Patented Nov. 3, 1953 2,658,024

UNITED STATES PATENT OFFICE 2,658,024 METHOD FOR PRODUCTION OF PRODIGOSN Roger L. Harned, Terre Haute, Ind., assignor to Commercial Solvents Corporation, Terre Haute, Ind., a corporation of Maryland No Drawing. Application April 26, 1951, Seria No. 223,58 10 Claims. (C. 195-96) 2 Myinvention relates to the antibiotic prodigiosin. m0rceScenS in an aerated, agitated nutrient me and more particularly it relates to a method for dium which is more particularly described here the production of prodigiosin by submerged inafter. Surface cultivation of the organism fermentation of a Suitable nutrient medium by Serratid marceScens on my new medium can be the organism Serratia, mdrcescens. conducted but quantity production of the anti Prodigiosin is a thermo-stable red pigment pro biotic is severely limited when such a procedure duced in nutrient media by the Organism Serratia is employed. marceScenS. Although prodigiosin has been The Organism Serratia marcescens is produc known for a number of years, the antibiotic prop tive of a number of bacterial variants as de erties of the material were not recognized 0. Scribed by D. B. Reed in the Journal of Bac until Hettche (Arch. Hyg., 107, 348) first report teriology 34, 255. In producing prodigiosin ac edits bacteriostatic activity against Staphylococci Cording to my process, I prefer to employ a non and Bacillus anthracis in vitro in 1932. Since mucoid, rough Strain of the organism because that time it has been the Subject of considerable this type Strain does not produce the large quan study and the structure of the material has been 5 tities of mucilaginous slime produced by the identified as a tripyrryl methene. As an anti raucoid type Strains and therefore filtration of biotic, prodigiosin is active against Gram-posi the medium following fermentation is greatly. tive organisms but is essentially inactive against facilitated, this operation being practically im-. Gram-negative organisms. It also has been pOSSible following use of a mucoid type strain of found to poSSeSS antifungal activity and to be the Organism. fungistatic against the organism Coccidioides The nutrient medium which I employ in the immitis which causes coccidioido-mycosis or San production of prodigioSin is composed of a car Joaquin Walley fever. bohydrate-like material, a protein, and a salt. The antibiotic prodigioSin is produced by and The carbohydrate-like materials which I can use is contained Within the cells of the Organism in niny medium are the hexose alcohols such as Serratid marceScens When the Organism is grOWn mannitol, dulcitol and Sorbitol. The protein in nutrient media. In the past, prodigiosin has which I employ is any of a number of protein been produced almost entirely by Surface culture aceous soy products such as soy flour, soy grits, on expensive experimental media containing Such Soybean expeller meal, solvent extracted soy ingredients as agar, meat extract, brewer's yeast, bean meal, etc. The salt which I employ in my commercial peptOne mixtures, etc. Such media, new medium is magnesium sulfate. have been used for the production of prodigiosin In Compounding my new medium, I add from for experimental purposes and they are produc about 1 to about 3% of the hexose alcohol, from tive of only small yields of the antibiotic. An about 0.5 to about 1.5% of the proteinaceous soy inexpensive medium capable of producing high 35 product and from about 0.1 to about 0.25% yields of the antibiotic has been highly desired magnesium Sulfate to a Water base made up to by the art. the desired volume. I then adjust the pH of I have now discovered a proceSS for the pro the medium to a value between about 4.5 and 8, duction of prodigiosin by Submerged fermenta preferably about 5.0, with a suitable acid Such as tion which utilizes an inexpensive nutrient me 40 for example hydrochloric acid. My preferred ne dium and is capable of producing greatly in dium consists of about 3% of the hexose alcohol, creased yields of the antibiotic over that pre about 1.5% of the soy product, and about 0.125% viously obtained by the prior art. By employing magnesium Sulfate, my new process I am able to obtain markedly Following preparation of my new medium, I increased quantities of the antibiotic at a great 45 inoculate it with a culture of the organism Ser ly reduced cost and in addition the use of my new ratia marceScens using a strain such as that process for the production of the antibiotic facili described above. The medium is then allowed to tates subsequent recovery of the antibiotic from ferment and during fermentation air is supplied the medium in which it is produced. ProdigioSin to the medium at a rate ranging from about 0.5 can be recovered from the media, in Which it is 50 to about 1.0 volume of air per volume of medium produced by the method described in Copending per minute. The medium is preferably agitated Serial No. 196,711 by Urs F. Nager entitled during the fermentation since agitated media “Method for the Recovery of Prodigiosin.' give higher yields of prodigiosin than the non My new process consists primarily of the Sub agitated media, although, of course, prodigiosin merged cultivation of the organism Serratia 55 can be produced with fair success in non-agitated. 