IL30058A - Process for preparing antibiotic a10388(pyrrolnitrin) - Google Patents

Description

"PROCESS FOR PREPARING ANTIBIOTIC A1Q338" (PYRROLNITRIN) A 103158 ¾ρ« 3'»03Κ n33n V ?ffl * < This invention concerns an improved process for preparing by a bacterium, the antibiotic A10338.

The antibiotip ΑΙΟ33Θ known i.n the literature as py- rollnitrin, is a pale yellow crystalline solid melting at about , 0 12 C. It is insoluble in water and relatively insolyble in aliphatic hydrocarbon solvents, but Is relatively soluble in most other organic solvents such as alcohols, lower ketones, such as acetone, methyl ethyl ketone, and the like benzene, chloroform, esters, such as ethyl acetate, butyl acetate, and the like; and ethers, such as diethyl ether. It is stable over a relatively wide pH range, remaining substantially unchanged between about pH 2 and pH 10, and contains no tltratable groups.

Antibiotic A10338 exhibits an inhibitor action against the growth of a variety of microbial organisms, including both bacteria and fungi. It is especially useful in the treatment of fungal infections. Thus, for example, antibiotic A10338 is especially valuable for agricultural use in the treatment of plants infected with powdery mildew or anthracnose.

Antibiotic AIO338 has been produced by culturing the organisms Pseudomonas spp. A.T.C.C. 15925 and A.T.C.C. 15926 under aerobic conditions in a culture medium containing assimilable sources or carbon, nitrogen, and inorganic salts. These organisms have subsequently been Identified as members of the species Pseudomonas aureofaciens.

In accordance with this invention it has een found that eight novel strains of organisms of the genus Pseudomonas may be used to produce antibiotic A10338 by fermentation of a nutrient medium..

More particularly, the present invention provides -* a process for preparing antibiotic A10338 which comprises cultivating an organism selected from the group consisting of Pseudomonas fluorescens A.T.C.C. 17810, Pseudomonas fluorescens A.T.C.C. 17 16, Pseudomonas fluorescens A.T.C.C. 17 17, Pseudomonas fluorescens A.T.C.C. 17 19, Pseudomonas multivorans A.T.C.C. 17460, Pseudomonas multivorans A.T.C.C. 17478, Pseudomonas multivorans A.T.C.C. 17759, and PPsseeuuddoommoonnaass mmuullttiivvoorraannss A.T.C.C. 17760 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions until a substantial amount of antibiotic A10338 is produced by said organisms in said culture medium.

The culture medium employed to produce antibiotic A10338 from the various organisms useful In the process of this invention can be any one of a number of media. The antibiotic producing organisms are cappble of utilizing energy from a variety of sources. However, for economy in production, optimal yield, and ease of isolation of the antibiotic, certain culture media are preferred. Thus, for example, one of the preferred sources of carbohydrate in the fermentation is glycerol, although molasses, glucose, fructose, sucrose, starch, inositol, and the, like can also be employed. Pre-ferred sources of nitrogen are barley, peptones, soybean meal, amino acid mixtures and the like. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, ammonium, calcium, phosphate, chloride, carbonate and like ions.

Essential trace elements necessary for the growth and development of the organism used for the production of A10338 should also be included in the culture medium. Such trace elements commonly occur as impurities in the other con-stituents of the medium in amounts sufficient to meet the growth requirements of the organism.

The organisms employed to produce A10338 are tolerant of considerable variation in the growth conditions. Thus for example, the organisms will grow in a variety of media in which the initial pH can vary from about pH 5 to about pH 10. Prior to inoculation of the medium with one of the organisms, however, it is desirable to adjust the pH of the culture medium to between about pH 5.5 and about pH 7.5, depending upon the particular medium employed. The pH of the medium gradually changes as the fermentation proceeds and may either Increase or decrease during the growth period of the organism while the antibiotic is being produced, depending upon the medium employed. The final pH is determined, at least In part, by the initial pH of the medium, the buffers present in the medium, and the length of time for which the organism is permitted to grow.

Submerged aerobic culture in large tanks is preferably employed for the production of substantial amounts of antibiotic A10338, just as for other antibiotics. Small quantities of the antibiotic are conveniently obtained rom shake flasks and surface culture in bottles. The fermentation medium in the sterile tank can be inoculated with a bacterial cell suspension to initiate a fermentation. However, inasmuch as some growth lag is experienced when the fermentation tank is inoculated directly, an intermediate vegetative culture is employed.

