KR820001204B1 - Method for producing antibiotic c-15003 p-3 - Google Patents

Abstract

Antibiotic C-15003 P-3 is obtained with high yield by cultivation on a culture medium contg. valine and isobutyric acid. Thus, 40ml inoculant medium(pH = 7.0) is added to 200ml erlenmeyer flask followed by inoculation with Nocardia species No C-15003 and incubation on a rotary shaker. The resulting cells are washed with distd. water and resuspended to original volumn. 1ml of resulting material is inoculated into 40 ml culture medium and this main culture is incubated at 28≰C for 8days. The total yield of C-15003 was 16μg/mL and the ratio of P-3 corresponding to total material is 95 wt.%.

Description

Preparation of antibiotic C-15003 P-3

The present invention relates to a process for industrially advantageously preparing antibiotic C-15003 P-3.

Antibiotic C-15003 P-3 (hereinafter sometimes abbreviated as "P-3") is a novel compound obtained by culturing microorganisms of the genus Nokaldia isolated from samples such as soil. The manufacturing method is already filed (Japanese Patent Application No. 52-37166, Japanese Patent Application No. 52-37866, Korean Patent Application No. 1622 in 1977) The bacteria used in the above method is a kind of each component of the antibiotic C-15003 or Since more than that is produced and accumulated at the same time, if only a specific component thereof is to be collected, the separation and purification process is slightly complicated, and the yield is not necessarily high.

The present inventors have conducted various studies on a method for efficiently producing P-3 by simplifying the separation and purification process, and by adding a specific substance to the medium, the target P-3 is extremely efficient, or The present invention has been completed as a result of a new discovery that it can be produced in a state close to a single product, and further studies have been conducted accordingly.

That is, according to the present invention, the antibiotic C-15003 producing bacteria belonging to the genus Nokaldia is cultured in a medium to which isobutykill, a precursor of Co and / or a fatty acid having 4 carbon atoms are added, and the antibiotic C is cultured in the culture. -15003 is a method for producing antibiotic C-15003 P-3 characterized by specifically producing and accumulating and collecting P-3.

In the present invention, "antibiotic C-15003" or "C-15003" is a generic term including each of two compounds or a mixture of two or more of the four compounds represented by the following general formula (I).

, -CO-CH₂-CH₂-CH₃또는

을 나타낸다.)Wherein R is CO-CH ₂ -CH ₃ ,

, -CO-CH ₂ -CH ₂ -CH ₃ or

Is displayed.)

인 화합물을 "항생물질 C-15003 P-3"으로, R가 -CO-CH₂-CH₂-CH₃인 화합물을"항생물질 C-15003 P-3'" 혹은 단지 "P-3'로, R가

인 화합물을 "항생 물질 P-15003 P-4" 혹은 단지 "P-4"로, 각각 호칭하는 것으로 한다.Are those wherein R is -CO-CH ₂ -CH ₃ in the formula (I) as "antibiotic C-15003 P-2", or just "P-2", R,

Phosphorus compound as "antibiotic C-15003 P-3", R is -CO-CH ₂ -CH ₂ -CH ₃ compound as "antibiotic C-15003 P-3 '" or just "P-3' , R

Phosphorus compounds shall be referred to as "antibiotic P-15003 P-4" or only "P-4" respectively.

As a monarch which can be used for the method of this invention, for example, Actinomycetes No. C-15003 note (Hereinafter, abbreviated as "No. C-15003"), etc. are mentioned.

No. Method of Charing and High Tribe of C-15003

According to [International Journal of Sysetematic Bacteriology, Vol. 16, pp. 313-340, 1966], the results observed over 28 days at 28 ° C were as follows. same.

1) Morphological features

Parasitic hyphae are well elongated and branched in both agar and liquid media. Most of the diameter is 0.8-1.2 micrometers, and sometimes it may divide into a bacilli or a branched short mycelium.

Although it grows well on various sorting mediums, the mycelia are grown on parasitic mycelia, but often form a supernatant (50 to 020 µm × 200 × 1000 µm), which are often grown on the stomach.

Most of the shapes of the aerial mycelia are in a curved state or a straight state, and in rare cases, a gentle spiral state can be seen.

