NO123738B - - Google Patents

Description

Process for the production of pimaricin.

The present invention relates to a method for the biosynthetic production of a new antibiotic, which has been named pimaricin, and by means of a previously unknown species of streptomyces. For this purpose, a culture of Streptomyces natalensis nov. spee, whose properties will be described below, by the aerobic method and under suitable conditions

see, and the product pimaricin, whose properties

per also will be described in the following, is formed in the fermentation medium. The new antibiotic is advantageously recovered from the culture liquid and/or the mycelium by extraction with an alcohol which is only partially miscible with water, e.g. butanol or by treatment with methanol (a methanol solution of calcium chloride), concentration of the extract and purification by means of a solvent.

The microorganism which causes the formation of the present antibiotic was isolated from a soil sample from Pieter Maritzburg, in the province of Natal, South Africa. It is an actinomycete of the genus Streptomyces, and is called Streptomyces natalensis. It is deposited in the Type Culture Collection of the Northern Regional Research Laboratories

at Preoria, U.S.A. under catalog no. NRRL 2051. The microorganism has the following morphological and physiological characteristics:

Morphological characteristics:

The hyphae are branched and unevenly twisted, often practically straight and parallel to each other in the growth zone, have a thickness of 0.3 — 0.7 [x, usually have uniform thickness, but often local thickenings. The spordan- nende hyphae have the form of lateral branches with attachment to the aerial mycelium. The spores take the form of uneven wavy chains and rarely form loosely twisted spirals. The spores are separated from each other by short intermediate parts without protoplasm and have an oval, round or tangerine-like shape with a size of 0.7 — 1.2 x 0.7 — 0.8 |x.

The characteristics of the culture when grown on

the substrates below (after 7 days at 25° C, unless otherwise stated):

Uavre agar :

Good growth. The vegetative mycelium and the back are colorless. After 28 days a few colonies at the bottom and top of the tube. On the back, graph arget ("chamois"), after 57 days a little darker ("warm buff"). The colony is practically flat, some colonies somewhat higher in the middle. Pale mouse gray ("pale mouse gray") to light mouse gray ("light mouse gray") powdered and felted aerial mycelium, which after 28 days becomes medium gray ("one third gray") and after 57 days darker gray ("drab gray") . Earthy somewhat sour smell. No soluble pigments.

Potato agar:

Moderate growth. The vegetative mycelium has a light mineral gray ("light mineral gray") to pale brownish yellow ("pale olive buff") color. Reverse: cream color ("cream color"), after 57 days darker ("cream buff to chamois"). The colonies are moist and partially covered by a slightly raised aerial mycelium. The aerial mycelium, which after 7 days is white and felt, after 28 days medium gray ("drab gray") covers 30-70 per cent of the colony. No soluble pigments. Earthy unpleasant smell.

Potato:

Good growth. The vegetative mycelium is moist and somewhat raised and lumpy and has a pale pinkish buff to pinkish buff color, which after 28 days changes to a dark brownish olive color (deep olive buff”). After 28 days, the back side of the glass wall is brown ("chamois"). After 7 days no aerial mycelium. After 28 days a good deal of aerial mycelium, which is first white, then pale mouse gray ('pale mouse grey') and after 27 days medium gray ('drab grey'). The potato is not discolored after 7 days, after 57 days it is, in the same way as the liquid at the bottom of the tube, discolored and has acquired a dark brown-yellow color ("buffy brown").

Sabouraud glucose agar:

Medium growth. Vegetative mycelium, lumpy, raised, after 7 days a light brown olive color ("olive buff") which, on the back, after 28 days is darker ("deep olive buff to dark olive buff"). The back now clay-coloured. The aerial mycelium is white, short and felted and then pale mouse grey. No visible, soluble pigments after 7 days, but after 57 days the color is medium red-brown ("medium auburn").

Emerson agar:

Very good growth. Vegetative mycelium, olive colored ("olive buff"). The back is softer («cream buff to chamois»). No soluble pigments. Aerial mycelium short and felted, white. No soluble pigments. Strong earthy smell.

Starch agar:

Medium growth. The vegetative mycelium and the back are pale olive-yellow ("pale olive buff"). Colony slightly raised, about half covered by felty, white aerial mycelium. No soluble pigments. Strong earthy smell.

