BE555769A - - Google Patents

Description

       

   <Desc / Clms Page number 1>
 



   The present invention relates to a process for the biosynthesis of a novel antibiotic, pimaricin, using a hitherto unknown species of Streptomyces. For this purpose, a culture of Streptomyces natalensis nov. Spec. the properties of which are described below under suitable aerobic conditions and the pimaricin thus formed, the properties of which are also given below, is obtained from the fermented broth.

   The preparation of the antibiotic with the aid of the culture fluid and / or the mycelium can advantageously be carried out by extraction with alcohols miscible with water to a limited degree, for example butanol, or by treatment with methanol (methanolic solution of calcium chloride) then by concentration of the extract followed by purification with the aid of solvents.

 <Desc / Clms Page number 2>

 



   The microorganism producing the antibiotic in question was isolated from a soil sample obtained in Pieter Laritzburg, province of Natal, South Africa. It is an actinomycete of the genus Streptomyces now called Streptomyces natalensis.



   Its morphological and physiological characteristics are as follows: Morphological characteristics. Branched, irregularly twisted hyphae, often nearly straight and parallel in the zone of development, 0.3-0.7 thick, usually uniform in thickness but sometimes showing local thickenings. Hyphae sporulating in monopod lateral branches on the aerial mycelium. Spores in irregularly wavy chains, more rarely in chains coiled in loose spirals. Spores separated from each other by short intermediate pieces without protoplasm, oval, round or tangerine-shaped, 0.7 - 1.2 x 0.7 - 0.8.



  Culture characteristics (after seven days at 25 ° C, unless otherwise specified).



   TABLE 1 (The colors mentioned have been taken from "Color Standards and Nomenclature" (1912), by Ridgway. For the avoidance of doubt, the color designated by Ridgway as "Buff" is hereinafter referred to as "buff". B ", in contrast to the simple" buff ". The" Buff "shade corresponds to a light buff).



  Oat flour-agar Good development. Vegetative mycelium and colorless reverse. After twenty-eight days, a few colonies at the bottom and top of the tube. Back buff, a little darker (Warm Chamois B) after fifty-seven days. Almost flat colony, separate colonies slightly raised in the center. Mouse-gray aerial mycelium

 <Desc / Clms Page number 3>

   pale, light mouse-gray, powdery and felt, gray after twenty-eight days, dirty gray after fifty-seven days.



   Earthy odor with additional sour odor. No soluble pigment.



    Potato agar Moderate development ... Vegetative mycelium light mineral gray to buff B pale olive. Cream lapel ,. chamois B cream every month after fifty-seven days. Slightly raised moist colonies, aerial mycelium partially covering the colony. At the end of seven days, white and felted, at the end of twenty-eight days, dirty gray and covering 30 to 70% of the colony. No soluble pigment. Unpleasant earthy odor.



  Potato Good development. Vegetative mycelium moist, raised, lumpy, buff B pale pinkish to buff B pinkish, buff
B dark olive after twenty-eight days.



   Buff on the sides: glass.



   No aerial mycelium after seven days but fairly abundant after twenty-eight days, white at first, then pale mouse gray, dirty gray after fifty-seven days. The color of the potato is not altered after seven days, but it turns buff brown after fifty-seven days, like the fluid in the bottom of the tube. Earthy smell.

 <Desc / Clms Page number 4>

 



  Moderate development glucose agar. Mycelium vegeta, - Sabouraud tif lumpy, raised, buff B olive like the reverse after seven days, darker (buff B dark olive to buff
B blackish olive) after twenty-eight days. Back of clay color. Aerial mycelium at first white, short and felted, then pale mouse-gray.



   No soluble pigment visible after seven days but medium auburn after fifty-seven.



  Emerson's Agar Very good development. Veggie mycelium. tative chamois B olive. Darker reverse (chamois B cream to chamois).



   Short, felted aerial mycelium, white. No soluble pigment. Strong smell of earth.



    Starch-aar Moderate development. Vegetative mycelium and buff reverse B pale olive. Slightly raised colony, about half of which is covered with a felted white aerial mycelium. No soluble pigment. Strong smell of earth.



  Glucose-nitrate-agar Good development. Colonies round,, moist, gray, slightly raised in the center, 2-2.5 mm in diameter, somewhat paler than buff B pale olive.



   Aerial mycelium scanty, in concentric dots or rings, felted, white.



   No soluble pigment. Earthy smell.



  Sodium citrate- Very poor development. Round, moist agar colonies, slightly raised at the

 <Desc / Clms Page number 5>

 center, about 1 mm in diameter (paler than buff B pale olive). Pale olive chamois B reverse. No soluble pigment. No smell.



  Nutrient agar Good development. Damp, slightly raised colony, in very small damp, rough and dull tufts, with shallow and fuzzy cerebroid streaks, cream to creamy buff color. Unpleasant odor. No soluble pigment.



  Czapek Agar Very little development. Hyaline vegetative mycelium (13 days). No aerial mycelium.



   No soluble pigment.