When microscopic examination of the culture was performed, spores were not considered to be in a chain state. As a result, microscopic suspensions obtained from these culture surfaces were examined, and long ovals (0.8-1.2 μm × 4.8-68 μm) and ellipsoids (0.8) were observed. Many of the spherical spherical shape of (-1.2 * 1.0 * 2.0micrometer) was observed, and the surface was smooth by the observation by the electron microscope.

2) Cell composition

The strain was vibrated in a modified medium of ISP No. 1 at 28 ° C. for 66 to 90 hours to collect and wash the cells.

Rain the cells. Backker et al. [Applied, Applied Mierobiology, Vol. 12, p. 421, 1964] and M. blood. According to the method of the recreational barrier [Journal of Laboratory and Laboratory Medicine 71, p. 934, 1968], the diamino pimethyl acid and the sugar composition in the cell cells were examined. Meso form, the latter has been confirmed the presence of spots equivalent to galactose and arabinose.

3) Determination on the classification medium

This strain grows relatively well on all kinds of medium, and the parasitic hyphae are colorless to pale yellow at the beginning of the culture, and then pale yellow brown or yellow brown.

In addition, yellow to yellow brown soluble pigments are produced in various sorting media.

The aerial mycelium is powdery, generally grown to a seed level, and is colored to yellow or pale yellow brown.

The compositional phases of the strains on the various sorting media are as shown in the first table.

TABLE 1

4) Physiological Properties

Physiological properties of this strain are shown in Table 2. That is, the growth temperature ranges from 12 ° C to 38 ° C, and the temperature range where the mycelia grow on the agar medium (ISP Na. 2) is 20 ° C to 35 ° C.

TABLE 2

5) Availability of various carbon sources

Method of Freedam and Hightribe [Journal of Bacter iology, Vol. 56, p. 107, 1948], and the basis of 0.1% of yeast extract (Egret: Bacto) added to it. The medium was used to examine the availability of various carbon sources, and these results are shown in Table 3.

TABLE 3

6) Other Properties

Collect the cells by the method described in the above 2), these and J .. Maama et al. [Journal. Orb. Morecurea. According to Journal of Molecular Biology, Vol. 3, p. 208, 1961, DNA was prepared, and the G-C (guanine-cytosine) content of DHA was about 71 mol%.

Gram staining of the mycelia of this strain was positive.

No. described above. C-15003 S. Lord. a. Waxman, D. The Actinomycetes, Vol. 2, More. Weriams. And. Wilkins Camp, 1961, al. this. Hatching. And, Y. This basic piece, the body. Manual. Orb. It was searched according to the Bergey's manual of Determinative Bacteriology, 8th edition 1974 and other documents.

This strain is thought to belong to Group III of the genus Nokaldia, but no known strain has been identified among the known strains.

This strain No. C-15003 shares the FERM-P No. 3992, IFO-13726, to the Fermentation Research Institute (Japan). American, type. Cultour. TCC-31281 has been deposited with Collecson (The American Type Culture Collection, Maryland, U.S.A.).

As described above, the No. The C-15003 strain is a new species of the genus Nocaldia, but as a general property of the microorganism it can cause mutations either naturally or by mutagenic agents.

For example, a large number of mutant strains obtained by mutating by irradiation of radiation such as X-rays, gamma rays, ultraviolet rays or the like, culturing on a medium containing various drugs, or other means, or naturally occurring mutant strains may be mentioned. It is only a substantially separate species in comparison with the bacteriological properties or with the bacteriological properties described below, and furthermore, it produces P-2, P-3, P-3 'and / or P-4. Anything having properties can be used in the method of the present invention.

For example, No. C-15003 strain undergoing various mutation treatments, producing little soluble pigment, parasitic mycelium colorless, yellowish green, reddish brown to light red, mycelium as a bacilli or branched short mycelium There are many mutant strains which are easy to be divided and the mycelia of the mycelia and rarely grow white or the mycelia.

The valine added to the medium in the method of the present invention may be an ester thereof (e.g., methyl ester, ethyl ester) or a salt thereof (e.g. hydrochloride), or may be any of D, L and DL.

The isobutyric acid added to the medium may be an ester thereof (eg, methyl ester, ethyl ester) or a salt thereof (eg, sodium salt, potassium salt, calcium salt).