Glucose nitrate agar:

Good growth. Round, moist, gray colonies, slightly raised in the middle, approx. 2 to 2.5 mm's diameter, the color is pale olive brown («pale olive buff”). Aerial mycelium sparse, in the form of points or concentric rings, felted, white. No soluble pigments. Earthy smell.

Sodium citrate agar:

Very sparse growth: Round, moist, gray colonies, slightly raised in the middle, approx. 1 mm in diameter. Lighter than pale light brown olive color ("pale olive buff"). The back is a pale light brown olive color ("pale olive buff"). No soluble pigment.

Nutrition saga:

Good growth. The colony is moist, slightly raised, very small, in the form of very small, wet knotty and dull tufts, with small and indistinct oblong elevations, white to light yellow cream color ("cream color to cream buff"). Unpleasant smell. No soluble pigment.

Czapek agar (13 days):

Very sparse growth. Clear transparent vegetative mycelium. No aerial mycelium. No soluble pigment.

Bacto agar 2%:

Very sparse growth, first visible after 13 days. Clearly transparent vegetatively

mycelium. No aerial mycelium. No soluble pigment.

The organism which, according to the invention, is used for the production of pimaricin belongs to the genus Streptomyces and is not identical to any previously described Streptomyces species. As can be seen from the above detailed characteristics, it belongs to group 1 in Waksman's classification in Bergey's Manual (see also S. A. Waksman "The Actinomycetes", 1950, page 30).

Streptomyces natalensis nov. spee. can also be classified in Baldacci's "Neo-Ingri" series (see Symposium Actinomycetales, VI Congr. Intern. Microb. Rom, 1953, page 31). It is noted that the name Streptomyces natalensis nov. spee. is not exclusively limited to actinomycetes, which correspond in a stereotypical and rigorous way to the description given here, but that the expression also includes all related strains, which generally have the same specific properties and produce the new antibiotic, pimaricin. It also covers mutants of Streptomyces natalensis, which can be obtained from it by means of any means or substances which cause mutation, e.g. irradiation or treatment with substances with a toxic effect.

The new antibiotic, pimaricin, is mainly active against saprophytic and parasitic fungi and yeasts.

Table II gives an overview of pimaricin's effect on yeasts and fungi which have no pathological effect on humans. For each of these species, the pimaricin concentration in micrograms/ml in the nutrient that is just capable of stopping the yeast's growth is indicated.

Table III similarly indicates the relationship directly opposite a number of yeasts and fungi with a pathological effect on humans.

It appears from the above tables, among other things. a. that pimaricin has a pronounced effect on phytopathogenic fungi, e.g. Verti-cillium, Cladosporium and Fusarium. The effect on Candida albicans is also very strong.

Another very important feature of the new antibiotic is that pimaricin has a very small phytotoxic effect, in contrast to most other fungicidal antibiotics known to date. The new antibiotic is therefore very suitable for use in arable and horticulture and for combating plant diseases, all the more so because it diffuses easily and works systematically.

Pimaricin will thus penetrate into e.g. peas (Pisum sativum) after immersion in a dilute aqueous solution. This is evident from the fact that pimaricin can be extracted from the cotyledons and sprouts of the treated peas.

Table IV a and b shows the internal disinfecting action. Mycelium fungi (among these Ascochyta pisi) are killed in the seed.

Incoming samples showed that primaricin in a concentration of 75 parts per million, which is an effective fungicide, has no harmful effect on the pea's ability to germinate and the plant's growth (see Table IV c and d).

All data in these tables relate to the pea variety Eminent, which is extremely sensitive to Ascochyta pisi.

Experiments with rats and mice show a very low toxic effect. To determine pimaricin's acute toxic effect on white rats, the rats were given a material containing 985 micrograms/mg, and the result was recorded after 7 days.

With pimaricin in water, f01-like results were obtained:

Pimaricin does not irritate the skin or mucous membranes.

In a pure state, pimaricin is a white crystalline amphoteric compound, the salts of which can be prepared in the usual ways. With FeCl., it gives no color reaction, but with conc. phosphoric acid a pink unstable color. Concentrated hydrochloric acid and sulfuric acid produce a blue and olive-green discoloration. Bromine water is decoloured. Because of this and also with regard to the infrared and ultraviolet spectrum (see below), pimaricin must be considered one of the so-called polyene antibiotics. It is very slightly soluble in water, namely 8 mg in 100 ml at 20° C. It is more easily soluble in organic solvents, especially in alcohols with 1 to 6 carbon atoms, e.g. methanol and n-butanol and in cellulose solvents. Furthermore, it is soluble in pyridine, dimethylformamide, dimethylacetamide, glacial acetic acid and alkali hydroxide. In aliphatic coal water substances, such as pentane, hexane, cyclohexane and the like, it is practically insoluble.

