IL31374A - Preparation of pleuromutilin and its use in animal feeds - Google Patents

Description

PREPARATION OP PLEUR OMUT I LIN AND ITS USE IN ANIMAL FEEDS .A.23514 Pile 16881 .XII.68 The present invention relates to the production of an antibiotic now shown to be identical with the known antibiotic pleuromutilin but previously named BC 757 by us. It also relates to animal feeds containing the antibiotic!, !Dhe application is divided out of Application No. 23314i The antibiotic produced according to the present invention has an empirical molecular formula C22H34^5 (molecular weight 378) is readily soluble in alcohol, ether; butyl acetate acetone, methylisobutylke one, tetraglycol; and chloroform, but difficultly soluble in water; petroleum ether and cyclohexanej melts at 167°C1*crystallized from HgO : has an optical rotation|VJ D e f 32 (10$ in chloroform), and exhibits characteristic absorption maxima in the ultra-violet speotrum at 205 and 293 mp(in other) or 209 πιμ -^d 290 πιμ (in 60 ethanol), respectively, and characteristic absorption bands in the infra-red spectrum (potassium bromide pressings) at 3400, 3OOO, 1235 and 1095 and in a methylene chloride solution at 3??0,36lO, 173O, 1740 and 1630 cm"1. Recent tests have shown that · the antibiotic, previously named by us BC 757, is identical with the known antibiotic pleuromutilin which is described in Proc* Nat. Acad.' Sci. 1951f Vol.37, page 570(Prederick avanagh et al. It has also been found tha BO 757 P.A.23314 File 16881 .XII.68 According to the present invention there is provided a process for producing the antibiotic comprises fermenting a particular strain of Olitopilus paseeekerianus (Pil.) Sing, (belonging to the family of Tricholomatacaea in the order Agaricales of the class Basidlomycetee) or a Pleuromiltilin - producing mutant or variant thereof in an aqueous nutrient medium in the presence of sources of assimilable nitrogen and carbon, for example carbohydrates.

Olitopilus passecfeerlanus (Pil.) Sing, is a known species of fungus which is listed in the "New Check List of British Agarics and Boleti" compiled'by Dennis, Orton and Hora, and published in I960 by the British Mycologlcal Society, A culture of the above mentioned strain has been deposited with the Fermentation Division of the Northern Regional Research Laboratories (WRRL), Peoria, Illinois, U.S.A., where it is included in the culture collection under the number NRRL 3100.

The antimito ic may be isolated by conventional methods for the isolation of pleuromutilin from a fermentation broth, for example as described in Proc. Nat, Aoad. Scil, 1951»Vol 37, page 570.

The following Table gives a summary of the productivity of the strain HRRL 3100 as compared with the strains described in the literature, Strain Yields: mg.of isolated pleuromutilin from 1 litre : ; culture.

.XII.68 Drosophil substrata small amounts together drosophilin A,C and D (Drosophilin B « pleuro mutilin) Clitopilus passeckeriahus 2390 crude product,corres(Pil.)Sing* NRRL 3100 ponding to I960100 pure substance It has been found and confirmed b^numerous feeding tests that the antibiotic Pleuromutilin obtained as described above is a useful additive to animal feeds for improving the utilization of the feed by the animal and increasing the rate of growth and that it affords advantage over antibiotics hitherto used as feed supplements.

Accordingly the invention also provides ready-mixed feeds containing an amount of pleuromutilin sufficient to increase the animal growth rate and, feed supplements containing pleuromutilin for preparing such ready-mixed feeds The feed supplements of the invention may also contain the sual additives incorporated in feed mixtures, such as protein concentrates, fish meali dried mycelia particularly from the production of penicillini dried blood, mineral substances or vitamins* The antibiotic pleuromutilin need not be completely isolated when it is to be used in feed supplements. Instead tfflQ whole of the culture solution resulting from a fermentation of. the antibiotic-producing microorganism may be evaporated to dryness in vacuo and the resulting cake powdered and used for producin a feed or feed supplements Ρ.Δ.23314 Pile 16881 .ΧΙΙ.68 The difficultly water-soluble Pleuromutilin is only slightly resorbed in the intestine. The antibiotic bacitracin, however, which is a substance that is not resorbed in the intestinal tract, is alraady being used in the animal feed-producing industry. High resorption is therefore clearly not essential to the efficaoy of an antibiotic in an animal feed.

A particular advantage of the antibiotic Pleuromutilin is that it is entirely stable both as a pure substance and in admixture with fish meals which often considerably attack antibiotic additives.

The storage keeping properties of both a feed supplement containing a 10 concentration of Pleuromutilin as well as a mixed protein concentrate with a content of 150 γ and a ready fattening feed with a oontent of 21γ of Pleuromutilin per gramme were tested at 24 and 37°Ci. At intervals of 1 month samples were taken over a period of six months, the antibiotic being extracted and tested. A fall in the antibiotic content was not found to have occurred.

For achieving an optimum rate of gain and an improved utilization of the feed by farm animals an addition of the antibiotic Pleuromutilin in concentrations that are conventional in the case of antibiotics hitherto used is sufficient. In other words, ready mixed feeds according to the invention should contain the following quantities of Pleuromutilin per kg. of the mixture P.A. 23314 Pile 16881 .XII.68 and for calves and beef cattle the feed supplements according to the invention should be fed in quantities to provide the animal with between 25 and 70 mg. of Pleuroiautilin per day.

