BE544260A - - Google Patents

Description

       

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   The present invention relates to the pretreatment of fermentation media, nutrients, and aqueous, to improve the yield of tetracycline obtained from these media, and it constitutes an improvement or a modification of the invention according to the main patent.



   It is known that tetracycline is obtained by the fermentation of an aqueous nutrient medium by tetracycline-producing r-cro-organisms belonging to the group of Streptomyces, as explained in the main patent, It is also known that chlorotetracycline is obtained at the same time as tetracycline when there are chloride ions in the medium.



   One of the objects of the present invention is to provide a fermentation medium from which a substantially chlorotetracycline-free tetracycline can be obtained, which is highly desirable from a medical point of view.



   In accordance with the present invention, there is provided a process for the pretreatment of a fermentation medium, nutrient and aqueous, to improve the yield of tetracycline obtained from this medium when fermented by a producing microorganism. tetracycline tor from the Streptomyces group; The method consists of bringing this aqueous medium into contact with an ion exchanger to reduce the content of chloride ions. The treatment is preferably continued until the content of chloride ions in the aqueous medium is less than 50 parts per million, and preferably less than 10 parts per million.

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   The microorganisms used include strains, variants or mutants of S. aurefaciens, particularly those which give relatively high yields of tetracycline in the fermentation liquor, particularly those which give more than 500 micrograms per cm3. As an example, there may be mentioned an organism called UV-8, which is a mutant of the Texas microorganism. This mutant is a new strain which produces such yields in the fermentation medium, and it is a heretofore undescribed strain, strain UV-8, when grown on Waksman agar (Journal of Bacteriology; 339-341, 1922), shows a strong production of mycelium, initially 7: chadish, turning yellow, and gradually becoming covered with a powdery white aerial mycelium, which sporulates white afterwards.

   Old cultures are jet black with small patches of white mycelium. This black vegetation is made up of a mass of bodies with short chains which break easily. These bodies vary in size and shape, ranging from approximately 0.5 to 4.5 microns, and can vary in shape from an oval to a slightly angular spherical globe. The average size is somewhat larger than that found in S. aureofaciens (NRRL-2209), and there is a greater variety in size and shape.



   Many different strains of streptomyces have been observed which have the ability to produce both chlorotetracycline and tetracycline, and it is believed that they can all be reasonably classified as Streptomyces aureofaciens, despite the differences. quite marked in their growth habits and nutrient requirements, as reported by Duggar et al .; in "Annals of the New York Academy of Sciences",

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 volume 60, pages 71 to 101, published October 29, 54; yet some might, for one reason or another, claim that their tetracycline-producing microorganisms belong to a different species.

   In view of this possible conflict of terminology, and given the general nature of the present invention, Applicants wish to include here the use of all harmful microorganisms which exhibit the tetracycline production qualities indicated above.



   The preparation of fermentation media containing less than 10 parts per million chloride ions is relatively easy, when the medium is a synthetic medium "* that is, a medium in which the carbon requirements for fermentation are satisfied by relatively pure materials, such as saccharose, and in which the nitrogen is supplied by ammonium sulphate or other highly purified chemicals. Unfortunately, these synthetic media are very expensive, and lack some essential principles of the metabolism of the fermentation process to achieve high yields of tetracycline.



  For this reason and others imperfectly known, it is preferable to use, in the preparation of fermentation media, materials of natural origin, for example, corn maceration liquor, fermentation liquor of animal origin. , digested casein, etc. Not only are these materials less expensive than synthetics, but they give much higher yields of the desired antibiotic.



  Unfortunately, these materials contain appreciable amounts of chloride ions, and when tetracycline is prepared by fermentation, excessive amounts of chlorotetracycline are over time obtained.

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   Natural materials, such as corn maceration liquor, can be diluted with water to a suitable concentration to pass them through a layer of ion exchangers, and the acid can be removed. chloride ion to the extent deemed necessary. The additional amounts of nitrogen, carbon and mineral elements required for the fermentation medium can then be added to the deionized maize solution, and the fermentation medium is then ready for use in the fermentation medium. the process. Corn maceration liquor contains appreciable amounts of chloride ions, in the range of 0.15 to 0.5% by weight, and more care should be taken in the processing of a material. of this kind, than in the case of other natural substances used in the preparation of the fermentation medium.