The addition amount of the above additive is generally about 0.01 to 1.0% (W / V%), more preferably about 0.1 to 0.5% (W / V%) with respect to the medium.

As long as the production of the antibiotic P-3 to be added is continued, for example, it may be at the beginning of the culture, or may be a suitable time after the start of the culture. The medium used for the cultivation of the antibiotic C-15003 producing bacterium may be liquid or solid as long as it contains a nutrient source that can be used by the strain, but it is more appropriate to use a liquid medium when treating a large amount. In addition to the addition of the additive used in the present invention to the medium, No. C-15003 Shareable sources of mobilizable carbon sources, digestible nitrogen sources, inorganic substances, and micronutrients.

Examples of carbon sources include glucose, chopped sugar, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, etc. Dry yeast, soybean meal, corn steep liquor, peptone, cottonseed meal, fructose, urea, ammonium salts (eg ammonium sulfate, ammonium softened, ammonium nitrate, ammonium acetate, etc.) and the like are used.

In addition, salts containing sodium, potassium, calcium and magnesium, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid and salts of organic acids such as acetic acid and propionic acid are suitably used.

Other amino acids (e.g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g., B ₁ , B ₂ , nacotinic acid, B ₁₂ , C, E, and the like), nucleic acids (eg, purine, pyrimidine and derivatives thereof), and the like.

Of course, an inorganic or organic acid, an alkali, a buffer, or the like may be added for the purpose of adjusting the pH of the medium, or an appropriate amount of fat or oil or surface active agent may be added for the purpose of defoaming.

The means for culturing may be a stationary culturing or a means such as a vibrating culture or aeration stirring culture method.

It goes without saying that it is preferable to carry out the so-called heartworm aeration culture for mass treatment.

Naturally, the conditions of the culture are not constant depending on the condition of the medium, the composition, the type of strain, the means of the cultivation, and the like, but these are usually selected at the initial pH near the neutral at a temperature of 20 ° C to 35 ° C.

In particular, the temperature in the incubator is preferably 23 ° C. to 30 ° C., and the initial pH is 6.5 to 7.5.

The incubation period is not constant according to the above conditions, but it is better to incubate until the desired concentration of antibiotic is maximized.

The time required is about 2 to 10 days in the case of vibration culture using a liquid medium or aeration stirring culture.

As a specific experimental example of the above-mentioned P-3 manufacturing method, NaC-15003 stock was prepared from soluble starch (3%, ammonium chloride 0.2%, magnesium sulfate 0.05%, potassium phosphate 1.09%, potassium diphosphate 2.09% ferrous sulfate 0.01). Medium (I) in% or dextrin 5%, corn steep liquor 3%, peptone 0.1%, calcium carbonate in medium (II) was added to the above-mentioned various additives and cultured. In the fourth table, the case of medium (II) is shown in the fifth table.

In this case, the total production of C-15003 was tested using Tararomyces avellaneus IFO-7721, and the test medium [3.5 g of sodium diphosphate, 0.5 g of potassium phosphate 0.5, yeast extract (manufactured by dIF CO Co., Ltd.). ) 5 g, glucose 10 g, agar 15 g, distilled water 1000 ml, pH 7.0] was measured by a paper disc using P-3 as a standard.

In addition, the fraction of the produced P-2, P-3, or P-4 was extracted by adding the same amount of ethyl acetate to the culture filtrate containing them, concentrated and dried, and then solidified. Was dissolved in ethyl acetate to prepare a sample for bacterium chromatography.

As a developing solvent, ethyl saturated acetate was used as a developing solvent using the glass plate of silica gel 60F254 (made by Melk).

The production amount and production rate were measured at a concentration and area of absorption of 254 nm using a Shimadzu 2-wavelength chromatography scanner CS-910.

In this case, the detection amounts of P-2, P-3, and P-4 were calculated as 100%.

These results are shown in the 4th table | surface and the 5th table | surface.

That is, No. When C-15003 strain was cultured normally, the amount of P-3 produced was about 60 w / w%, and the yield decreased in the purification step of isolating this, but as indicated in the experiment, For example, by adding valine to the culture medium, most of the product becomes only the desired P-3 and can be efficiently collected.

