i 23 DK 172818 B1

The media listed in Table II are described below. After adjusting the pH, the media was autoclaved for 20 minutes at 121 ° C.

The quantity declarations always refer to 1 liter of water. With 5 chemically defined media, dedonized water was used.

Medium: (2) Glucose agar medium:

Peptone 10 g Meat extract 8 g 10 NaCl 3 g

Na2HPO4 2 g

Glucose 10 g (separate autoclaved)

Agar 20 g pH 7.2 (3) Yeast malt medium: Yeast extract 4 g

Malt extract 10 g

Glucose 4 g 20 Years 20 9 pH 7.3 (4) Oxide medium for bacteria: Meat extract 10 g

Peptone 10 g 25 "NaCl 5 g

Agar 20 g PH 7.2 (5) Nutrient Fluid 8 g 30 (Company Difco)

Agar 20 g PH 7.2 (6) Minimum medium for bacteria (HOTTER et al., 1966): 35 D-Glucose 8 g

Di-ammonium tartrate 4 g 24 B1

NaCl 5 g K2HPO4 2 g

MgSO 4 x 7 H 2 O 1 g 1 CaCl 2 0.2 g 5 MnSO 4 x H 2 O 0.01 g

Ferrioxamine B 0.02 g

Agar 20 g pH 7.2 (7) Medium for Clostridium pasteurianum: Meat extract 3 g Yeast extract 3 g

Malt extract 3 g

Peptone 20 g D-Glucose 5 g

Ascorbic acid 0.2 g

Agar 20 g ™ pH 7.0 (8) Brewer thioglycolate medium for Propionibacterlum 20 aenes:

Thioglycolate medium 40.5 g Agar 20 g pH 7.2 Epidermin is well tolerated and no topical activity has been observed in topical use.

It is a great advantage that the resistant mutant Staphylococcus epldermidis DSM 3095 is resistant to the epidermin produced by the mutant. The strain NCIB 30 11536 has no such resistance to the low molecular weight antibiotic produced by this strain. j The novel clone DSM 3095 was won as follows:

The adaptation was carried out in 100 ml side Erlenmeyer 35 flasks with lateral insert and 10 ml nutrient solution 25 (Brain-Heart-Infusion). The starting culture was seeded with 0.5 ml of a 12 hour old culture. To the flasks containing increasing Epidermin concentrations, 0.5 ml of the each preceding 5 well-grown culture served as seed material. The growth assessment was performed photometrically at 578 nm. The culture, which, at a concentration of 1.0 mg / ml of liquid culture, was still well grown, was diluted in saline and plated on a plate containing 0.5 mg / ml of Epidermin.

10 As a comparison value, an undiluted 12 hour old culture of the comparative known NCIB 11536 from the literature was plated. After 24 hours of incubation at 37 ° C, 94 single colonies appeared in the 10 ° ® diluted adapted culture. The diluted plated comparative strain on plates 15 containing 0.15 mg / ml of Epidermin still did not grow after 48 hours of incubation. The resistant colonies were selected and screened in plate medium 2. After a pre-test for production by 20 mm diameter cut-outs and activity tests on Micrococcus luteus ATCC 9341, producing clones were tested in liquid medium.

Comparison of the resistant strain with the strain known in the literature: a) Minimal inhibition concentration by plate diffusion test: On test plates down the two strains, the minimal inhibition concentrations listed in Table IV were found.

Table IV

Minimum inhibitory concentrations of Epidermin in plate diffusion testing with NCIB 11536 and DSM 3095.

Strain ^ pg / ml 35 ---- NCIB 11536 10 DSM 3095> 1000 26 DK 172818 B1 b) __ Growth_02_productign: Growth and production progress of the two strains were compared in nutrient solution (3% meat extract, 2% malt extract, 0.37 % Ca (OH) 2). See Pig. 12th

5 Growth course: Determination of number of live germs

Production course: Activity against Micrococcus luteus ATCC 9341 by plate diffusion test.

In the resistant strain, better production is observed.

10

Sl_.§SSSifeiiitS £ -2JiS £ _l2E_5Ei§S £ lSi2 »i-.f2 £ §l £ §iii9§_Y5lSSi“ phases:

The self-inhibition experiments of the producer strain with the antibiocum and the study of the resistance of the selected clones were carried out in a biophotometer (Eppendorf photometer with circulator).

These experiments could be carried out in detail, for example, as follows: A 12 hour old culture of Staphylococcus 20 epidermidis NCIB 11536 or of the selected resistant strain DMS 3095 was each grafted 0.5 ml onto fresh medium.