   Casein, which is a very useful material for preparing fermentation liquors, is liable to contain large amounts of chloride ions, depending on the methods of preparation. Frequently, amounts of chloride ions of up to 0.30% are encountered. The enzymatic digestion products of casein, also useful for the preparation of fermentation media, may contain even higher concentrations of chloride ions, and these products should be treated in accordance with the information of the present invention, if given. used in the preparation of fermentation liquors for obtaining tetracycline.



   Fermentation liquor of animal origin, meat powder, and digested fish powders, as well as various other materials, particularly of animal origin, contain large quantities

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 chloride ions, and solutions prepared from these materials should be treated in accordance with the information of the present invention to reduce their chloride ion content.



   It will be understood, of course, that the constituents of the fermentation medium which are in insoluble form cannot pass through layers of ion exchangers without rapidly obstructing them by insoluble deposits. The process of the present invention contemplates the treatment of soluble constituents of the media mentioned herein. Highly viscous substances also have difficulty passing through ion exchange layers, and therefore must be diluted with water to a fluidity which allows sufficient flow through the layer.



   In some cases where insoluble materials need to be added to the fermentation medium, they can be washed with deionized water to remove chloride ions before adding such materials to the fermentation medium. Obviously, the nature of the material will determine the suitable treatment. For raw starch, for example, which often contains considerable amounts of chloride ions, it can be washed in deionized water, added to the fermentation medium, and then cooked or otherwise treated to make it assimilated by the fermentation microorganism. By proper silution, the soluble starch can be passed through the ion exchanger layer to remove chloride ions.



   Soybean meal often contains excessive amounts of chloride ions for the preparation of tetracycline, and when such materials are used in large amounts, they must also be treated.

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 by the ion exchange technique as described above. The same observations apply to other materials of natural origin which are used to prepare fermentation liquors, when their content of chloride ions is too high.



   Tetracycline producing strains of Streptomyces aureofaciens which produce more than 500 micrograms of antibiotic per cm3 are preferable. In such a case, the fermentation medium may contain 10 parts per million chloride ions, although smaller amounts are preferable. On the other hand, the improvements made to the selection of strains of Streptomyces producing tetracycline have led to notable increases in the yield of tetracycline which can be obtained, so that it is now possible to easily obtain 5000 micrograms of tetras. cyclin per cm3, and even more.

   This is achieved not only by selecting particularly strong strains which produce tetracycline, but also by carefully selecting the materials which constitute the fermentation medium, and by restricting the content of chloride ions. The present invention therefore contemplates the use of strains of S. aureofaciens capable of producing amounts of tetracycline greater than 500 micrograms per cm3, in fermentation media containing natural materials of the type commonly used now in other countries. fermentation process, but which have been treated to remove as many chloride ions as possible.

   In this way, it is possible to obtain crude fermentation media in which only a small proportion of the total antibiotic is present as chlorotetracyclin.

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   As stated above, it is permissible, although undesirable, for the total antibiotic present in the fermentation medium to be in the form of chlorotetracycline up to 8-10%. So, as can be seen, a fermentation medium containing 5000 micrograms of tetracycline per cm3, could contain up to 500 micrograms per cm3 of chlorotetracycline, which could occur in the presence of 40 to 50 parts per million ions. chlorides in fermentation media, when the microorganism is an efficient user. If the particular strain used in the process does not fully utilize the chloride ions, the fermentation liquor may contain greater amounts of chloride ions.

   In such a case, many natural materials do not need to be previously treated in accordance with the process of the invention, since their content of chloride ions is already sufficiently low. However, the most effective substrates for high antibiotic yields include corn maceration liquor, which, as noted above, has a very high chloride content, and fermentation liquors containing appreciable amounts of this. material can improve if we treat them.



   As is common in other fermentation processes for the production of antibiotics, fermentation liquors contain available sources of carbon, nitrogen and mineral salts. Many of these sources are relatively free from chloride ions, and are used to supplement the above materials which are treated by ion exchangers in accordance with the present invention. They include in particular sucrose, glucose, dex-

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 trine, alcohols / sugars, citric acid, starch, flour of cottonseed, corn, soybean, peanut and other carbonaceous and nitrogenous substances which are used in varying amounts, at 0.5 to 5% by weight or more based on the total point of the fermentation medium.