2,658,024 3r - 4. media. In carrying out the fermentation I can of air per volume of medium per minute and the employ temperatures ranging from about 25 to fermentation carried out at 28°C. The anti-foam. about 35° C. although I prefer to use a tempera agent used was 0.25% of a 3% Solution of octa ture of about 28° C. It should be noted that the decanol in mineral oil. One of the fermenters agitated media tend to foam during the fermen 5 was agitated and one was not. The resultS are tation and therefore I have found it advanta shown in the following table. geous to add an anti-foam agent to the medium Table II to counteract this tendency. Any of the usual anti-foam agents such as for example octadeca Agitated Nonagitated 10 nol, corn oil, substituted oxazolines, etc., can be mg, prod- mg. prodil used, one qualification of the anti-foam agent Sample No. Age, hours Eastgiosin hy-giosin SESS; hy being inertness toward prodigioSin. drochloride drochloride The following examples are offered to illus per liter per liter trate my invention but it is to be understood that 1------7 0. 0 I do not intend to be limited to the Specific 5 2------3 77 ------3------19 284 ------amounts, proportions, Substances, and procedures 24 394 285 shown therein. 3. 410 ------37 636 310 EXAMPLE 43 680 344 A 1200 gallon portion of a medium consisting 48 720 474 of 3% sorbital, 1.5% Soy flour, and 0.125% mag nesium sulfate was placed in a 2,000 gallon Stain EXAMPLE less steel fermenter provided with agitator. The A series of experiments was conducted in 10 fermenter was inoculated with a rough, non liter jars wherein a 1200 ml, portion of a medium mucoid Strain of the organism Serratia nar consisting of 3% sorbitol, 1.5% Soy flour, and ceScens developed as follows: an agar Slant cul 2 5 0.125% magnesium Sulfate was placed in each ture of the organism was washed with 10 ml, of jar. The pH was not adjusted. The jars were sterile Saline Solution into one liter of sterile then inoculated with a non-mucoid rough strain inoculum medium consisting of 1% peptone, 1% of the organism Serratia marceScens and the me Sorbitol, and 0.125% magnesium Sulfate, the pH 30 dium fermented for 48 hours at a temperature of having been adjusted to 5.0 with hydrochloric 28° C. Aeration was at the rate of 1 volume of acid, in a 4 liter flask, and incubated for 16 hours air per volume of medium per minute. The re with reciprocal shaking at 28° C. The contents sults are shown in the following table. of the flask were then transferred to 30 gallons of the regular fermentation medium in a 100 Table III gallon non-agitated tank and grown for 16 hours 35 Micrograms prodigiosin at 28 C., air being Supplied at the rate of 2 hydrochloride per milliliter volume of air per volume of medium per minute. Jar The Contents of the tank Were then transferred 24 hrs. 30hrs. 48 hrs. to the medium in the fermenter whereupon fer mentation was carried out at a temperature of 400 494 600 28 C., air being supplied to the fermenter at a 366 446 530 410 466 620 rate of A volume of air per volume of medium 354 444 530 per minute. The anti-foam agent used was 0.25% 366 426 530 of a 3% solution of octadecanol in mineral oil. 376 426 535 The following table shows the results of the fer mentation through 35 hours at which point the EXAMPLE IV prodigiosin was recovered. The following series of experiments shows war iOuS media compositions and their effect on the Table I yield of prodigiosin. The experiments were car Milligrams ried out in 1 liter Erlenmeyer flasks containing prodigiosin 200 ml. of medium of the composition shown to Sample No. Age, hours hydro chloride each of which was added 0.125% magnesium sul per liter fate. The pH of all the flasks was adjusted to 5.0 With hydrochloric acid. Fermentation was 0. 55 conducted on a reciprocal shaker at a tempera 16 43 19 253 ture of 28° C. Each flask was inoculated with a 506 non-mucoid rough strain of the organism Ser 27 570 31 574 ratic marceScens developed for 16 hours at 28°C. 35 574 in a medium consisting of 1% sorbitol and 1% 60 peptOne. The following table shows the results EXAMPLE of the series of experiments. A 50 gallon portion of a medium consisting of Table IV 3% sorbitol, 1.5% soy flour, and 0.125% magne sium sulfate was placed in each of two 100 gallon 65 Micrograms prodigiosin stainless steel tanks. The tanks were then inocu hydrochloride per milliliter lated with an agar slant culture of a rough, non Flask Medium mucoid strain of Serratia marcescens, washed 24hrs 30 hrs. 48 hrs. with 10 ml. of sterile saline into 1 liter of sterile inioctlum medium consisting of 1% peptone, 1% 5------1% sorbitol, 4% soy flour.-- 88 110 160 Sorbitol, and 0.125% magnesium sulfate, the pH 1------a-do------1% sprbitol, 1% soy four---- 100107 17715 250160 ------a-do;------150 200 230 having been adjusted to 50 with hydrochloric 3------2% sorbitol, 1% soy flour---- 45 '97 286 acid, in a 4 liter fiask, and incubated for 16 hours 4------*:::::------s------172 95 276 With reciprocal shaking at 28°C. Aeration was 7-...-- 3% SOrbitol, 1%% soy flour. 