These cultures were allowed to grow high up to an extinction of 0.043 at 578 nm (cuvettes with a layer thickness of 1 cm), after which they were distributed in 7.5 ml aliquots in the biophotometer cuvettes (layer thickness 2 cm).

The density of the cell suspension was adjusted to a 90% transmission in the bio-photometer. The incubation took place at 37 ° C and with maximum aeration.

Epidermin was added in the form of aqueous solution at various growth stages.

At both strains, different Epidermin concentrations were added initially and in the middle of the logarithmic phase. The results are shown in FIG. 13 and 14.

With the concentrations tested so far, no • i could be observed in the resistant strain

A

27 DK 172818 B1 lysis.

The antibiotic Epidermin in question, like the culture wash from which it is isolated, exerts a bactericidal effect that leads to lysis of the cells.

As regards the bactericidal action and the broad spectrum of action against gram-positive bacteria, the subject polypeptide antibiotic Epidermin is particularly active in the treatment of infections caused by gram-positive bacteria. Epidermin is particularly valuable for combating skin infections such as eczema, impetigo, cellulitis and especially acne.

The excellent activity against some important Propioni bacterium acnes strains is shown in Tables II and III.

It can be said that Epidermin, normally produced by organisms that colonize the human skin, exerts better activity in protecting the skin than other usual antibiotics.

The aforementioned pharmaceutical formulations containing the antibiotic of this invention together with 20 pharmaceutically usual adjuvants and carriers can be used orally, parenterally, enterally or topically.

Such formulations, but especially topical formulations, may be present as solutions, emulsions, gels, lotions, ointments, creams or powders.

The following examples show the preparation of such pharmaceutical uses.

Example 2

Tincture 30 In 100 g of tincture is contained

Epidermin 1.0 g

Ethanol (94.5 vol%) 56.0 g 1,2-propylene glycol 40.0 g

Demineralized water 3.0 g 35

Preparation:

The epidermin is dissolved in the ethanol / 1,2-propylene glycol / water mixture and the solution is sterilized.

DK 172818 B1 28

Example 3

Lotion I 100 g lotion contains: in Epidermin 1.00 g 5 1,2-propylene glycol 7.00 g

Alkyldimethylbenzylammonium chloride (Benzalkon A®) 0.15 g

Sorbitan monopalmitate (Splan 40 ®) 0.40 g

Sorbitam Acrogol Palmitate (Tween 40 ®) 1.20 g of Acetic Acid Decyl Ester (Cetiol V ®) 2.40 g

Mixture of cetyl and λ stearyl alcohol (Lanette 0 ^ 1.60 g

Cetylplamitate 0.80 g Demineralized water to 100 g

Preparation:

The above-mentioned amounts of alkyl dimethylbenzyl ammonium chloride, sorbitan monopalmitate, sorbimacrogol plamitate, oleic acid decyl ether, cetyl and stearyl alcohol 20 and cetylplamitate are stirred in 75 ml of water, the filtered solution of Epidermine is dissolved in water and 1,2-propylene glycol.

Example 4 Gel 5 100 g Gel contains:

Epidermin 9

Polyethylene glycol ether of lauryl alcohol (Brij 35®) 1.0 g of 1,2-propylene glycol 5.0 g of Acrylic Acid Polymerize (Carbopol 934 ®) 1.2 g of p-Hydroxybenzoic acid methyl ester 1.6 g of p-Hydroxybenzoic acid propyl ester 0.4 g

Perfume q.s.

Sodium liquor to pH 6.5

Demineralized water to 100 g in • 4

..... J

29 DK 172818 B1

Preparation:

The aforementioned amounts of auxiliaries are stirred in 75 ml of water. The epidermin is dissolved in a mixture of 1,2-propylene glycol and the remaining water, and this solution is also stirred. The finished gel is further homogenized.

TegnlnqsforklarInger:

FIG. 1: Amino acid chromatogram for the Epidermin acidic total hydrolyzate; Ninhydrin staining, λ = 570 nm.

FIG. 2: Gas chromatogram for the N-pentafluoropropionyl-amino acid-n-propyl ester of the acidic total hydrolyzate of Epidermin on Chirasil-Val.

Temperature program: 3 min. 85 ° C isotherm, which after 4 ° C / min. to 200 ° C. Carrier gas H2 (0.92 bar).

FIG. 3: UV spectrum of Epidermin in water, pH 3 (C * 0.15 mg / ml).

FIG. 4: IR spectrum of Epidermin in a potassium bromide tablet.

FIG. 5: 1 H NMR spectroscopy of Epidermin (20 mg / 0.5 ml of D 1 -dimethylformamide, 400.16 MHz).