By avoiding the growth lag in this manner, more efficient utilization of the fermentation equipment is realized, Accordingly, It is desirable first to produce a vegetative inoculum of the organism by Inoculating a relatively small quan- tity of a culture medium with the organism, and when a young, active vegetative inoculum has been obtained, to transfer the vegetative inoculum aseptically into a large fermentation tank. The medium in which the vegetative inoculum is produced can be either the same as or different from the medium utilized for the large scale production of A10338.

The organisms which produced A10338 will grow over a wide temperature range between about 25°C. to about 37°C.

Maximum growth, however, occurs between about 27° C. to about 3 °C, and it is preferred to conduct the fermentation at a temperature between about 27°C. and 30°C, As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium during fermentation. For efficient growth of the organism and conser quent efficient production of A10338, the volume of air employ ed in the tank production of the antibiotic is preferably upwards of about 0.1 volume of air per minute per volume of culture medium. Most efficient growth and optimal yields, of the antibiotic are obtained when the volume of air used is at least one-half volume of air per minute per volume of culture medium, and preferably substantially more.

The concentration of antibiotic activity in the culture medium can readily be followed during the course of the fermentation by testing samples of the culture medium for their Inhibitory activity against the growth of an organism known to be Inhibited in the presence of this antibiotic. A species of the organism Neurospora, designated as Neurospora spp. M 5-846, has been found to be suitable for this purpose. The testing of the samples can be carried out by the well-known turbidimetric or cup-plate methods, In general , maximum production of A10338 occurs within about two to five days after inoculation of the culture medium when submerged aerobic culture or shake-flask culture is employed, and within about five to ten days when surface culture is employed.

The antibiotic activity produced during the fermentation of A10338 occurs both ;Ln the antibiotic broth, and In the cells. Accordingly, isolation techniques employed in the production of A10338 are designed to permit maximum recovery of the antibiotic from both sources. Thus, the cells of the organism producing the antibiotic and redissolved solids are removed from the fermentation broth by conventional means, such as filtration or centrifugatlon, usually after the dilution of the broth by the addition of a water miscible organic solvent such as methanol which is effective in recovering the antibiotic retained by the cells. The antibiotic is contained in the filtered broth and can be recovered therefrom by employing well-known extraction techniques. The extracting solvent can be any one of a number of solvents which is immiscible with water and in which the antibiotic is readily soluble, chloroform being especially preferred. The treatment of the solvent extracts containing the antibiotic will vary slightly, depending upon the organism employed for preparation of A10338.

In general, isolation of antibiotic A10338 Is accom- plished as follows: The whole broth, diluted with an organic solvent such as methanol, Is filtered with the aid of a commercial filter aid and the filtrate Is concentrated and adjusted to about pH 10 with base, although the broth may be subjected to the Isolation procedure at the harvest pH, which is generally about pH 6. 9. The concentrated filtrate is extracted with an immiscible solvent for the antibiotic.

The following specific examples, will more fully illustrate the process of this invention, but are not to be con-strued to be the exclusive embodiments thereof.

EXAMPLE 1 A culture of Fseudomonas fluorescens A.T.C.C. 17810 is produced by growing the organism on a nutrient agar slant having the following composition: Peptone Agar Slant Medium Peptone (Difco) 10 g.

Agar 20 g.

Deionized water, added to make a final 1 liter volume of The medium Is sterilized by autoclaving at 121°C. for minutes. The pH of the medium after sterilization is between about pH 7 and about pH 7.2. The slant is inoculated with cells of Pseudomonas fluorescens A.T.C.C. 17810 and is Incubated at about 30°C. for about 48 hours. Sterile water is then added to the slant, and the slant is scraped gently to remove the organisms and to provide an aqueous cell suspension. The cell suspension is transferred to a sterile container and is adjusted to about 50 percent light transmission at a wave length of 525 mu by the addition of sterile water. The cell suspension so ob- tained.is employed to inoculate the production medium.