At room temperature, pimaricin is a very stable compound. A solution in water (15 mg in 100 ml) retains its full activity for 7 days at pH — 7.0 at 25° C. At pH = 2, the same solution loses 50

percent of its activity during 3 days at the same temperature and at pH = 10 during the course

of 6 days. At higher temperatures, the solution is less stable. Thus, a solution with the above pimaricin concentration in water and pH = 2 and at 90° C will lose 90 per cent of its activity within 15 minutes, at pH = 6.5 15 per cent and at pH = 9 50 per cent. of its activity. In methanol, pimaricin is stable at higher (25° -60° C) temperatures. Pimaricin's antibiotic effect was tested microbiologically with Saccharomyces cerevisae — Holland's variety as a test organism in relation to a pure standard preparation.

Pimaricin has no melting point, but shows incipient decomposition at above 150°C.

The specific rotation

(c = 0.083 percent in 100 percent methanol). The substance's molecular weight is approx. 727, and is assumed to have the following empirical formula C3.,H-0NOU. The elemental analysis was as follows: 57.7 percent C, 7.27 percent H, 1.95 percent N and 33.01 percent O.

The ultraviolet absorption spectrum of pimaricin has maxima at 290, 304 and 318 m|A with a "peak" at about 280 and a minimum at about 250 rri|i (C = 3.93 micrograms/ml in methanol). A sample of pimaricin pressed into a potassium bromide plate (C = 0.5 per cent) shows the following infra-red absorption (in cm-<1>): 3460, 2985, 1721, 1637, 1577, 1441, 1401, 1381, 1275 , 1269, 1238, 1192, 1176, 1136, 1109, 1066, 1006, 988, 948, 887, 855, 844, 803, 794 (for the curvilinear wavelengths the absorption is strong to very strong.)

Fig. 1 shows graphically the infrared spectrum of pimaricin in a potassium bromide plate.

For the production of pimaricin, Streptomyces natalensis is grown aerobically in stationary cultures or immersed in a liquid nutrient medium under sterile conditions in closed vessels equipped with stirring devices, which during cultivation are supplied with sterile oxygen or air for aeration.

The duration of cultivation, the temperature and the other conditions that must be met in order to obtain good yields of pimaricin are determined experimentally. They will be discussed in more detail below.

The nutrient medium can consist of the usual substances. It must contain a carbon source and a fermentable organic and/or inorganic nitrogen source, but also mineral salts, e.g. phosphates, potassium and sodium salts and trace metals should be present. But the starting materials used in the execution of the invention are often contaminated with these mineral salts, so no additional addition is necessary.

Both soluble and insoluble carbohydrates can be used as a carbon source, e.g. glucose, sucrose, lactose and starch. Sugar alcohols, e.g. glycerol, can also be used. The amount of the carbon source in the medium can vary greatly and depends on the nature of the carbohydrate used and the medium's other components, and usually amounts to approximately between 0.15 and 5 percent of the medium's weight.

A large number of substances can be used as a nitrogen source. One can mention here hydrolysed or non-hydrolysed casein, corn meal, peptone, meat extract, soya flour (defatted or not), peanut flour, fish meal and nitrates. The choice of nitrogen source will usually depend on the medium's other constituents, which in turn are chosen so that the antibiotic substance can be produced as economically as possible. In this connection, it can be mentioned that small amounts of nitrogen-containing starting material, e.g. yeast extract, "distillers' solubles", glue water etc. are able to increase the pimaricin yield in certain media. Also lipoid-containing substances, such as animal and vegetable fats, e.g. soya oil and fish oil) are capable of increasing yields to a significant extent.

The fermentation time is strongly dependent on the composition of the nutrient medium. It usually varies between 48 and 120 hours, but can also be continued, e.g. up to 14 days, if the greater dividends obtained thereby justify such an extension from a financial point of view.