   To increase efficiency and economy, mixtures of these materials are often used in conjunction with the naturally occurring products mentioned above, which have been treated with ion exchangers to remove excess amounts of gas. chloride ions.



   Inorganic salts are also added, commercially, to the fermentation medium, in varying amounts, to promote the growth of the microorganism and to achieve high levels of tetracycline. Among the salts, there may be mentioned ammonium phosphate, potassium phosphate, magnesium sulfate, calcium carbonate, and various trace elements including cobalt, copper, zinc, manganese, iron, chromium, etc. The use of these trace elements to promote fermentation is well known to those skilled in the art, and any additional description appears unnecessary.



   As will be seen, when treating aqueous solutions containing nutrients for the fermentation process to remove chloride ions, the non-selective uptake of anions which some anion exchangers have tends sometimes to remove some of the desirable anions contained in the fermentation liquor. For example, under certain conditions phosphate ions can be removed from solution. Likewise, the sulfate anion can also be removed.

   To avoid this possibility, and to reduce the. load imposed on the ion exchanger column,

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 it is preferable that the major part of the mineral elements, for example the phosphates and sulphates, as well as the trace elements, are added after the removal of the chlorides by the ion exchange treatment.



   The main purpose of the ion exchange treatment is, of course, to remove excess chloride ions from naturally occurring materials which contain them in excessive amounts. Under certain conditions, it may also be desirable to remove certain cationic groups prior to fermentation. Accordingly, mixed layers can be used which remove both cations and anions. The use and control of these mixed ion exchange layers is within the skill of the technician, and there is no need to expand further:
The ion exchangers contemplated in accordance with the present invention include, among the water-insoluble substances, those which have the property of adsorbing, or otherwise removing, chloride ions from aqueous solutions.

   They are generally synthetic resins containing free amino groups, for example phenol-formaldehyde-polyamine condensates,
 EMI9.1
 melamine-guanidine-formaldehyde resins, and polyalkylene-polyamine resins. The exact chemical nature of these substances and how they adsorb chloride ions are not fully known. However, they can be purchased from different manufacturers, specifying that they are intended to remove chloride ions from aqueous solutions. Various types are available on the market.



   Substances which simply adsorb chloride ions can be used in the present invention, but this would not be industrially applicable, because they could not be reused. The mofechange "

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 is used, in connection with the ion exchangers of the present invention, to denote a material which can be reactivated, when it has adsorbed chloride ions from a solution, in order to remove the chloride ions thus ad - sorbed and reuse the resin to prepare new fermentation media.



   Some layers of ion exchangers may function to remove only anions from the solution. In others, there may be replacement. For example, in an ion exchange resin operating on the sulfate cycle, its chloride ions are removed and sulfate ions are released to replace them in solution. Although not usually necessary, these sulfate ions can be adsorbed by a second layer of ion exchangers operating simultaneously with the first. The same may be true of other ions which may be involved. For these reasons, it is important, after the ion exchange treatment, to adjust the anionic requirements of the nutrient media, especially phosphate.



   After having treated with ion exchange resins some of the constituents of the fermentation media, one can then add to the treated liquid the other necessary materials which are used in the fermentation process, sterilize the whole, and teach. start with tetracycline-producing microorganisms. The fermentation is then carried out in the usual manner.



   During fermentation, it is desirable to cultivate the organism under immersion conditions, with suitable aeration and agitation, for example in a flask placed on a shaking machine, or

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   in one. stirred fermentation apparatus equipped with a nozzle to continuously produce a stream of air.



   The temperature does not appear to be critical, within the range of
25 to 35 C, although the range of 30-33 C is preferred. The initial pH of the medium should be close to neutral, although a little antibiotic is obtained with initial pHs as low as 5.0 or as high as 8.5.



   Buffers such as calcium carbonate and salts of organic acids such as citrates, acetates and lactates are useful in maintaining the pH in the proper range. In addition, organic acids can serve as sources of carbon in the metabolism of microorganisms. The use of an anti-foaming agent is desirable in large-scale Lion fermenters, but in this fermentation the foam is not a particularly difficult problem, and is easily prevented by using ordinary anti-foaming agents. , such as octaaecanol in lard oil, or another suitable commercial defoamer.