A medium having the following composition is employed for production; Glycerine 30.0 g Magna yeast 20.0 g Gluten meal 20.0 g Corn steep liquor 10.0 g Potassium dihydrogen phosphate 21 . 8 g Disodium hydrogen phosphate 1 .3 g dodecahydrate Sodium chloride 3.0 g Magnesium sulfate heptahydrate 0.5 g Ferrous sulfate heptahydrate 0.5 g Tap water, added to make a final volume of 1 liter The production medium Is sterilized by autoclavlng at 121°C. for about 25 minutes. The final pH after sterilization is between about pH 7.2 and. about pH 7.4. For shake-flask production, 250 ml. Erlenmeyer flasks containing 60 ml. of the above medium per flask are inoculated with 1 .5 percent (volume/volume) of the cell suspension obtained as described above. The flasks are incubated at a temperature between about 26 and about 30° C. for about 96 hours on a rotary shaker having an agitation rate of 250 rpm and a 2-inch throw. During the period of growth of the organism while the antibiotic is being produced, the pH of the medium drops slightly so that the final pH at the end of the fermentation is between about pH 6.7 and about pH 6. 9, The culture broth obtained by this process, after dilution with methanol and filtration to remove solids, is found to contain the antibiotic A10338 produced by the above organism. ,.

EXAMPLE 2 Antibiotic A10338 was prepared by the method of Example 1 using the organism Pseudomonas. fluorescens A.T.C.C. 17416.

EXAMPLE 3 Antibiotic Al0338 was prepared by the method of Ex-ample 1 using the organism Pseudomonas multlvorans A.T.C.C. 17460.

EXAMPLE 4 Antibiotic A10338 was prepared by the method of Ex: ample 1 using the organism Pseudomonas multlvorans A.T.C.C. 17478.

EXAMPLE 5 Antibiotic A10338 was prepared by the method of Example 1 using the organ!sm Pseudomonas mu 11vorans A.T.C.C. 17759, EXAMPLE 6 Antibiotic A10338 was prepared by the method of Example 1 using the organism Pseudomonas multlvorans A.T.C.C. 17760.

EXAMPLE 7 Antibiotic A10338 was prepared by the method of Example 1 using the organism Pseudomonas fluorescens A.T.C.C. 17 17 and a production medium consisting of :'· Glycerine 30.0 g, Monosodium glutamate 10.0 g.

Ammonium chloride 3.0 g, Glycine 1,0 g.

Potassium dihydrogen phosphate 21,8 g.

Disodium hydrogen phosphate l4.3 g. dodecahydrate Magnesium sulfate heptahydrate 0„5 g.

Zinc sulfate heptahydrate 0,05 g.

Ferrous sulfate heptahydrate 0.5 g.

Delonized water, added to make a final volume of 1 liter EXAMPLE 8 Antibiotic A10338 was prepared by the method of Example 7 using the organism Pseudomonas fluorescens A.T.C.C. 17 19.

EXAMPLE 9 Antibiotic A10338 was prepared by the method of Example 8 using the organism Pseudomonas multlvorans A.T.C.C. 17460.

EXAMPLE 10 Antibiotic A10338 was prepared by the method of Example 8 using the organism Pseudomonas multlvorans A.T.C.C. 17478, EXAMPLE 11 Antibiotic A10338 was prepared by the method of Ex-ample 8 using the organism Pseudomonas multlvorans A.T.C.C. 17759.

EXAMPLE 12 Antibiotic A10338 was prepared by the method of Ex^ ample 8 using the organism Pseudomonas multlvorans A.T.C.C. 1776O,

Claims (2)

30058/ 2 CLAIMS : (pyrrolnitrin)

1. A process for preparing antibiotic A 10338/which comprises cultivating an organism selected from the group consisting of Pseudomonas fluorescens A, T. C. C. 17810, Pseudomonas fluorescens A. T. C. C, 17416, Pseudomonas fluorescens A. T. C. C. 17417, Pseudomonas fluorescens A. T. C. C. 17419, Pseudomonas multivorans A. T, C. C. 17460 Pseudomonas multivorans A. T. C. C. 17478 Pseudomonas multivorans A, T. C. C. 17759, and Pseudomonas multivorans A, T? C, C. 17760 in a culture medium containing assimilable sources of carbon, nitrogen, and inorganic salts under submerged aerobic conditions until a substantial amount of antibiotic A10338 is produced by said organisms in said culture medium and recovering said antibiotic from the culture medium.

2. The process for preparing antibiotic A10338 substantially as herein described with particular reference S. HOROWITZ & CO. J AGENTS FOR APPLICANTS