    Bacto-agar 2% Very little development, 'invisible for thirteen days. Hyaline vegetative mycelium. No aerial mycelium. No soluble pigment.



   As has already been said, the microorganism used according to the invention for the preparation of pimaricin belongs to the genus of Streptomyces and is different from all the Streptomyces species described. Due to the characteristics detailed above it belongs to group 1 of those described by Waksman in the Bergey manual (see also S.A.



    Waksman, "The Actinomycetes", 1950, p. 30).



   Streptomyces natalensis nov. Spec. can also be classified in the "Keo-Ingri" series by Baldacci (cf. Actinomycetales Symposium, VI International Congress of Microbiology, Rome, 1953, p. 31). It should be noted that the name of Streptomyces natalensis nov. Spec. is not exclusively attributed to the actinycetes answering in a stereotypical and rigorous way to the given description but that it also applies to the sub-

 <Desc / Clms Page number 6>

 related ches having generally the same specific properties and producing pimaricin, which can be regarded as subspecies, varieties, races, forms, serological or other type groups, variants, phases, products of mutation spontaneous, modifications, etc. of this species.

   It also covers the mutation products of
 EMI6.1
 Streptomyces natalensis obtainable from this species using substances or means which induce mutation, such as irradiation or substances with toxic action.



   Pimaricin, a new antibiotic, is mainly active against fungi and saprophytic and parasitic yeasts.



   Table II summarizes the antibiotic properties of pimaricin with respect to yeasts and fungi which are not pathogenic to the human body; there is indicated, for each species, the amount of antibiotic in micrograms (Ù) per cm3 of nutrient medium exactly inhibiting the development
Table III mentions the inhibition of a certain number of yeasts and fungi pathogenic to humans.



   TABLE II
 EMI6.2
 
<tb> Test <SEP> Microorganism <SEP> Concentration <SEP> of <SEP> the <SEP> pimaricin
<tb>
 
 EMI6.3
 ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯ in y; Icm3
 EMI6.4
 
<tb> Saccharomyces <SEP> cerevisiae <SEP> - <SEP> H <SEP> 0.15
<tb>
<tb> Saccharomyces <SEP> cerevisiae <SEP> - <SEP> KLL <SEP> 0.9
<tb>
<tb> Pichia <SEP> membranifaciens <SEP> 2.5
<tb>
<tb> Schwanniomyces <SEP> occidentalis <SEP> 2.5
<tb>
<tb> Aspergillus <SEP> niger <SEP> 1.8
<tb>
<tb> Aspergillus <SEP> fumigatus <SEP> 1,2
<tb>
<tb> Cladosporium <SEP> cucumerinum <SEP> 0.9
<tb>
<tb> Verticillium <SEP> dahliae <SEP> 1,2
<tb>
<tb> Fusarium <SEP> spec. <SEP> 1,2
<tb>
<tb> Penicillium <SEP> chrysogenum <SEP> 0.6
<tb>
<tb> Tricho'derma <SEP> spec. <SEP> 1,2
<tb>
 

 <Desc / Clms Page number 7>

 
 EMI7.1
 T.T:

  ¯U III
 EMI7.2
 Test microorganism Concentration of piluaricin
 EMI7.3
 
<tb> in <SEP> / cm3 <SEP> on <SEP> agar <SEP> of <SEP> Sabouraud
<tb>
<tb> Candida <SEP> albicans <SEP> 6 <SEP> - <SEP> 12
<tb> (3 <SEP> strains)
<tb>
<tb> Candida <SEP> tropicalis <SEP> (1) <SEP> 3 <SEP> - <SEP> 12
<tb> (:::, <SEP> strains) <SEP>
<tb>
<tb> Trichosporon <SEP> cutaneum <SEP> (1) <SEP> 12
<tb>
 
 EMI7.4
 Pityrosporum spec.

   (1) 12
 EMI7.5
 
<tb> Candida <SEP> parapsilosis <SEP> (1) <SEP> 12
<tb>
 
 EMI7.6
 Histoplasma capsulatum 3
 EMI7.7
 
<tb> Sporotrichum <SEP> Schenckii <SEP> 6
<tb>
 
 EMI7.8
 Trichophyton sulfuriùum 3 Trichophyton ment agr p phyt es 50
 EMI7.9
 
<tb> (3 <SEP> strains) <SEP>
<tb>
 
 EMI7.10
 Trichophyton violaceum 6
 EMI7.11
 
<tb> Trichophyton <SEP> rosaceum <SEP> 12.5
<tb>
<tb> Trichophyton <SEP> Schnleineii <SEP> 6
<tb>
<tb> Trichophyton <SEP> rubrum <SEP> 12.5
<tb>
 
 EMI7.12
 Trichophyton interdigttale 25 lliicrosporum Janosun, 12.5 E idermo h ton floc: .sum 12.5 Hormodendrum .22Ep.2, ctwu 6 (1) This yeast is not pathogenic but has been isolated from a medical material.