   The seed intended for fermentation can be prepared with a culture obtained on jellies seeded with S. aureofaciens. A suitable medium for jellies is Wacksman's agar, having the following composition:
Glucose 10 g / 1
Peptonse 5
PO4H2K 1 S04Mg.7H20 0.5 agar-agar 20
The jelly culture can be transferred to shaker flasks which can be used as small scale laboratory fermentation apparatus.

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 the, or-to produce a seed intended to inoculate more extensive fermentations.



   For large scale tank fermentation, jelly culture is used to inoculate a suitable liquid medium, into a shaker flask which is shaken on an alternating shaker machine at temperatures ranging from 26 to 35 C. Usually uses a second stage of shaker flasks, in conjunction with submerged tank fermentations, to increase the volume of stirred seed to equal 1.5-2.5% of the volume of liquid in the tank. The reaction of the stirrer flask medium is initially between pH 6.5 and 7.5, and as growth occurs, a continuous drop in pH is observed, down to 3.8. However, a physiological age such as that indicated by a pH of about 5.0 is apparently the most favorable time to transfer the seed.



   This, occupying 1.5 - 2.5% of the liquid volume of the tank, is transferred aseptically to the fermentation apparatus, and it is cultivated for 2 to
3 days with continuous agitation and aeration. Proportions of 0.5 - 1.5 volumes of free air per volume of liquid can be used, depending on the size of the apparatus.



   Foaming of the wort can be controlled by the addition, under aseptic conditions, of an antifoaming agent such as lard oil containing 2% octadecanol.



   To recover the tetracycline in the fermentation wort, it is possible to use ordinary methods developed in connection with other antibiotics, for example extractions with known solvents, with or without carriers, or adsorptions. Among the known solvents, such as butanol, ethyl acetate and

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 chloroform, the use of which involves subsequent purification steps, butanol is best for the extraction of tetracycline. These known methods are applicable with sufficient success in the case of fermentation musts in which little antibiotic substance other than tetracycline is formed, the aim then being simply to achieve the separation between the antibiotic and the ordinary inactive substances. - not classified as impurities.

   In the case where there is appreciable formation of an antibiotic substance other than tetracycline, for example when the quantity of chloride ions is not kept to a minimum and there is appreciable formation of chlorotetracycline, these known methods are less effective if it is desired to recover the highly concentrated tetracycline in the final product, eliminating other antibiotic substances, (Antibiotics are here considered as antagonist substances of E. coli).



   Preferably, the recovery is carried out using a particular group of quaternary ammonium salts which selectively precipitate tetracycline from the wort, at alkaline pH (pH 8 to 11), as the quaternary ammonium salt of. tetracycline. After having filtered this salt, it is then stirred with a small quantity of water and a relatively large quantity of chloroform, which has the effect of dissolving the quaternary ammonium salt of the tetracycline in the mixture. chloroform phase of the slurry. The aqueous and chloroform phases are then preferably separated, in order to remove all the impurities' dissolved in the water.

   The tetracycline is then extracted with an aqueous acid solution at a pH of about 1 to 2.5, with formation of the acid salt of tetracycline; which goes into solution in the phase

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   aqueous, where it precipitates as a crystalline product when the pH is brought to the range 3 to 7, precipitation starting at the pH lower of these two digits.



   Particularly useful quaternary ammonium salts in this recovery process are alkyl dimethylammonium chlorides and dialkyldimethylammonium chlorides in which the alkyl group contains from 8 to 18 carbon atoms, inclusive.



   EXAMPLE I
A medium based on corn maceration liquor is prepared as follows; A corn maceration liquor having a solids content of about 50% (by weight) is taken, diluted with distilled water to give a final concentration of 2%.



   The resulting diluted material is passed through a column packed with 30 liters of a mixture consisting of 2 volumes of Amberlite IR-45 as exchange / anionic resin, and 1 volume of Amberlite IRC-50 as exchange / cationic resin. The flow liquid is only started to be collected when the resistivity has risen to 5000 ohms per centimeter, and it is continued until the resistivity is lowered below 3000 ohms per centimeter.