TABLE 4

TABLE 5

As described above, in order to purify and produce P-3 accumulated and accumulated in the culture, a method of separation and purification commonly used for collecting such microbial metabolites is suitably used.

First, since the substance is neutral fat-soluble, organic solvents which are not mixed with water are used, for example, fatty acid esters such as ethyl acetate, amyl acetate, allohols such as butanol, halogenated hydrocarbons such as chloroform, and methyl isobutyl ketone. do.

Extraction is performed near neutral, Preferably, it is performed using ethyl acetate in the culture filtrate adjusted to pH7.

The extract is washed with water, concentrated under reduced pressure, and a non-polar solvent such as petroleum ether or nucleic acid is added to collect crude material containing the active ingredient.

Since a large number of outputs other than the antibiotic C-15003 are identified on the T LC, a purification process is then used.

That is, various adsorption chromatography is effective as a purification method normally used, and a carrier generally used, such as silica gel, alumina, macroportus nonionic adsorption resin, etc., can be used as an adsorbent. In the case of using silica gel, development starts from a non-polar solvent such as petroleum ether and nucleic acid, and precipitates polar solvents such as ethyl acetate, acetone, ethanol and methanol, or development from combined halogen hydrocarbons such as dichloromethane and chloroform. Then, P-3 is eluted, separated and collected by adding polar solvents such as alcohols such as ethanol and methanol, ketones such as acetone and methyl ethyl ketone.

When macroporous adsorbent resins are used, lower alcohols or lower ketones, mixtures of esters and water, etc. are used to elute P-3.

As lower alcohols, for example, as lower ketones such as methanol, ethanol and butanol, for example, acetone, methyl ethyl ketone, and ethyl acetate may be used as esters.

The example shows that crude substance II is dissolved in 60% methanol water, passed through a DIION HP-10 (manufactured by Mitsubishi Chemical Co., Ltd.) column for adsorption, washed with 70% methanol water, and eluted with 90% methanol water. -3 is eluted.

In any method, the fraction of P-3 obtained is concentrated under reduced pressure, and 5-8 times of ethyl acetate is added to the dried product, and the crystals of P-3 precipitate.

According to the method of the present invention, the production rate of P-3 is very high, and the production amount also increases several times. Therefore, the method of the present invention is extremely industrially advantageous in terms of the increase in the yield and the ease of separation and purification.

The physical and chemical properties of P-3 obtained in Example 4 are shown in Table 6.

TABLE 6

P-3, P-3 'and P-4 are novel compounds, while P-2 is reported by Kuupchan et al. In elemental analysis, specific light intensity, UV absorption spectrum, infrared absorption spectrum, mass spectrum, etc. , Orb, American, and Journal of America Chemical Society, Vol. 97, pp. 5294, 1975, the same compound as maytansinol, propionate.

[Biological activity]

A) antimicrobial bioactivity

Using tril thiacaase and soy agar medium (manufactured by BBL) as the assay medium, the ability to inhibit the development of the microorganisms indicated next was examined by a free method.

That is, growth inhibition ability was examined by impregnating 0.02 ml of a solution of 300 µg / ml of P-3 on the following microbial microbial plate medium (Toyo Corporation, thin, 8 mm in diameter). As a result, it did not show activity against the following microorganisms.

Sierricha, Escherichia cde, Proteus, proteus valgaris, Proteus, mirabicis, Pseudomonas, pseu domonas aeruginosa, Staphylococcus, aure Stap-hylococcus aureus, bacillus, Bacillus subtilis, bacillus, cereus, crebesgera, Klebsiella pneumoniae, Cerratia. Serratia marcescens, Mycobacterium arium.

On the other hand, using the agar plate of assay medium [3.5g sodium diphosphate, 0.5g potassium phosphate 0.5g, yeast extract (Dipco) 5g, glucose 10g, agar 15g, distilled water 1000ml, pH7.0], Tyranomyces, When P. aerellaneus (Talaromyces avellaneus) was tested and its viability was examined, P-3 showed growth inhibition at 3 ㎍ / ml.