   From the foregoing it emerges, inter alia, that pima- ricin has a clear and remarkable action against the fungi
 EMI7.13
 phytopathogens such as Verticillium, Olados122 and Fusarimit The inhibition of Gandida albicans is also very remarkable,
Another important feature is the very mild phytotoxic effect of pimaricin, as opposed to most of the fungicidal antibiotics known heretofore. Thus, this new antibiotic is very suitable, among others, for applications in agriculture and horticulture, to combat

 <Desc / Clms Page number 8>

 plant diseases, especially as it diffuses easily and acts systematically.



   Thus, pimaricin penetrates, for example, in the seeds of peas (Pisum sativum), after being soaked in an extended aqueous solution. This is verified by the fact that pimaricin can be extracted from the cotyledons and the germs of the seeds thus treated.



   Tables IV a and IV b also show an internal antiseptic action. The mycelium of funi (among others Ascochyta pisi) is killed in the seed.



   TABLE IV a
 EMI8.1
 
<tb> Concentration <SEP> in <SEP> pimaricin <SEP> in <SEP>% <SEP> of <SEP> seeds <SEP> with <SEP> mycelium
<tb>
<tb> parts <SEP> by <SEP> million <SEP> (1) <SEP> (2)
<tb>
<tb>
<tb> 150 <SEP> 0
<tb>
<tb>
<tb>
<tb>
<tb> 75 <SEP> 3
<tb>
<tb>
<tb>
<tb> 37.5 <SEP> 11
<tb>
<tb>
<tb>
<tb> 18.7 <SEP> 25
<tb>
<tb>
<tb>
<tb> unprocessed <SEP> 80
<tb>
 TABLE IV b
 EMI8.2
 
<tb> Concentration <SEP> in <SEP> pimaricin <SEP>% <SEP> of <SEP> plants <SEP> diseased
<tb>
<tb>
<tb> (p.p.m.) <SEP> (1) <SEP> Test <SEP> I <SEP> Test <SEP> II
<tb>
<tb>
<tb>
<tb>
<tb> 150 <SEP> 0 <SEP> 0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 75 <SEP> 0 <SEP> 0.7
<tb>
<tb>
<tb>
<tb>
<tb> 37.5 <SEP> 1 <SEP> 2
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 18.7 <SEP> 2 <SEP> 3.6
<tb>
<tb>
<tb>
<tb>
<tb> unprocessed <SEP> 15 <SEP> 24
<tb>
 (1)

   Aqueous solution in which the seeds have been soaked for twenty-four hours at 20 C.



  (2) Development of the mycelium on filter paper.



   Detailed trials have shown that pimaricin at a concentration of 75 p.p.m., which is a very effective fungicide, does not interfere with the germination of pea seeds and

 <Desc / Clms Page number 9>

 the development of the plant (see Tables IVc and IVd).



     . TABLE IV c
 EMI9.1
 
<tb> Concentration <SEP> in <SEP>% <SEP> seeds <SEP> germinated <SEP> Length <SEP> of <SEP> roots
<tb>
<tb>
<tb>
<tb> pimaricine <SEP> p.p.m. (1) <SEP> (2) <SEP> in <SEP> mm <SEP> after <SEP> 3 <SEP> j. <SEP> (2)
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> unprocessed <SEP> 100 <SEP> 23
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 25 <SEP> 100 <SEP> 24
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 50 <SEP> 100 <SEP> 22
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 75 <SEP> 100 <SEP> 22
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 100 <SEP> 100 <SEP> 23
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 125 <SEP> 100 <SEP>.

   <SEP> 22
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 150 <SEP> 96 <SEP> 29
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 200 <SEP> 98 <SEP> 14
<tb>
 (1) Aqueous solution in which the seeds have been soaked; for twenty-four hours at 20 0.



  (2) Seeds placed on wet filter paper.



   TABLE I V d
 EMI9.2
 
<tb> Seeds <SEP> of <SEP> peas <SEP> soaked <SEP> in <SEP> concentration <SEP> in <SEP> pimaricin
<tb>
<tb> in <SEP> water <SEP> 75 <SEP> p.p.m. <SEP> (1)
<tb>
<tb>
<tb>
<tb> Germination <SEP> (number) <SEP> 126 <SEP> 128
<tb>
<tb>
<tb>
<tb>
<tb> <SEP> length of <SEP> the <SEP> crashes
<tb>
<tb> in <SEP> cm <SEP> at <SEP> end <SEP> of <SEP> two
<tb>
<tb> month <SEP> 31.8 <SEP> 33.8
<tb>
<tb>
<tb>
<tb> Number <SEP> of <SEP> terminals <SEP> by
<tb>
<tb> crash <SEP> 2.8 <SEP> 2.8
<tb>
 (1) Aqueous solution in which the peas were soaked for twenty-four hours at 20 C.



   All the results given in the tables relate to the race "Eminent", which is very susceptible to Ascochyta pisi.



   Experiments performed in mice and rats indicate very low toxicity.



   To determine the acute toxicity of pimaricin in the white rat, a 985 (Ó / mg product was administered to rats which were sacrificed after seven days.