   To 1000 cc of the deionized corn maceration liquor solution, the following components are added to prepare a medium suitable for the preparation of semen and for the production of tetracycline, to obtain a medium containing about
17 parts per million chloride, although lower chloride content is preferable:

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 sucrose 30.0 g P04HK2 15.0
PO4H (NH4) 2 5.0
SO4Mg.7H2O 2.0
CO3Ca 1.0
KBr 0.5 S04Zn.7H20 0.05 S04Cu.5H20 0.003
SO4Mn.4H2O 0.0025
For the preparation of a seed, volumes of 50 cm3 of the above medium are dispensed in 250 cm3 Erlenmeyer flasks, and 400 cm3 volumes in 2 liter flasks.

   A dry spoonful of spores from the surface vegetation of the tetracycline organism, grown on a tilted semi-solid medium, is used to inoculate the 50 cc volumes which are shaken at 30 C for 72 hours on a reciprocating shaking machine - (82 cycles per minute with a displacement of 73 mm). At the end of this time, 1.0 cc of the resulting mycelium suspension is used to inoculate 400 cc of medium which is stirred for 28 hours. Four 400 cc volumes were combined under aseptic conditions and used to inoculate the fermentation apparatus.



   The fermentation medium is prepared in 10 liter apparatus as follows: the constituents are dissolved, at the concentrations indicated above, in 43 liters of the deionized corn maceration liquor solution, to obtain a final total volume of 57 liters. The medium was sterilized at 121 ° C. by steaming at 1.05 kg / cm 2 directly into the batch for 30 minutes. The volume supplied by the steam condensate gives the difference

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 desired between the initial 43 liters of solution comprising maceration liquor of deionized corn, sugar and salts, and the final desired 57 liters.

   After having cooled to 30 ° C., the mixture is inoculated with the four volumes of 400 cm3 of combined seed, mentioned above, it is stirred and aerated at a rate of 1.6 volumes of air per volume of medium, for 38 hours. At this time, by reaction of the wort, the pH, which was originally at 5.9, dropped to 4.1. The wort titrates 20 micrograms of antibiotic per cm 3 if oxytetracycline is used as a standard, and contains an appreciable amount of tetracycline.



   Corn maceration liquor solutions are deionized at concentrations of 1.5 and 2.0% with resin mixtures containing 3 volumes of anionic resin Amberlite IR-45 and 1 volume of cationic Amberlite IRC resin. -50 (about 10 operations). An operation is carried out with a mixed Rohm & t Haas MB-1 resin (2 volumes of anionic resin IRA-400 plus 1 volume of cationic resin IR-120). The 1: 3 ratio of resin volume to flow liquid volume is found to be satisfactory. ; A flow rate of 0.01 cm3 of solution per cm3 of resin per minute gives better resionization than 0.016 cm3 of solution / cm3 of resin / minute. The use of two resin columns in series achieves good deionization.

   When the chloride ions in the deionized solution are titrated in this manner, 3.5 to 29 parts per million are found.



   EXAMPLE II
A corn maceration liquor having a solid content of about 50% (by weight) is taken, and diluted with distilled water to give a solid content.

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 final concentration of 1.5%. The diluted material is passed through a column packed with a mixture comprising 3 volumes of Amberlite IR-45, anionic exchange resin, and 1 volume of Amberlite IRC-50, cationic exchange resin. Collecting the flowing liquid only begins when the resistivity has risen to 5000 ohms per centimeter, and continues until the resistivity has dropped below 3000 ohms per centimeter.

   The following materials are added to the deionized corn steep liquor solution to prepare a medium suitable for seed propagation and tetracyclin production: sucrose 2.0% PO4H2K 1.5%
PO4H (NH4) 2 0.5 S04Mg.7H20 0.2 CO3Ca 0.1 S04Zn.7H20 50.0 parts per million S04Cu.5H20 '3.0 parts per million S04Mn.4H20 2.5 parts per million
KBr 0.05%
For use in the preparation of seed as well as fermentation flasks, this medium is dispensed in 100 cm3 portions into 500 cm3 Erlenmeyer flasks, and the autoclave is sterilized for 20 minutes at 121 C under. 1.05 kg / cm2.

   The chloride content of the medium after sterilization is 17.7 parts per million.



   A seed is prepared by inoculating vials with a lyophilized mixture of spores and mycelium of Streptomyces aureofaciens (strain 201-15)

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 and incubating the vials at 26 ° C for 48 hours on a reciprocating dryer. 5% seed volumes of 40 hours were used to seed the fermentation flasks, which were then incubated under the same conditions for 48 hours.