FIG. 6: 1'C NMR spectrum of Epidermin (40 mg / 0.5 ml of 12C, 2H-dimethylformamide, 100.6 MHz, 45360 pulses).

FIG. 7: HPLC Chromatogram for Epidermin on u Bondapak (300x3.9 mm). Mobile phase: A * acetonitrile / 0.01M KH2PO4 (10/90), B »acetonitrile / 0.01M KH2PO4 (70/30); linear gradient from 10% B to 100% B in 30 minutes, flow rate 2 ml / min.

FIG. Δ C NMR spectrum of the fragment P2 (12 mg / 0.5 ml of 12 C, 2H-dimethylformamide, 100.6 MHz, 38300 pulses).

FIG. 9: Isolation of Epidermin.

FIG. 10: Comparison of media in terms of number of live germs (full lines) and activity 30 DK 172818 B1 versus Micrococcus luteus (dashed lines), ▲ Brain-Heart Infusion o 3% meat extract, 2% malt extract, 0.25% CaCO ^ • 3% meat extract, 2% malt extract, 0.37% 5 Ca (OH) 2>

FIG. 11: Progress of cultivation on a 10 liter scale • pH Course A- Number of live germs o Activity towards Streptococcus pyogenes 10 ATCC 8668.

FIG. 12: Comparison of strain known and resistant clone DMS 3095 in terms of number of live germs (full lines) and activity against Micrococcus luteus ATCC 9341 (dotted lines).

o Tribe known from literature Æ. Resistant clone

FIG. 13: Epidermin addition at different growth stages for Staphylococcus epidermidis NCIB 11536: 20 Curve 1: Normal growth course

Curve 2: Addition of 10 µg / ml Epidermin at the beginning of the logarithmic phase leads to lysis.

Curve 3: Addition of 10 µg / ml Epidermin at the middle of the logarithmic phase leads straight to lysis.

Curve 4: Addition of 2.5 µg / ml Epidermin at the middle of the logarithmic phase leads to growth retardation.

FIG. 14: Epidermal Addition in Different Growth Phases for Resistant Clone DSM 3095:

Curve 1: Normal growth course, identical to strain NCIB 11536.

Curve 2: Addition of 120 µg / ml Epidermin 35 at the beginning of the logarithmic phase results in only a slight growth delay.

Curve 3: Addition of 360 µg / ml Epidermin at the middle of the logarithmic phase also leads here only to a growth retardation.

FIG. 15: Gas chromatogram for the N-pentafluoropropionylamino acid-n-propyl ester of the total hydrolyzate of H1 at Chirasil-Val. Temperature program: 5 3 min. 80 ° C isothermal, then 4 ° C / min.

to 200 ° C, carrier gas Hj.

Claims (9)