   The following results were obtained using suspensions

 <Desc / Clms Page number 10>

 aqueous pimaricin. mg / kg LD50, oral 1,500 LD50, intramuscular route> 2,000) Average values) on 40 rats LD50, hypodermic route) 5,000) LD50, intraperitoneal route 250
Pimaricin does not have an irritating effect on the skin and mucous membranes, so it can also be applied to humans.



   In the pure state, pimaricin is a white crystalline compound of amphoteric nature, the salts of which can be prepared in the usual manner. It does not give a reaction, colored with FeCl3 but it gives, with concentrated phosphoric acid, an unstable pink color. Concentrated hydrochloric acid and concentrated sulfuric acid produce a blue or olive-green color. This antibiotic bleaches bromine water. This fact, as well as the infrared and ultra-violet spectra (see below) suggest that pimaricin is one of the so-called polyene antibiotics. Its solubility in water is very slight, i.e. 8 mg in 100 cm3 of water at 20 C.

   It is more soluble in organic solvents such as alcohols, glycols, ketones, in particular alcohols with 1 to 6 carbon atoms, such as normal butanol, as well as in "Cellosolves".



  It is further soluble in pyridine, dimethylformamide, dimethylacetamide, glacial acetic acid and alkali hydroxides. It is practically insoluble in aliphatic hydrocarbons such as pentane, hexane, cyclohexane, etc.



   At room temperature, pimaricin is a very stable compound. An aqueous solution at 5 mg / 100 cm3 retains its full activity for seven days at pH 7 at 25 C. At pH 2, this solution loses 50% of its activity in three days at this same.

 <Desc / Clms Page number 11>

 temperature. At pH 10 and 25 C, it loses 50% of its activity in six days. At high temperatures, the solution is less stable. Thus, the above-mentioned aqueous solution of pimaricin, at 90 ° C., loses, in fifteen minutes, at pH 2, 90%, at pH 6.5, 15 and at PE 9, 50% of its activity. Pimaricin in methanolic solution is still stable at higher temperatures (25 to 60 C).

   The antibiotic activity of pimaricin was tested microbiologically with Saccharomyces cerevisiae, Dutch strain, as a test microorganism, compared to a normal pure preparation.



   Pimaricin does not show a melting point, but it does; begins to decompose at around 150 C. The specific optical rotation Ó 25D is equal to +250 (conc. 0.083% in absolute methanol). The molecular weight of the substance is approximately 727. Its probable crude formula is C35H50NO14. Elemental analysis gives the following values: C = 57.77%, H = 7.27%, N = 1.95%, 0 (calculated) = 33.01%.



   The ultraviolet absorption spectrum of pimaricin is characterized by maxima at 290, 304 and 318 m with shoulder at 280 m and minimum at about 250 m (Conc. = 3.93Ù / cm3 in methanol). A sample of pimaricin pressed on a potassium bromide plate (conc. = 0.5%) 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, 803, 794 (for the underlined values the absorption is from strong to very strong ). (See attached diagram).



   To prepare pimaricin, Streptomyces natalensis is grown aerobically in stationary cultures or submerged in a fluid nutrient medium, under sterile conditions, in closed containers fitted with stirring devices and in which, for of aeration, you can breathe in oxygen, -

 <Desc / Clms Page number 12>

 gene or sterile air during cultivation.



   Cultivation time, temperature and other conditions which must be met in order to obtain good yields of pimaricin are readily determined by experience. These conditions are discussed more fully below.



   The nutrient medium may consist of the usual substances; it should contain a source of fermentable organic and / or mineral carbon and nitrogen, the additional presence of inorganic salts, such as phosphates, potassium and sodium salts, and traces of various metals also being desirable. However, the raw materials used in practice are often sufficiently contaminated with these mineral salts that further additions are unnecessary.



   As the carbon source, soluble or insoluble carbohydrates can be used, such as glucose, sucrose, lactose or starch. Sugar alcohols, such as glycerin, are also suitable. The amount of carbon source present in the medium can vary widely depending on the nature of the carbohydrate used and the composition of the medium.



  It is generally about 0.5 to 5% by weight of the medium.



   The source of nitrogen can be made up of a wide variety of substances. Mention may be made of hydrolysed or non-hydrolyzed casein, corn steep liquor, peptone, meat extract, defatted or non-defatted soy flour, arachi flour, fish meal. , nitrates. The choice of the nitrogen source usually depends on the composition of the medium, which is itself chosen so as to obtain the antibiotic in the most economical manner. It may be mentioned in this connection that small amounts of nitrogenous raw materials such as yeast extract, soluble products from distilleries, soluble fish products, etc., considerably improve the yields of pimaricin in particular media. -

 <Desc / Clms Page number 13>

 bind.

   Also, lipids such as fatty oils, of vegetable as well as animal origin (soybean oil, fish oil for example) are likely to improve the yield in a substantial way.