    'Antibiotic activity is demonstrated by saturating a 13 mm paper disc with filtered wort, placing the disc on an agar-agar tray seeded with Escherichia coli, and incubating at 30 ° C. - before 6 p.m.



   400 cm3 of this mixture are treated with 2.4 g of oxalic acid and 1.6 g of ammonium oxalate. After stirring for 10 minutes, the mixture is filtered. The filtrate is then stirred with 80 cm3 of ethyl acetate and the pH is adjusted to 8.5. The solvent phase which contains the major part of the active product is stirred with 5 cm3 of water and adjusted to pH 2. When diluted to 1/500 with the lower phase of the butanol-water system containing
2.5% acetic acid, absorption maxima in the ultraviolet at 270 ... and 360 millimicrons are obtained.



   Analysis by ', countercurrent distribution (48 transfers, 2.5% butanol-acetic acid) of this concentrate, shows that it contains tetracycline.



   This is achieved at the end of dispensing by diluting each of the lower phases with methanol and measuring the optical density at 370 millimicrons. Most of the product is found in tube 19. The peak tube numbers for chlorotetracycline and oxytetracycline are 26 and 16, respectively.



   EXAMPLE III A stirred flask fermentation is carried out, identical in all respects to that described above, except that the anionic and cationic resins used

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 are respectively Amberlite IRA 400 (2 volumes) and Amberlite IR 120 (1 volume). The medium contains
13.3 parts per million of chloride after sterilization.



   The antibiotic activity of the mixture was demonstrated by the paper disc and E. coli tray method.



   When this mixture is processed to concentrate the antibiotic activity as described in Example II above, it is seen that the UV absorption and countercurrent distribution data are substantially the same as for medium prepared with Amberlite IRC 50 and IR 45 resins.



   EXAMPLE IV
To prepare a seed for use in 10 liter fermenters, 50 cc volumes of the medium described in Example II are dispensed into 250 cc Erlenmeyer flasks. and volumes of 400 cm3 in 2 liter vials. A dry spoonful of spores from the superficial vegetation of S. aureofaciens (strain T-5), cultivated on inclined semi-solid medium, is used to seed the volumes of 50 cm3, which are stirred at 30 C for 72 hours on a reciprocating shaker machine.



  At the end of this time, 1.0 cc of the resulting mycelial suspension was used to inoculate 400 cc of medium, which was stirred for 28 hours. Four 400 cc volumes were combined aseptically and used to inoculate the fermentation apparatus.



   To prepare the fermentation medium, the corn maceration liquor solution is made up to a concentration of 2% and deionized with the 3: 1 mixture of Amberlite IRC 50 and IR 45 resins.

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  The other constituents of the medium described in Example II are added to this deionized solution, in quantities such that, after 30 minutes of sterilization at 121 ° C. by direct injection of steam at 1.05 kg / cm2 into the furnace, the volume supplied by the steam condensate gives a total volume of 57 liters of medium per 10 liter fermenter.



   After cooling to 30 ° C., the fermenters are inoculated with seed volumes of 1600 cm3, stirred mechanically at 155 - 165 revolutions per minute, and aerated at 1.6 volumes of air per volume of medium for 38 hours. . The musts from two of these fermentations contain 81 and 116 micrograms of antibiotic per cm 3, respectively, when titrated with S. lutea using oxytetracycline as a standard.



   96 liters of the mixture obtained by combining the batches described above are taken, treated with 920 g of oxalic acid and the pH adjusted to 3.5. Hyflo is added, the mixture is filtered and the cake is washed with 4 liters of water. The filtrate and the washing liquid are combined, extracted at pH 8.5 with 20 liters of ethyl acetate containing 1 liter of Emulsept
 EMI20.1
 (N - / "'lauroylcolaminoformalmethyl¯7-pyridine). The solvent extract is then stirred at pH 2, with four successive portions of 140 cm 3 of water. These aqueous concentrates are combined and the mixture is stirred. The solids are adjusted to pH 7.8 The solids which separate are filtered off, washed with water and then dried in a desiccator under vacuum to give 4.19 g, assaying 904 micrograms per mg. an activity yield of 41%.