In addition, tetrahimena and Pythaymena pyriformis W strains were used as test microorganisms, and assay medium [Triftos. 20 g of peptone (dipco), 1 g of yeast extract, 2 g of glucose, 1000 ml of distilled water, 1 molar phosphate buffer pH7.0, 10 ml] was incubated at 28 ° C. for 44 to 48 hours, and the antibiotic was diluted by liquid dilution assay. The ability to inhibit microbial development of the material was examined. As a result, C-15003 P-3 was confirmed to inhibit the growth of this microorganism at 1 µg / ml.

Anti fungal activity is shown in Table 7.

As is clear from Table 7, P-3 inhibits the growth of phytopathogens.

A circular filter paper impregnated with 0.02 ml of 1000 μg / ml solution of P-3 was placed in a medium into which the microorganisms of Table 7 were respectively implanted, and the diameter of the low support was measured.

TABLE 7

B) antitumor activity

The therapeutic effect of P-3 (9 days continuous intraperitoneal administration) on tumor cells P 388 (1-10 ⁶ cells / mouse, mice, intraperitoneal transplantation) was investigated.

As a result, the antitumor effect of the mouse by these substances was 200% at 0.00625 mg / k g / day administration was confirmed as compared to the control.

C) toxicity

In the venus toxicity test using mice, the intraperitoneal injection of P-3 showed a LD ₁₀₀ value of 0.625 mg / kg and an LD ₀ value of 0.313 mg / kg.

As mentioned above, P-3 has a strong growth potential against filamentous fungi and protozoa, and thus is also useful as an antiseptic or an antiprotozoal agent.

In addition, P-3 is expected to be useful as an anti-tumor agent because it has a life-saving effect on mammals (eg mice) with tumors.

P-3 can be advantageously used as an antiseptic and an antiprotozoal agent, for example when examining bacterial conditions such as soil, activated sludge or animal body fluids. In other words, when separating useful bacteria from the soil, or when testing the action of bacteria other than protozoa or fungi in the operation and analysis of the activated sludge method used for wastewater treatment, the fungus or protozoa does not develop in the sample. Can selectively develop bacterial state.

Specifically, the test sample is added to a liquid or solid medium, and 0.1 ml of an aqueous solution containing 1% methanol of 10 to 100 µg / ml of the antibiotic is added per 1 ml of the medium, followed by culturing.

P-3 can also be used as an antibacterial agent used for the treatment of plant diseases caused by the microorganisms shown in Table 7.

As a specific melting example, P-3 was dissolved in 1% methanol water so as to have a concentration of 0.5 µg / ml to 5 µg / ml. For example, it can be used for the treatment of rice nodules, rice rot, rice paddy disease, and the like. .

An Example is given to the following and this invention is demonstrated to it further in detail.

In addition, percentages refer to weight / volume% unless otherwise indicated.

Example 1

40 ml of the seed medium (0.1% glucose, 2.0% Baek-Triptone, 1.2% Baek-Yeast extract, pH7.0) were dispensed into a 200ml Erlenmeyer flask, and after sterilization, Nocal diason no. c-15003 (IFO 13726, ATcc 31281; FERMP-NO.3992) was inoculated.

This was incubated for 48 hours in a rotary vibrator (200r.p.m) at 29 ℃ was used as species culture.

The culture was sterilized three times with sterile distilled water, and then the bacterial body was returned to the original amount with sterile distilled water, and 1 ml of this medium was used as a main medium (3% soluble starch, 0.2% ammonium chloride, 0.05% magnesium sulfate, and 1 potassium phosphate). 1.09%, dipotassium phosphate 2.09%, ferrous sulfate 0.001%, and L-valine 0.1%) were inoculated to carry out main culture.

The main culture was performed by dispensing, sterilizing, and inoculating a 40 ml medium into a 200 ml Erlenmeyer flask, and then performing an eight-day period at 28 ° C. on a rotary vibrator (200 r.p.m).

The total yield of C-15003 was 16 μg / ml, about 95 w / w% of P-3.

Example 2

The seed medium shown in Example 1 was prepared, and 500 ml of the seed medium was dispensed into a 2000 ml flask and sterilized.

This was inoculated with the same strain as Example 1, and cultured for 48 hours in a reciprocating vibration (110 r.p.m) at 28 ° C to adjust the seed culture for inoculation.

Seed medium (Glucose 2.0%, soluble starch 3.0%, corn steep liquor 1.0%, soybean flour, 1.0%, polypeptone 0.5%, salt 0.3%, calcium carbonate 0.% pH7.0 ) 100 L was prepared, sterilized at 121 ° C. for 20 minutes, cooled, and inoculated with 500 ml of seed culture.