   The duration of fermentation depends to a large extent on the composition of the nutrient medium. It usually varies between forty-eight and one hundred and twenty hours but it can be continued for a longer period if desired, for example up to fourteen days, if the increase in antibiotic yield thus obtained justifies the additional expense. involves the prolongation of the fermentation cycle.



   The temperature at which the fermentation is carried out can in principle vary between 15 and 30 C. Preference is given to. temperatures from 26 to 28 C.



   To achieve the optimum conditions for proliferation of the microorganism and yield of pimaricin, the pH can vary in particular during the first phase of the fermentation, between 5 and 8 for example. After sterilization, the nutrient medium is preferably adjusted to a pH between 6 and 7. Fermentation is preferably carried out at a pH between 6.5 and 8, since in this case the highest yields are obtained. High -.- The pH can be kept constant during fermentation by the addition of an alkali hydroxide or an acid at regular intervals under sterile conditions.

   However, calcium carbonate is usually used as a buffer in proportions which vary between 0.2 and 1% by weight of the medium. The amount of air that is passed through the medium during fermentation under sterile conditions depends largely on the shape of the vessel, the speed and the shape of the agitators. Generally, this amount varies between 0.1 and 4 liters per liter of nutrient medium and per minute.

 <Desc / Clms Page number 14>

 



   Preferably forty-eight to seventy-two hour pre-cultures of Streptomyces natalensis are used as the seed material for the main fermentation medium. In order to obtain good yields and to avoid fluctuation of the results, the inoculation is preferably carried out with the aid of amounts of culture representing 1 to 7% by volume of the nutrient medium contained in the main fermentation tank. Obviously, in the case of large fermentation tanks, several stages of preculture must be carried out. However, it is also possible to keep a portion of the culture, for example 10%, in the main fermentation tank for the production of a new culture. The fermentation, as a whole, can also be carried out in a continuous manner.



   Pimaricin can be removed from the culture medium in several ways. In accordance with the invention, it is possible to take advantage of the solubility of the antibiotic in organic solvents miscible with water to a limited extent, such as butanol.



  It is possible, for example, to apply the following method. When a sufficient quantity of active medium has been obtained, its pH is adjusted to about 10 so as to extract the active substance from the "mycelium", which is then separated by filtration. The medium can then be depleted, for example using normal butanol, at a pH of between 3 and 10. If the fluid is acidified, the acid used is preferably phosphoric acid. . Any precipitate which may form is separated and, if necessary, also spent. The extract is concentrated by azeotropic distillation and the crude active substance eventually crystallizes. This can be purified by selective precipitation, for example in glacial acetic acid, pyridine, dimethylformamide, etc.

   In fermentations giving yields greater than 700 Ù / cm3, the yield of pimaricin

 <Desc / Clms Page number 15>

 can be increased very noticeably by carrying out the exhaust-. ment of the mycelium, an operation in which it is advantageous to use, as vehicle, methanol, the solvent power of which is increased by the addition of 1 to 3% of calcium chloride.



  Example 1 - Preparation of the seed culture.
From a tube culture of Streptomyces natalensis nov.spec. for example on oatmeal-agar, exhibiting good sporulation, small quantities of conidia are transferred ,. under sterile conditions, in shaken flasks with a capacity of about 2 liters, into which 500 cc of a fluid nutrient medium having the following composition has been introduced.
 EMI15.1
 
<tb>



  Peptone <SEP> 0.5
<tb>
<tb>
<tb>
<tb> Liquor <SEP> concentrated <SEP> of <SEP> steeping
<tb>
<tb> of the <SEP> maize <SEP> (50 <SEP>% <SEP> dry <SEP> material) <SEP> 0.6 <SEP>%
<tb>
<tb>
<tb>
<tb> Glucose <SEP> 1.0 <SEP>%
<tb>
<tb>
<tb>
<tb>
<tb> Ordinary <SEP> salt <SEP> 1.0 <SEP>% <SEP>
<tb>
 (pH adjusted to 7 using an alkaline hydroxide).



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



     Example II - Preparation of culture.-
1 liter of the seed culture prepared in accordance with Example 1 is transferred under sterile conditions into a fermentation vessel fitted with a stirrer and a device for blowing sterile air, this vessel containing
15 liters of a culture medium having the following composition:
 EMI15.2
 
<tb> Glucose <SEP> 3.0%
<tb>
<tb> Liquor <SEP> concentrated <SEP> of <SEP> steeping
<tb> of <SEP> but <SEP> (50 <SEP> dry <SEP> material) <SEP> 0.2
<tb>
<tb> Ammonium <SEP> <SEP> 0.5 <SEP>%
<tb>
<tb> <SEP> potassium chloride <SEP> 0.4 <SEP>%
<tb>
<tb> Primary <SEP> potassium <SEP> <SEP> <SEP> <SEP> 0.02 <SEP>%
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 0,

  8%
<tb>
 

 <Desc / Clms Page number 16>

 
The pH of this culture medium is adjusted to 6.9 by means of an alkali hydroxide. After 48 hours of incubation at 27 ° C., with aeration and constant agitation, the culture was found to contain 610 micrograms of pimaricin per cubic centimeter.