   The ultraviolet absorption of this material in 0.1 M sulfuric acid is determined. Maximum values are found at 266 and

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 360 millimicrons, minima at 232 and 301 millimicrons.



  Five milligrams are distributed between chloroform and pH 7 phosphate buffer. Most of the activity is found in the lower phase. Under these conditions, the oxytetracycline becomes concentrated in the upper phase.



   50 g of the product are dissolved in a minimum of 1N hydrochloric acid, and the solution is seeded with a few crystals of chlorotetracycline, and the mixture is left to stand at room temperature. Crystallization begins after 15 minutes. The mixture is placed in a cooler overnight. The next day, the crystalline solids are separated and dried on a porous porcelain tray. The ultraviolet absorption spectrum of these crystals in solution in 0.1 N sulfuric acid shows maxima at 267 and 360 millimicrons, and minima at 234 and 301 millimicrons. A solution of crystals in concentrated sulfuric acid containing boric acid gives a lilac-pink color.

   Oxytetracycline and chlorotetracycline under analogous conditions give cherry-red and violet colorations, respectively,
EXAMPLE V
We take 17.8 kg of Amberlite IR-4B, a weakly anionic exchange resin of the phenol-formal-dehyde-polyamine type sold by Rohm and Haas, it is neutralized with 1 N sulfuric acid, it is packed in a 15 cm column. to a height of 1.90 m, and washed with water until the pH of the flowing liquid is 2.0. 15.0 kg of corn maceration liquor is diluted with 45 liters of water, and the mixture is filtered at 50 ° C. The cake is washed with 15 liters of water. We pass

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 the corn maceration solution through the column at a rate of 40 liters per hour.

   A positive chloride test is obtained in the elution liquid after having treated the equivalent of 13.5 kg of liquor. The treated, chloride-free liquor is adjusted to pH 4.0 with concentrated ammonium hydroxide and is then ready for use.



   A fermentation medium is prepared with the following constituents: Corn maceration liquor treated with resin: 20 g / 1 (solids) corn starch, "fluidity" degraded by sulfuric acid 55 g / 1 calcium carbonate 8.0 g / 1 cottonseed flour 3.5 g / 1 ammonium sulfate, special low in chlorides 6.0 phosphoric acid (85%) 0.24 lard oil 1.0% (by volume) oligo- elements SO4Fe.2H2O 41 mg / l S04Zn.7H20 100 mg / 1
SO4Mn.4H2O 50 mg / 1 tap water, q. s. for 1000 cm3
The medium is sterilized for 15 minutes at
1250 C and then diluted with 3 volumes of tap water. This medium contains approximately 24 parts per million chloride ions.

   It is then inoculated with
2% of its volume of strain S. aureofaciens S-77 containing (calculated by weight in solid form) 1% corn maceration liquor, 3% dextrin, 0.625% calcium carbonate and 0.2% ammonium sulfate. The fermentation is carried out at 26-27 C for 112 hours with mechanical stirring and aeration, at the rate of. approximately 0.5 volume of air per volume of medium and per half

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 nute.



   At harvest, the fermented wort is found to contain more than 5000 micro grams of tetracycline per cm3, with an amount of chlorotetracycline less than 5% of this amount.



   To regenerate the column, 1N sulfuric acid is passed through it, at a rate of
100 l / h, until the flow liquid no longer contains chloride ions (200 l of acid is required).



   Water is passed at a rate of 100 l / h until the pH rises above 2.0. It takes 450 liters of water. The column is then ready to serve again. This column can be reused fifteen times without reducing its capacity for removing chlorides, or reducing the nutritive character of the liqueras maceration of corn obtained.



   EXAMPLE VI
A column similar to that of Example V was prepared using IRA-400, a strongly anionic ion-exchange resin of the phenol-formaldehyde-polyamine type, manufactured by Rohm and Haas. Maize maceration liquor is passed through the column as in Example V, and a fermentation medium is prepared, sterilized and inoculated with the same strain of tetracycline-producing S. aureofaciens. as above, and the fermentation is carried out in a similar fashion. This particular fermentation medium contains about 18 parts per million chloride ions.



   When we harvest the must, we find. that it contains 3510 g / cm3 of tetracycline, and 255 micrograms of chlorotetracycline per cm3.