The fermentation temperature can in principle vary between 15 and 30° C, but temperatures between approx. 26 and 28° C.

With optimal growth of the microorganism and optimal yield of pimaricin, the pH can vary, particularly during the first fermentation phase, e.g. between 5.0 and 8.0. After sterilization, the pH of the nutrient medium is preferably set to a value of between 6 and 7. The fermentation is preferably carried out at a pH between 6.5 and 8, as this achieves the best yields. The pH can be kept constant during the fermentation by adding alkali hydroxide or acid at regular intervals, and under sterile conditions. Usually, however, calcium carbonate is used as a kind of buffer in amounts from 0.2 to 1.0 percent by weight calculated on the medium. The amount of air which, under sterile conditions, is introduced into the medium during fermentation depends to a large extent on the shape of the vessel, the speed and the shape of the stirrers. Usually the quantity varies between 0.1 and 4 liters per liter of nutrient medium per minute.

Precultures of Streptomyces natalensis from 48 to 72 hours old are preferably used as inoculation material in the main fermentation vessel. In order to obtain good yields and avoid varying results, the inoculation is preferably carried out with culture amounts of from 1 to 7 volpst. of the nutrient medium in the main fermentation vessel. It is clear that with large fermentation vessels one must use a few steps of pre-cultures. But it is also possible to save a part, e.g. 10 percent of the culture in the main fermentation vessel for the production of a new culture. The entire fermentation can also be carried out continuously.

Pimaricin can be produced from the culture liquid in many ways. According to the invention, one can take advantage of pimaricin's solubility in organic solvents, which can only be mixed with water to a certain extent, e.g. butanol. But can e.g. proceed as follows: After you have obtained a sufficiently active liquid, this is adjusted to a pH of approx. 10 to release the active substance from the so-called mycelium, after which this is filtered off. The liquid can then be extracted, e.g. with n-butanol, at a pH between 3 and 10. If the liquid is acidified, this is preferably done with phosphoric acid and any resulting precipitate is removed and extracted if necessary, the n-butanol extract is concentrated by azeotropic distillation and the active raw substance crystallizes out . This can be purified by selective precipitation in e.g. glacial acetic acid, pyridine, dimethylformamide etc. 1. When fermenting with yields greater than 700 micrograms/ml, the pimaricin yield can be increased significantly by extraction of the mycelium. In this extraction, it is advantageous to carry out the extraction with methanol, in which 1-3 per cent calcium chloride is dissolved, in order to increase the solubility.

Example 1.

(Preparation of the inoculation culture).

From a tube with a culture of Streptomyces natalensis nov. spee. on e.g. oat flour agar on which good spore formation has taken place, small amounts of conidia are transferred under sterile conditions to 2 liter shaking bottles, which are filled with 500 ml of liquid nutrient medium. The nutrient medium consists of

After incubation with constant shaking at 26°C for 48 hours, the culture is ready for inoculation into the main fermentation medium.

Example II.

(Creating the Culture)

One liter of the inoculation culture prepared according to example 1 is transferred under sterile conditions to a fermentation vessel, which is equipped with a stirrer and a device for blowing in sterile air and contains 15 liters of a culture medium of the following composition:

The above culture medium is adjusted to a pH value of 6-9 with alkali hydroxide. After 8 hours of incubation at 27° C with constant aeration and stirring of the culture, it turned out that it contained 610 micrograms per ml.

Other cultural media with which you can achieve equally large dividends can have the following composition:

With this medium, with 4% inoculation culture and 72 hours of incubation and aeration at 26° C, 590 micrograms of pimaricin per ml of liquid.

With this medium, 640 micrograms of pimaricin per ml of fermentation liquid.

In this medium, after inoculation with 3% inoculation culture and 148 hours incubation and aeration at 26° C, 535 micrograms of pimaricin per ml. fermentation liquid.

In this medium, after inoculation with 3% inoculation culture in a 15 liter fermentation balloon and incubation and aeration for 168 hours at 28° C, 690 micrograms of pimaricin per ml. fermentation liquid. After treatment of the mycelium, which had been squeezed out and stirred in a portion of water, with diluted alkali hydroxide to pH = 10, 20.4 g of pimaricin was found in the mycelium filtrate.

Example III.

(Isolation of crude pimaricin).