The tests hereunder described illustrate the us© of the antibiotic Pleuromutilin in feed supplements and ready mixed feeds.

EXAMPLE I a) A ready mixed feed for fattening pigs. 100 g. of Ple\u?omutilln were thoroughly mixed in a a ball mil.'' with 900 g. of a dry mycelium from the production of penicillin by fermentation. The feed supplement thus obtained was processed into a protein concentrate containing per kg. 150 mg. of Pleuromutilin, 500 g. of blood meal* 250 g« of fish meal* 91 g. of extracted peanut meal* 80 g, of extraoted soybean meal, 35 g. of phosphate of lime* 31 g» of carbonate of lime, 5 g« of iodized cattle salt and 7.5 g* of a conventional mixture of vitamins and trace elements. The ready mixed feed used during the initial fattening period consisted of 860 g. of wholemeal barley and 140 g.' of the said protein concentrate, whereas for the second fattening period it consisted of 530 g. barley, 420 g. of wholemeal of wholemeal /maize and 0 g. of protein concentrate per kg* of feed. b) Fattening test.

The following pig fattening tests were carried out with a ready mixed feed containing Pleuromutilin and prepared as described under a) :*·.

P.A. 23314 ! File 16881 . II; 68 48 piglets each weighing 15kg* were equally, fed until their weight had risen to an average of 29 kg. Thereafter and until their live weight had reached about 50 kg. they were fed with a feed containing 18$ of raw protein. 24 trial animals received the above mentioned ready mixed feed containing 21 mg. of Pleuromutilin per kg., whereas 24 oontrol animals received an analogous feed which instead of Pleuromutilin contained the same quantity of chlorotetracycline per kg. From the time the live weight reached 50 kg. to the time of slaughtering the animals received a feed containing 12 of raw protein, with an additio of 7.5 mg. of Pleuromutilin per kg. to the trial feed and an addition of 7.5 mg. of chlorotetracycline '.,;·,χ -per kg, to the control feed. The results of the test are compiled in the following Tablet- Control Group Trial Group Gain during fattening 1557 kg, 1586 kg. period total Gain during f ttening 64,875 kg, 66 ,983kg, period per animal Feed tilization during 4,194 3,979 the fattening period The ollowing example illustrates the production of the antibiotic pleuromutilin(BO 757) .

EXAMPLE 2 : 60 co. of sterile nutrient solution in a 500 cc, wide-necked Erlenmeyer flask are inoculated with 3 co. of a suspension obtained by rinsing a culture of Clitopilus paBseckerianu Nutrient medium. for agar slant: bean flour 10.0 g. m2Po4 0.5 g.

FeCl-,(l# solution) 1.0 oc. 3 yeast extract " o.l g. malt extract 50.0 g. peptone 1.0 g, agar 1 »0 g.

Make up to 1000 cc. with distilled water, natural pH» Nutrient solution for fermentation: . glucose 50.0 g/1 beer yeast autoiLysate N 1*0 g/1 KH2P04 1.0 g/1 Ca(N03)? 0,5 g/1 NaOl 0.1 g/1 PeS04 . 7 H20: 0*05 g/1 pH 6.0 before sterilization.

After fermenting for 120 hours at 24 C. on a rotating shaking machine stroke 40 mm., (250 rotations per . minute), the contents of the 500 cc. Erlenmeyer wide-necked flask became viscous. 10 cc. of this are used for the inoculation of 200 cc. of the above sterile fermentatio nutrient solution in a 2 1 Erlenmeyer wide-necked flask. Incubation is effected under the same conditions as during the first preliminary step.

File 16881 .XII.68 used to inoculate a 10 litre stainless steel submerged culture tank containing 3i8 litres of the above-mentioned sterile fermentation nutrient medium. The submerged culture tank was equipped for vortex fermentation with a plate stirrer adjusted to rotate at 800 rpm. Aeration was from above, the admission pipe terminating directly below the cover. The air entered at a rate of 0;2 litres/minute agains a back pressure of Qi5 atm. guage At the end of a fermentation time of 120 hours at 24°C the content of the antibiotic had reached 740 τ/oc.

The whole of the culture solution containing the antibiotic was evaporated at room temperature in a vacuum until dry and the resultin cake was ground fine.

Prom 10 litres of the crop 200 g. of dry powder containing g. of Pleuromutllin were thus obtained.- This produot was used for the antibiotic feed supplement.

Claims (7)

P.A.2¾|L4# Pile 16881 30. II.68 WHAT IS CLAIMED IS::

1. A process for produoing the antibiotic pleuro-? mutilin which comprises cultivating Olito ilus passeck-erianus (Pil ) Sing. WHRL 3100 or a pleuromutilin-pro-ducing mutant or variant thereof in an aqueous nutrient medium containing assimilable sources of carbon and nitrogen.

2. A process for the production of pleuromutilin substantially as herein described with reference to Example 2;

3. '. Pleuromutilin-, whevener obtained by a process as claimed in Claim or 2*.

4. '. An animal feed or feed supplement containing an animal growth rate increasing amount of the antibiotic pleuromutilin;.'

5. An animal feed or (feed supplement containing an animal growth rate increasing amount of the antibiotic pleuromutilin whenever produced by a process according to Claim 1 or 2*.

6. An animal feed or feed supplement according to Claim 4 or 5 In which from 2.5 to 50 mg. of pleuromutilin is present per kg. of feed or feed supplement.

7. An animal feed or feed supplement containing pleuromutilin substantially as herejjibefore described Attorneys for Applicants