     EXAMPLE VII
A mixed layer of exchanged resin is prepared.

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 cationic-anionic, with equal parts of IRA-
400 and IR-120, the latter being a strongly cationic sulphonic acid type resin sold by Rohm and Haas, and treated with 1N ammonium sulphate in a manner similar to that indicated for the preparation of the column in Example V.



   The corn maceration liquor is treated as indicated above to remove chloride ions through this column, and a fermentation medium is prepared in the same manner as in Example V. The diluted medium is then inoculated and sterilized, with S. aureofaciens strain S-77, and the fermentation is carried out under the conditions described in Example V. The fermentation medium, at the start of the fermentation, contains approximately
28 parts per million chloride ions. The fermented wort was found to contain 4670 micrograms of tetracycline per cm3 and 390 micrograms of chlorotetracycline per cm3.



   EXAMPLE VIII
Corn maceration liquor is diluted with water, and treated with silver nitrate to remove chloride ions in the form of silver chloride. The chloride-free maceration liquor was incorporated into a fermentation medium similar to that of Example VII, and inoculated with S. aureofaciens, and the fermentation carried out as before. On analysis, the fermented wort was found to contain 3600 micrograms of tetracycline per cm3 and 270 micrograms of chlorotetracycline per cm3.



   EXAMPLE IX
In experiments a shaker flasks, using the fermentation media of Example V in which the medium is enriched with 200 mg / l of acid

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 phosphoric acid, 2830 micrograms per cm3 of tetracycline and 210 micrograms per cm3 of chlorotetracycline are obtained.



   When the fermentation is carried out as in the previous paragraph, using untreated corn maceration liquor at a rate of 1% of total solids, the fermented wort is found to contain about 1600 micrograms of tetracycline per cm3 and 1600 micrograms of chlorotetracycline per cm3, showing that the use of the chloride ion available in corn maceration liquor produces higher proportions of chlorotetracycline.



   EXAMPLE X
The anion exchange resin can function in various forms, such as sulphate, nitrate, hydroxyl etc. When the process described in Example V is repeated with the ion exchange resin on the nitrate cycle, prepared by treatment with 1 N nitric acid instead of sulfuric acid, two different operations give 3590 micrograms of tetracycline per cm3 and 210 micrograms of chlorotetracycline per cm3 in a first operation, and 4460 micrograms of tetracycline per cm3 and 230 micrograms of chloro - tetracycline per cm3 in a second operation.



   In the above operations, the fermentation liquor contains about 15 to 30 parts per million chloride ions and it is fully utilized by the microorganism S. aureofaciens to produce all the chlorotetracycline which is possible given the content of the medium in chlorides. But given the high yields of antibiotic obtained, it is observed that the proportion of chlorotetracycline in these operations, in which exchange resins are used

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 of ions to treat the corn maceration liquor is less than 8% in all cases.


    

Claims (1)

CLAIM ':) 1. Process for the preliminary treatment of an aqueous nutrient fermentation medium, with the aim of improving the yield of tetracycline obtained with a tetracycline-producing microorganism belonging to the group of Streptomyces, in which process the aqueous medium is brought into contact with a crions exchange material to reduce its content of chloride ions. 2. A process according to claim 1, wherein the treatment is continued until the chloride ion content is less than 50 parts per million, and preferably less than 10 parts per million. 3. A process according to either of claims 1 and 2, wherein phosphate ions are added to the medium after treatment / with ion-exchanging material;:;. 4. A process according to either of claims 1, 2 and 3, wherein a natural medium is used such as corn maceration liquor, digested caseins, and fermentation liquors. animal origin. 5. The method of claim 4, wherein the medium is diluted with water to a concentration of less than about 5% by weight relative to the dry weight. 6. A method according to either of claims 1 to 5, wherein the ion exchange material is an anion exchanger, or a mixed layer of anion and cation exchangers. 7. Process according to one or the other of the claims <Desc / Clms Page number 27> dations. 1 to 6, in which the microorganism is S. aureofaciens. 8. A method of pretreatment of an aqueous nutrient fermentation medium, in order to improve the yield of tetracycline, in substance, as described above. 9. Pretreated aqueous nutrient fermentation medium obtained by the process according to any one of claims 1 to 8.