Four liters of a pre-fermented culture liquid containing 590 micrograms/ml pimaricin was adjusted to pH = 10.0 with 10% caustic soda and then freed from mycelium by filtration with diatomaceous earth as a filter medium (2%). The filtered culture, which had a volume of 3.7 liters and an activity of 570 micrograms/ml, was acidified with phosphoric acid to pH = 3.0. The acidified filtrate was extracted with resp. 750, 350 and 350 ml of n-butanol. The butanol funnel was washed three times with 120 ml of 4% borax solution and finally twice with 120 ml of water. The butanol funnel then contained 1400 micrograms/ml. After azeotropic evaporation of the extract to 100 ml, 0.64 g of not completely pure pimaricin was separated in the form of crystals (activity 900 micrograms/ml). From the rest of the extract, 1.42 g of impure pimaricin (activity 395 micrograms/ml) was obtained by continued evaporation. The yield was 48 per cent, calculated on the total activity of the fully fermented liquid.

Example IV.

(Isolation of crude pimaricin).

15 liters of fully fermented culture liquid, which contained 610 micrograms/ml of pimaricin, was adjusted to pH = 10.3 with 35 percent potash and then freed from mycelium using 50 g of Hyflo as a filtering agent. The filtrate was then acidified to pH = 2.8 with phosphoric acid. The precipitate was filtered off, this time with 10% Hyflo. The precipitate was stirred for half an hour with 100 ml of n-butanol. After a period of time, what had settled on the bottom was washed with 100 ml of water. The entire amount of butanol extract was washed three times with 10 ml of 4% borax solution, then three times with 10 ml of water and evaporated in vacuo to 40 ml at 44° C. After cooling to 0° C, suction and drying, 0 .51 g of pale yellow, crystalline pimaricin with an activity of 850 micrograms/mg. The filtered culture filtrate was then treated in the manner described in example 3. The following two fractions were obtained: 3.21 g (act. 890 micrograms/mg) and 6.8 g (act. 223 micrograms/mg.

Total dividend 51.5 per cent.

Example V.

(Isolation of crude pimaricin).

Ten liters of completely fermented culture medium (act. 640 micrograms/ml) were

adjusted to pH = 10 with 15 percent potash. The mycelium was centrifuged off and the centrifuge set to pH = 6.9 with 10 n hydrochloric acid and then extracted with resp. 2000, 1000 and 1000 ml isoamyl alcohol. The isoamyl extracts were combined and washed three times with 300 ml of 4% sodium carbonate solution and then with 300 ml of water. The extract treated in this way was evaporated azeotropically to 100 ml. 2.62 g of pale yellow, crystalline pimaricin with an activity of 900 micrograms/mg is thereby secreted. Dividend, 36.8 per cent.

Example VI.

(Isolation of crude pimaricin).

Twenty liters of a completely fermented culture of Streptomyces natalensis (act. 700 micrograms/ml) was adjusted to pH = 10 with 20% caustic soda. The mycelium was filtered off using Hyflo (200 g). The clear culture filtrate was extracted with resp. 4000, 2000 and 1000 ml of n-butanol. The extracts were combined and washed twice with 500 ml of 4% borax solution and then twice with 500 ml of water. After adjusting the extract treated in this way to pH = 6.8, it was azeotropically evaporated in vacuo to 250 ml. 9.85 g of pale yellow, crystalline pimaricin (act. 913 micrograms/mg) is thereby excreted.

Example VII.

(Isolation of crude pimaricin from mycelium).

Fifteen liters of a completely fermented culture of Streptomyces natalensis nov. spee. was adjusted to pH = 4.1 with glacial acetic acid. The solution was added and stirred together with 200 g of Hyflo as a filtering agent. The mycelium was then pressed to remove as much water as possible and then weighed 1700 g.

1 g of this mycelium in this condition was

extracted for one hour with 40 ml of methanol to determine the number of micrograms of pimaricin. The volume of the methanol extract was increased to

100 ml and then measured spectrophotometrically.

The methanol solution contained 120 micrograms/ml.

The pimaricin content in the entire amount of mycelium was 20.4 g. The pressed mycelium was extracted for half an hour at room temperature with 8 l of methanol, in which 2 per cent calcium chloride was dissolved. The extract was diluted with 1 1 of water and freed from methanol in vacuum. In this way, crystalline pimaricin was secreted, which after aspiration, washing and drying in vacuum weighed 14.73 g and had an activity of 900 micrograms/mg = 65 per cent of the theoretical yield.