32 DK 172818 B1 1. Nature: Colorless powder. An antibiotic active polypeptide, designated Epidermin, characterized in that it has the following amino acid composition: Asn (1), Pro (1) »Gly 5 (2), Ala (2), Ile (2) 'Phe (2)' Light (2), Lan (2), β-Me-Lan (1), Dhb (1), Tyr (1), S- (2-aminovinyl) -D-cysteine (1) with the following primary structure: meso-Lanthionine CH , - S - CH, II H-Ile-Ala-NH-CE-CO-Lys-Phe-Ila-NH-CH-CO - (S) (R> CH, f1-Methyllanthionine (S) CH-S-CH, Ί I UNK-CH-CO-Pro-Gly-HN-CH-CO-Ala-Lys- (S) (R> 20 ~ _ <pH 3 meso -Lanthionine CH CK, - S -CH, || (SI I (2) -HN-C-CO-Gly-HN-CK-CO-Phe-Asn-KN-CE-CO-Tyr-HN-CH-CO -NE-CH 25 (Z) (S) I (£) || CK2-S - CH, S- (2-Aminovinyl) -D-cysteine in which the nearest N-terminus halves of the individual thioether amino acids each have D configuration. 2. Solubility: Very well soluble in mixtures of water / glacial acetic acid or methanol / glacial acetic acid, soluble in lower alcohols, insoluble in chloroform, acetone, diethyl ether, petroleum ether. Epidermin according to claim 1, characterized in that it has the following additional parameters: '35 3' 33 DK 172818 B1 Process for the preparation, isolation and purification of the antibiotic Epidermin, according to claim 1 or 2, characterized in that Staphylococcus epidermidis DSM 3095 is aerobically grown at 34-37 ° C in a complex nutrient solution consisting of 2 to 4% of a nitrogen source, such as meat extract, 1-3% of a sugar or sugar alcohol as carbon source and 0.25-1% of a carbonate and / or 0.25-0.5% of a hydroxide of an alkaline earth metal, after which the culture growth, after 34 B1 removal of the cells and inorganic salts, to isolate the active substance formed, is either extracted with n-butanol at pH 8, then the butanol extract is evaporated, the residue is dissolved in methanol, and the solution by ether precipitation is freed from the lipidic companions, or treated. with acrylic ester or polystyrene-based polymers, wherein the adsorbed Epidermin is dissolved from the methanol / conc. hydrochloric acid (99: 1), whereupon the solution is neutralized with ammonia and evaporated in vacuo, and the thus obtained isolates are separated to separate further low molecular peptides, amino acids and salt gel chromatographs (Sephadex LH-20 with methanol / acetic acid (95: 5)) and then subjected to a multiplicative countercurrent distribution according to Craig in which a liquid-liquid distribution is first made with the system n-butanol / ethyl acetate / Ο, ΙΝ acetic acid (3: 1: 3), leaving Epidermin sitting at start and then by another distribution with the neutral system 2-20 butanol / 0.05N ammonium acetate (1: 1), isolation of the pure Epidermin is obtained and the Epidermin is isolated in the form of colorless powder from the eluate by freeze-drying. 3. Color reaction to silica gel plates: Ninhydrin, chlorine / TDM = 4,4'-bis (dimethylamino) diphenylmethane), orcin / sulfuric acid, anisaldehyde / sulfuric acid. Destruction-free detection in UV light at 254 nm and by spraying with water. Process according to claim 3, characterized in that 2-4 weight of a meat extract is introduced into the culture medium as nitrogen source, a mixture of all 20 amino acids at amino acid concentrations of 2 g / liter for each, or 2-4 % casamic acid in the presence of tryptophan to which carbon source is introduced 1-3% by weight malt extract, maltose, galactose, lactose, mannite, glucose, glycerol or combinations of lactose with, maltose or galactose with maltose, furthermore 0.25-1% by weight calcium carbonate or 0.25-0.5% by weight of calcium hydroxide, and optionally further vitamins such as biotin, nicotinic acid, thiamine, pyridoxine, HCl and calcium pantothenate are added and the pH before cultivation is adjusted to 6.0-7.0 , and the production process is followed by continuous sampling. 4. Thin-layer chromatography: Silica gel finished plates 60 F254 (Merc ^) System A: Chloroform / methanol / 17% ammonia (2/2/1) RF = 0.73 System B: Chloroform / methanol / 17% ammonia (70 / 35/10) RF = 0.30 System C: n-Butanol / ice blanket / water (4/1/1) RF = 0.05 The epidermin obtained by the method of claim 3. 5. HPLC: See Fig. 7. 6. Epidermin-derived Ten Mating and Epldennine-resistant strain # Staphylococcus epidermidis DSM 3095. 6. Stability: Stable from pH52 to pH = 7, at higher pH values, strong activity decline occurs. 7. Stapylococcus epidermidis DSM 3095 # 5 characterized by # having the following characteristics: Gram staining positive Cell size: diameter 0.5-0.8 µm Colony size: approx. Mm mm 10 Colony appearance: smooth, shiny, middle slightly raised Colony color : greyish-white Cells in culture: often single cells or two together, rarely 15 'larger heap Hemolysis: strong cultures depicted on platelets showed strong hemolysis Anaerobic growth: strain also grows under 20 anaerobic conditions Lysozyme sensitivity: cells up to 2 mg / hil lysozyme-resistant Lysostaphin sensitivity: cells are already at 20 µg / ml sensitized to lysostaphin Epidermin-Resistance: detected up to 1 mg / ml Other resistances: in liquid culture, strain shows a pronounced resistance to Streptomycin (up to 1 mg / ml) and Spectinanycin (0.5 mg / ml) Increased NaCl content: the strain still grows well at 15 wt% sodium chloride. where the colonies or the extinct cultures are sticky. 36 DK 172818 B1 7. Molecular mass: In the region of 2160 (without anions} Antibacterial drug preparations, characterized in that they contain an Epidermin content according to claim 1 or 2 in addition to conventional carriers and / or excipients. 8. Ultraviolet absorption spectrum: In aqueous solution, long-wave maximum at 267 nm (Fig. 3). 9. Infrared absorption spectrum: IR spectrum of a sample in a potassium bromide tablet (Fig. 4). 10. Nuclear Magnetic Resonance Spectra: 1 H-NMR Spectrum: Fig. 5, C-NMR Spectrum: Fig. 6. Use of Epidermin according to claim 1 or 2, for the control of bacteria. "v9 -« lU .m