260 23 330 Supplied to the medium at the rate of 1 volume 15 2,658,024 5 6 EXAMPLE W to about 3% of a hexose alcohol selected from the The following Series of experiments was con group consisting of Sorbitol, mannitol, and dulci ducted using 1% mannitol as the carbohydrate tol, from about 0.5 to about 1.5% of a proteins along With 1% soy grits and 0.125% magnesium aceous SOy product, and from about 0.1 to about Sulfate with the pH adjusted to 5.5 with hydro 0.25% magnesium sulfate and isolating the pro chloric acid. One liter Erlenmeyer fiasks each digiosin thus produced. Containing 200 ml. of the medium were employed, 6. A process for the production of prodigiosin the medium being inoculated with a non-mucoid Which comprises growing a prodigiosin producing rough Strain of the organism Serratia, marcescens. Strain of Serratia marcescens in an agitated, Fermentation was carried out on reciprocal shak O aerated nutrient medium containing a hexose ers at a temperature of 28° C. The following alcohol Selected from the group consisting of dul citol, Sorbitol and mannitol, a proteinaceous SOy table shows the results obtained. product, and magnesium sulfate, the pH of the Table V medium being adjusted to a value between about 5 4.5 and 8.0 and isolating the prodigiosin thus OO Micrograms prodigiosinhy duced. drochloride per milliliter Flask 7. A process for the production of prodigiosin which comprises growing a prodigiosin producing 16 hrs. 30 hrs, 54 hrs. strain of Serratia marcescens in an agitated nu

20 38 90 50 trient medium containing a hexose alcohol se 24 275 287 lected from the group consisting of dulcitol, sor 22 220 263 30 30 325 bitol and mannitol, a proteinaceous soy product, 30 285 30 and magnesium Sulfate said medium being aer. atted at a rate between about 0.5 and 1.0 volume The Scope of my invention is outlined in this 25 of air per volume of medium perminute and iso Specification and the attached claims and I intend lating the prodigiosin thus produced. for all equivalents and variations apparent to 8. A process for the production of prodigioSin One skilled in the art to be specifically included Which comprises growing a non-mucoid, rough, herein. prodigiosin producing strain of Serratia marl What I clain is: 30 Cescens in an aerated, agitated nutrient medium 1. A proceSS for the production of prodigiosin containing a hexose alcohol selected from the which comprises inoculating with an active cul group consisting of dulcitol, sorbitol and manni ture of Serratia, mdrcescens an aerated nutrient tol, a proteinaceous soy product, and magnesium Inedium containing a hexose alcohol Selected from Sulfate and isolating the prodigiosin thus pro the group consisting of SOrbitol, mannitol, and duced. dulcitol, a proteinaceous soy product, and mag 9. A process for the production of prodigiosin neSiun Sulfate, allowing the medium to ferment which comprises growing a non-nucoid, rough, at a temperature favorable for prodigiosin pro prodigiosin producing strain of Serratia mar duction and growth of the organism until the CeScens in an agitated nutrient medium contain desired prodigiosin concentration has been ob ing Sorbitol, Soy flour, and magnesium Sulfate, tained. and isolating the prodigiosin thus produced. 2. A process for the production of prodigiosin 10. A process for the production of prodigiosin Which comprises growing a prodigiosin producing Which comprises growing a non-mucoid, rough, Strain of Serrottia, n.drcescens in an aerated nu prodigiosin producing strain of the organism Ser trient medium containing a hexose alcohol Se ratia marcescens in an agitated nutrient medium lected from the group consisting of Sorbitol, man containing 3% sorbitol, 1.5% soy flour, and initol, and dulcitol, a proteinaceous Soy product, 0.125% magnesium sulfate, the pH of the medium and magnesium Sulfate and isolating the pro being adjusted to 5.0 with hydrochloric acid, and digiOSin thus produced. Said medium being aerated at a rate of 1 volume 3. A process for the production of prodigiosin of air per volume of medium per minute and iso Which comprises grOWing a prodigiosin produzing lating the prodigiosin thus produced. Strain of Serratid marceScens in an aerated, agi ROGER HARNED. tated nutrient medium containing a hexose aco hol selected from the group consisting of dulcitol, References Cited in the file of this patent SOrbitol and mannitol, a proteinaceous Soy prod UNITED STATES PATENTS uct, and magnesium sulfate and isolating the pro Number Name Date digiosin thus produced. 2,498,165 Johnson ------Feb. 21, 1950 4. A process for the production of prodigiosin 2,567,698 Darker ------Sept. 1, 1951 Which comprises growing a prodigiosin producing . 2,586,762 Finlay et al. ------Feb. 19, 1952 Strain of Serratia marceScens in an aerated, agi tated nutrient medium containing a hexose alco OTHER REFERENCES hol Selected from the group consisting of dulcitol, Florey: Antibiotics, vol. I, pages 558-562, RS SOrbitol and mannitol, Soy flour and magnesium 161-A55. Sulfate and isolating the prodigiosin thus pro Baron.: Handbook of Antibiotics, 1950, Rein duced. hold Pub. Corp., pages 8 to 11. RS-161-B3, Sci. 5. A process for the production of prodigiosin Lib. which comprises growing a prodigiosin producing Wier et al.: 'A Clinical Trial of Prodigiosin in Strain of Serratid marceScens in an aerated, agi Disseminated Coccidioidomycosis,' Am. Jour. of tated nutrient medium containing from about 1 the Medical Sciences, volume 224, pages 70-76.