   Other media, with which yields of the same order of magnitude can be obtained, have the following composition:
 EMI16.1
 
<tb> A- <SEP> Peanut <SEP> flour <SEP> 2 <SEP>%
<tb>
<tb> Starch <SEP> of <SEP> apple <SEP> of <SEP> earth <SEP> 1 <SEP>%
<tb>
<tb> Liquor <SEP> concentrated <SEP> of <SEP> steeping
<tb> of the <SEP> but <SEP> (50 <SEP>% <SEP> dry <SEP> material) <SEP> 2 <SEP>%
<tb>
<tb> Ordinary <SEP> salt <SEP> 0.5 <SEP>%
<tb>
<tb> Magnesium <SEP> <SEP> <SEP> 0.1 <SEP>% sulfate
<tb>
<tb> Primary <SEP> potassium <SEP> <SEP> <SEP> <SEP> 0.05 <SEP>%
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 0.6 <SEP>%
<tb>
 
Potassium hydroxide up to pH 6.5.



   Using this medium, after seeding at 4% and incubation and aeration for 72 hours at 26 ° C., 590 micrograms of pimaricin per cubic centimeter of fermentation fluid were obtained.
 EMI16.2
 
<tb>



  B <SEP> - <SEP> Molasses <SEP> of <SEP> beet <SEP> (content <SEP> in
<tb> sugar <SEP> approximately <SEP> 50 <SEP>%) <SEP> 4.0%
<tb>
<tb> Liquor <SEP> of <SEP> soaking <SEP> of <SEP> but <SEP> concentrated <SEP> 2.0 <SEP>% <SEP>
<tb>
<tb> Lactose <SEP> 1.0 <SEP>% <SEP>
<tb>
<tb> Sodium <SEP> <SEP>, <SEP> 10 <SEP> aq. <SEP> 0.1 <SEP>%
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 0.5 <SEP>%
<tb>
 
Potassium hydroxide up to pH 7.1.



   With the aid of this medium, after inoculation at 5% and 120 hours of incubation and aeration at 27 ° C., 640 micrograms of pimaricin per cubic centimeter of fermentation fluid were obtained.
 EMI16.3
 



  Peptone (Difco ") 0 t 5 fi: gjctra4it of meat (IIDifco Il) 0 s5

 <Desc / Clms Page number 17>

 
 EMI17.1
 
<tb> Glucose <SEP> 1.0%
<tb>
<tb> Ordinary <SEP> salt <SEP> 0.5 <SEP>%
<tb>
   Using this medium, after seeding at 3% and 148 hours of incubation and aeration at 26 ° C., 535 micrograms of pimaricin per cubic centimeter of fermentation fluid were obtained.
 EMI17.2
 
<tb>



  D- <SEP> Soybean <SEP> coarse <SEP> <SEP> <SEP> 5.0 <SEP>%
<tb>
<tb>
<tb> Soybean <SEP> <SEP> oil <SEP> 0.5 <SEP>%
<tb>
<tb>
<tb>
<tb> Liquor <SEP> concentrated <SEP> of <SEP> steeping
<tb>
<tb> of <SEP> but <SEP> (50 <SEP>% <SEP> of <SEP> dry <SEP> material) <SEP> 0.2 <SEP>%
<tb>
<tb>
<tb>
<tb> Glucose <SEP> 1.0 <SEP>%
<tb>
<tb>
<tb>
<tb> Primary <SEP> potassium <SEP> <SEP> <SEP> <SEP> 0.02 <SEP>%
<tb>
<tb>
<tb>
<tb> Ammonium <SEP> sulphate <SEP> 0.5 <SEP>%
<tb>
<tb>
<tb>
<tb> Carbonate <SEP> of <SEP> calcium <SEP> 1.0 <SEP>%
<tb>
 
Using this medium, after inoculation at 3% in a 15 liter tank and fermentation and aeration for 168 hours at. 28 C, 690 micrograms of pimaricin are obtained per cubic meter of fermentation fluid.

   After treatment, with the aid of extended alkali hydroxide, of the mycelium previously separated by suction and stirred with a fairly large quantity of water, at pH 10, it was possible to find, in the filtrate of the mycelium, 20.4 g of pimaricin, Example III - Isolation of crude pimaricin.
4 liters of fully fermented fluid fermentation medium containing 590 Ù / cm3 of pimaricin were adjusted to pH 10 with sodium hydroxide and freed of the mycelium by filtration using Kieselguhr as a filter aid (2%). The culture filtrate, the volume of which was 3.7 liters, activity 570 Ù / cm3, was acidified with phosphoric acid to pH 3. The acidified filtrate was successively depleted by means of portions of n -butanol of 750, 350 and 150 cm3.

   The butanol extract was then washed three times with 120 cm3 of a 4% borax solution, followed by new.