Example VIII.

(Purification of pimaricin).

Ten grams of impure pimaricin (act. 890 micrograms/mg) were dissolved in 80 ml of glacial acetic acid under careful heating, after which undissolved impurities were filtered out as quickly as possible. The clear yellow-brown filtrate was dissolved in 1,500 ml of water and the solution adjusted to pH = 6.3 with 33% caustic soda. After cooling, the separated crystals were fractionated, washed twice with a total of 500 ml of water and suctioned off. The crystals were also washed on the filter with 200 ml of water and dried in vacuum over phosphorus pentoxide. The very pale yellow product obtained weighed 7.07 g and had an activity of 960 micrograms/mg.

The above treatment was repeated with 60 ml of glacial acetic acid and 1000 ml of water. The fabric was washed three times with 200 ml of water. The product then weighed 5.35 g and had an activity of 995 micrograms/mg. Total yield 59.8 per cent.

Example IX.

(Purification of pimaricin).

Ten grams of impure pimaricin (act. 890 micrograms/) were dissolved in 100 ml of dimethylformamide under gentle heating. The insoluble impurities were filtered off and the filter washed with 30 ml of dimethylformamide. The brown, clear filtrate was added to 250 ml of water, and the antibiotic substance separated in the form of crystals. The product was sucked off, the filter washed with 100 ml of water and the crystal mass dried in vacuum. The product obtained weighed 9.24 g. After repeated treatment in the same way, a pale yellow crystalline product was obtained which weighed 7.9 g and had an activity of 985 micrograms/mg. Total dividend 87.5 per cent.

Example X.

(Preparation of the sodium salt).

2.5 g of pimaricin (act. 985 micrograms/mg) was suspended in 20 ml of methanol with stirring and 0.145 g of sodium hydroxide dissolved in 0.3 ml of water was added to the suspension. The pimaricin, which first dissolved, crystallized within a few minutes in the form of the sodium salt. After stirring for 30 minutes and standing for 24 hours at 0° C, the crystal mass was suctioned off and washed with 5 ml of ethanol and 10 ml of diethyl ether. After drying in vacuum, the sodium salt, which was present in the form of white, needle-shaped crystals, weighed 2.16 g (content 995 micrograms/mg) = 87.3 percent of the calculated activity.

Other salts can be prepared in a similar way.

Claims (5)

1. Process for the production of an antibiotic, pimaricin, characterized in that Streptomyces natalensis nova species is grown under aerobic conditions, preferably submerged, in an aqueous nutrient medium, from which pimaricin is extracted, which is a substance with antibiotic properties against saprophytic and parasitic fungi and yeasts, and which in its pure state is a white crystalline substance, which begins to decompose at approx. 150° 25 C, has a specific rotation a — = + 250° C (in a concentration of 0.083 per cent in 100 per cent methanol), and gives no color reaction with ferric chloride, forms a pink unstable color with phosphoric acid, decolourises bromine water, is slightly soluble in water, more soluble in alcohols and soluble in pyridine, dimethylformamide, dimethylacetamide, glacial acetic acid and alkali hydroxides and practically insoluble in aliphatic hydrocarbons, and shows maximum absorption of ultraviolet light at 290, 300 and 318 m^i with a peak at approx. 280 and a minimum of approx. 250 mji, and in a potassium bromide plate shows the following infrared absorption (in cm-<1>): 3460, 2985, 1721, 1637, 1577, 1441, 1401, 1381, 1275 1269, 1238, 1192, 1176, 1136, 1109, 1066, 1006, 988 948 887 855 844, 794.03 2. Method according to claim 1, characterized in that the cultivation takes place at a temperature between approx. 15 and approx. 30° C, preferably between approx. 26 and approx. 28° C for a period of from approx. two to five days. 3. Method according to claims 1-2, characterized in that the cultivation takes place in an aqueous, nutrient-containing carbohydrate solution with a pH value of 6.5 to 8. 4. Method according to claims 1-3, characterized in that the cultivation takes place in an aqueous medium containing a soluble carbohydrate and a source of assimilable nitrogen. 5. Method according to claims 1-4, characterized in that the extraction of pimaricin comprises extracting the antibiotic with butyl alcohol at a pH value of approx. 3.