Vertical culture was carried out for 48 hours at a temperature of 28 ° C., aeration rate of 100 L / min and 200 revolutions / minute.

10 l of this seed culture was transplanted into 100 l of main medium (5% dextrin, 3% corn steep liquor, 0.1% peptone, 0.3% L-valine, 0.5% calcium carbonate, pH 7.0) in a 200-l stainless steel fermentation tank, and 28 ° C. The culture was carried out for 4 days at aeration rate 100 L / min and agitation 150 revolutions / minute.

P-3 was about 98 w / w% in C-15003 produced at a total yield of 12 gu / ml of C-15003.

Example 3

50 liters of acetone was added to 95 liters of the culture obtained in Example 2, followed by stirring for 30 minutes, and 2 kg of ipro supercell (manufactured by Jones & Manville Products, USA) was added and stirred well.

The mixture is filtered through a pressure filter and 13.5 L of the filtrate is obtained.

50 L of water and 90 L of ethyl acetate were added to the filtrate, and the filtrate was extracted and this operation was repeated twice.

The ethyl acetate layers were combined, washed twice with 80 L each, 1 kg of anhydrous sodium sulfate was added, concentrated to reduced pressure to 200 ml after drying, petroleum ether was added, and the precipitate precipitated was filtered out (35 g).

50 ml of ethyl acetate was added to the obtained crude material, followed by stirring. The insoluble material was filtered off, 10 g of silica gel (0.05 to 0.2 mm, manufactured by West Germany Melk Co., Ltd.) was added to the filtrate, followed by stirring, and ethyl acetate was distilled off under reduced pressure. (500 ml) is simple, 500 ml of n-hexane, 500 ml of n-hexane ethyl acetate (3: 1), n-hexane ethyl acetate (1: 1), and 2 liters of ethyl acetate saturated water are poured out, and the eluate is 500 ml each. Fraction.

1 ml of each fraction was concentrated and firmly added, and 0.1 ml of ethyl acetate was added thereto. The solution was put at a position of 2.5 cm from the bottom of a silica gel glass plate (Kiegel Gel 60 F ₂₅₄ 0.25 mm 20 x 20, manufactured by West Germany Melk, Inc.) And about 17 cm with saturated acetyl ethyl.

After the development, the absorbed phase was irradiated with ultraviolet rays (2537 Å) to collect fractions with absorption near Rf 0.42, concentrated under reduced pressure to about 2 ml, and 20 ml of petroleum ether was added to the concentrated solution to obtain 9.1 g of crude crystals.

After heating and dissolving this in 20 ml of ethyl acetate, it cooled and precipitated, and the crystal | crystallization which precipitated was obtained 850 mg of P-3 crystals.

mp 189-190 degreeC (P-3: 97w / w%)

Example 4

20 g of the crude material obtained in Example 3 is dissolved in 200 ml of methanol, and 200 ml of water is added and dissolved.

1000 ml of Diaion HP-10 (product of Mitsubishi Chemical Co., Ltd.) were charged to a column having a diameter of 2.5, and adjusted by flowing 3 liter of 50% methanol water.

The sample solution prepared first was flowed, followed by 1 liter of 60% methanol, and fractions of 75% of the eluted solution eluted between 7.5 liters of 60% methanol and 7.5 liters of 95% methanol. Each fraction was separated by thin layer chromatography on silica gel. Detect by attaching to

Fraction No. 145-153 were collected and concentrated, 500 ml of water and 1 liter of ethyl acetate were added to dissolve, and the mixture was shaken and mixed in a separatory funnel, followed by mixing. The water layer was separated, washed twice with 300 ml of water, and the ethyl acetate layer was concentrated to dryness with forget-me-not. Crystals precipitate.

This crystal is filtered off and dried.

C-15003 P-3 880mg

Melting Point 188 ~ 190 ℃ (P-3: 95w / w%)

Claims (1)

The antibiotic C-15003 producing bacteria belonging to the genus Nokaldia was cultured in a medium to which valine and / or isobutyl acid were added. Method for producing antibiotic C-15003 P-3, characterized in that.