 <Desc / Clms Page number 18>

 veal twice in 120 cm3 of water. The butanol extract then contained 1400 Ù / cm of pimaricin. Azeotropic concentration of this extract at 100 cc gave 0.64 g of not quite pure pimaricin, crystalline (activity 900 / mg). A total amount of 1.42 g of impure pimaricin (activity 395 Ù / mg) was then obtained by evaporation of the remainder of the extract.

   The yield, related to the total activity of the completely fermented fluid, was 48.3%: Example IV - Isolation of crude pimaricin.
15 liters of a fully fermented broth containing 610 Ù / cm3 of pimaricin were adjusted to pH 10.3 with the aid of 35% potassium hydroxide, then the mycelium was separated therefrom by filtration, using 50 g of "Hyflo" as a filter aid. The filtrate was then acidified to pH 2.8 with phosphoric acid.

   The precipitate formed was then filtered off, this time with 10 g of "Eyflo". The precipitate was then stirred for half an hour with 100 cm 3 of n-butanol. After standing, the butanol extract was decanted and the deposit was washed with 100 cm3 of water. The total butanol extract was washed three times with 10 cm3 of 4% borax solution, then three times with 10 cm3 of water. It was then evaporated under vacuum at 44 ° C. to 40 cm3. After cooling to 0 C, vacuum suction and drying, 'was obtained 0.51 g of pale yellow crystalline pimaricin, with an activity of 850 Ù / mg. The filtrate was then treated as described in Example I.

   Two fractions were obtained, respectively 3.21 g (activity 890 / mg) and 6.8 g (activity 223 Ù / mg). The total yield was 51.5%, Example V - Isolation of crude pimaricin. -
10 liters of a fully fermented culture broth (activity 640 Ù / cm3) were adjusted to pH 10 using 15% potassium hydroxide. The mycelium was separated by centrifugation.

 <Desc / Clms Page number 19>

 and the clear filtrate was adjusted to pH 6.8 with 10 N hydrochloric acid. It was then extracted successively with 2,000, 1,000 and 1,000 cc portions of iso-amyl alcohol.

   The isoamyl extracts were washed successively three times with 30.0 cm3 of 4% sodium carbonate solution, then twice with 300 cm3 of water.



   The extract thus treated was subjected to azeotropic evaporation, up to 100 cm 3. 2.62 g of pale yellow crystallized pimaricin with an activity of 900 Ù / cm3 separated. Yield: 36.8%.



  Example VI - Isolation of crude pimaricin. -
The pH was adjusted to 10 using 20% sodium hydroxide.
20 liters of a culture broth completely. fermented from
Streptomyces natalensis (activity 700 / cm3), then the mycelium was separated by filtration using "Hyflo" (200 g). The clear filtrate was successively depleted with 4,000, 2,000 and 1,000 cc portions of n-butanol. The mixture of butanol extracts was washed twice with 500 cm3 of 4% borax solution, then twice with 500 cm3 of water. After adjusting the butanol extract thus treated to pH 6.8 with 10 N hydrochloric acid, the extract was subjected to azeotropic concentration in vacuo to a volume of 250 cm 3.

   9.85 g of crystallized pale yellow pimaricin with activity 913 Ù / mg separated. Yield: 60.8%.



   Example VII - Isolation of crude pimaricin from mycelium.
Was adjusted to pH 4.1, using glacial acetic acid,
15 liters of a fully fermented culture broth of
Streptomyces natalensis nov. Spec. Then 200 g of "Hyflo" was added as a filter aid and the solution stirred. The mycelium was then drained to the greatest possible dryness (total weight 1700 g). 1 g of mycelium in this state was depleted for one hour with 40 cc of methanol, so as to determine the number of micrograms of pimaricin.

 <Desc / Clms Page number 20>

 



  The methanolic extract was extended to 100 cm 3 and subjected to spectrophotometry. The pimaricin content of this solu-
 EMI20.1
 methaiolic ratio was 120 / cm3.



   The pimaricin content of the total mycelium was 20.4 g.
The drained mycelium was depleted for half an hour at room temperature with 8 liters of methanol in which 2% calcium chloride had been dissolved. The extract thus obtained was extended with the aid of one liter of water and there was some. removed the methanol in vacuo. A crystalline precipitate of pimaricin was thus obtained which, after filtering off, washing with water and drying under vacuum, weighed 14.73 g and whose activity was 900 Ù / mg. Yield: 65% of theory.



   Example VIII - Purification of pimaricin.-
10 g of impure pimaricin (activity 890 / mg) was dissolved with gentle heating in 80 cm3 of glacial acetic acid, and then the insoluble impurities were removed as quickly as possible by filtration. The clear yellow-brown filtrate was extended with 1500 cm3 of water and the pH of the solution thus obtained was adjusted to 6.3 using a solution of sodium hydroxide to 33 %.



   After cooling, the crystalline precipitate formed was separated by centrifugation and washed twice with a total amount of 500 cm 3 of water, after which it was filtered off under reduced pressure. The crystallized mass was washed again on a filter with 200 cm 3 of water, then dried under vacuum over phosphorus pentoxide. The weight of the very pale yellow product thus obtained was 7.07 g and its activity 960 Ù / mg.



   The above treatment was recommended using 60 cc of glacial acetic acid and 1000 cc of water. The substance was washed three times with 200 cc of water. The product thus obtained weighed 5.35 g and had an activity of 985 Ù / mg.

 <Desc / Clms Page number 21>

 



  Total yield: 59.8%.



    Example IX - Purification of pimaricin. -
It was heated gently in 100 cm3 of dimethyl-formamide
10 g of impure piuaricin of activity 890 Ù / mg, this solution was filtered to remove insoluble impurities and the filter was washed with a further 30 cm3 of dimethylformamide. To the clear brown filtrate was added 250 cc of water, which precipitated the antibiotic in crystalline form. The precipitate was filtered off under vacuum and the filter was washed again with 100 cc of water. The crystalline mass was then dried in vacuo. The product thus obtained weighed 9.24 g.

   The above treatment was repeated and a pale yellow crystalline product, weighing
7.9 g and having an activity of 985 Ù / mg. Total yield: 87.5%.



   Preparation of the sodium salt. 2.5 g of pimaricin of 985 Ù / mg activity were suspended, with mechanical stirring, in 20 cm3 of methanol. A solution of 0.145 g of sodium hydroxide in 0.3 cm3 of water was then added to the suspension. The pimaricin first dissolved and then separated after a few minutes as the crystallized sodium salt.

   After another half hour of stirring and twenty-four hours of standing at 0 ° C., the crystallized mass was filtered off with suction and washed with 5 cm3. of ethanol and 10 cm3 of diethyl ether. After drying in vacuo, 2.16 g of sodium salt crystallized in white needles were obtained. Activity: 995 Ù / mg, or 87.3% of the calculated activity.

** ATTENTION ** end of DESC field can contain start of CLMS **.


    

Claims (1)

CLAIMS. 1.- Substance having antibiotic properties against fungi and saprophytic and parasitic yeasts which, in the <Desc / Clms Page number 22> EMI22.1 pure, is a crystalline solid b7¯ :. c cc}; L: iuj-G..i ') 21, zi dCO; '11) Dare at about 15C C, po: s: c, a specific .otL.ce power 1 D5 = + 250 (at a concentration of O, C; j .; 'd * noe the: .. ¯ëi ¯o? ¯ absolute) and which shows an absorption T: laxil: H1 ..: l of: the first ultraviolet at 290, 304 and 316 T, l, 1 L'with a ';' 9at; .lGdlit at about 280 and a minimum at about 250 m and which, on a potassium bromide plate, shows the following absorption at infrared (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. 2. - Substance having antibiotic properties with regard to fungi and saprophytic and parasitic yeasts which. in its pure state, is a white crystalline compound which does not give a color reaction with ferric chloride, produces with phosphoric acid an unstable pink color, discolors water of, bromine, is slightly soluble in water and more soluble in alcohols and is soluble in pyridine, dimethylformamide, dimethylacetamide, glacial acetic acid, alkali hydroxides and aliphatic hydrocarbons, and which exhibits maximum absorption in ultraviolet light at 290 , 304 and 318 m with a shoulder at about 280 and a minimum at about 250 m and which, on a potassium bromide plate, shows the following absorption in infrared light (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. 3.- Process for the preparation of pimaricin, characterized in that a culture of Streptomyces natalensis is propagated in an aqueous nutrient medium under submerged aerobic conditions. 4. - Process for preparing pimaricin, characterized in that a strain of Streptomyces natalensis is propagated in <Desc / Clms Page number 23> an aqueous nutrient medium under submerged aerobic conditions at a temperature between about 15 ° C and about 30 ° C for a period of about two to five days. 5. - Process for the preparation of pimaricin, characterized in that a culture of Streptomyces natalensis is propagated in an aqueous nutrient medium under submerged aerobic conditions at a temperature between about 26 C and about 28 C for a period. from two to five days, and in recovering the pimaricin thus produced from the resulting fermentation liquor. 6. - Process for the preparation of pimaricin, characterized in that a culture of Streptomyces natalensis is propagated in an aqueous carbohydrate solution containing nutrients and having a pH of 6.5 to 8, under condi- aerobic conditions are submerged, at a temperature of between about 15 ° C and about 30 ° C for two to five days, and in that the pimaricin is recovered from the resulting fermentation liquor. 7.- Process for the preparation of pimaricin, characterized in that a culture of Streptomyces natalensis is developed under aerobic conditions in an aqueous medium having a pH of between 6.5 and 8 and containing a soluble carbohydrate and a source of assimilable nitrogen at temperatures in the range of from about 15 C to about 30 C for a period of about two to five days, the aqueous medium being thus fermented and pimaricin produced, and in that that we recover the pimaricin from the fermented liquor. 8. - Process according to claim 7, characterized in that the recovery of the pimaricin comprises the operative phase of extracting the antibiotic in butyl alcohol at a pH of about 3.