BE600777A - - Google Patents

Description

       

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 EMI1.1
 



  Process for the preparation of antimicrobial agents8. -----------------------------------------------
The present invention relates to a process for the preparation of novel compound * endowed with antimicrobial properties. More particularly, the invention relates to a process aiming to prepare by fermentation a novel composition of matter called here * antimicrobial agent M-188 ", to isolate it and to concentrate it starting from crude solutions such as broths. fermentation, and to purify it.The agent may be in the form of acid addition salts.



   One of the objects of the present invention is to prepare a new and useful antimicrobial agent which is active against various bacteria and various protozoa such as Elmeria tenelle. Another object is to prepare acid addition salts of this antimicrobial agent. Others

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 Aims and characteristics of the invention will become apparent to those skilled in the art on reading the following description.



   It has been found that by culturing under controlled conditions and on suitable culture media a previously unknown strain of the microorganism Streptomyces caeleatia, a new composition of matter is obtained herein referred to as antimicrobial agent M-188. The microorganism strain was isolated from a soil sample collected in Capitol City, Texas. In its morphological characteristics, the organism appears to be most closely related to Streptomyces caelestis, a known species. As discussed below, there are certain differences between the cultivation properties of the known organism and those of the new strain.

   This was therefore considered to be a strain of Streptomyces caelestis. A culture of the living organism has been deposited with the Northern Use Research and Development Division *, of the Agricultural Research Service. at the United States Department of Agriculture, Peoria, Illinois, and was listed under NRRL 2821.



   A careful study of the morphology and physiology of the new strain of Streptomyces caelestis shows that it can be distinguished from Streptomyces caelestis as fully described in British Patent No. 768,971. It has been found that xylose, arabinose, rhamnose, dextrose, galactose, mannose, fructose, sucrose, lactose, maltose, cellobiosa, dextrin, raffinose, glycerol and Sodium succinate are readily used by both organizations. Sorbose, dulcitol, sodium and potassium tartrate and soluble starch are not used by

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 neither of the two organizations.

   On the other hand, sorbitol, sodium citrate and salicin are used by the strain M-188 but not by the known Streptomyces caelestis.



  The use of the above various carbon compounds by the new strain was determined by the method of Pridham and Gottlieb, "Journal of Bacteriology" 56, 107-114 (1948). By direct comparison with a light microscope, the sporulating mycelium and the spores of the two organisms cannot be distinguished.



   Further direct comparisons of cultures on Pridham medium containing various carbon sources show that the aerial sporulating mycelium of Streptomyces caelestis strain M-188 is greenish blue in color, while that of known Streptomyces caelestis is sky blue in color.



  The following similarities and differences are noted between strain M-188 and the known Streptomyces caelestis:

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 EMI4.1
 
<tb> Medium <SEP> of <SEP> base <SEP> Compared <SEP> characters <SEP> Strain <SEP> M-188 <SEP> S. <SEP> caelestis
<tb>
 
 EMI4.2
 --------------- ------------------- ---------------
 EMI4.3
 
<tb> Gelatin <SEP> Growth <SEP> fairly <SEP> good <SEP> good
<tb> Aerial <SEP> white <SEP> pearly <SEP> gray-blue
<tb> Pigment <SEP> black <SEP> brownish <SEP> brown
<tb> dark
<tb> Liquefaction <SEP> negative <SEP> up to <SEP> the <SEP> depth <SEP> of the
<tb> pigment
<tb>
<tb> Milk <SEP> Growth <SEP> fairly <SEP> good <SEP> good
<tb> Coagulation <SEP> none <SEP> none
<tb> Peptonization <SEP> none <SEP> none
<tb> Reduction <SEP> none <SEP> none
<tb>
<tb> Broth <SEP> nutri- <SEP> Growth <SEP>

  fairly <SEP> good <SEP> to <SEP> good
<tb> tif <SEP> to <SEP> D-gluco- <SEP> good
<tb> se <SEP> Air <SEP> none <SEP> none
<tb> Pigment <SEP> none <SEP> havana
<tb>
<tb> Agar-agar <SEP> hu- <SEP> Growth <SEP> good <SEP> fairly <SEP> good
<tb> tritif <SEP> to <SEP> good
<tb> Aerial <SEP> - <SEP> ivory <SEP> or <SEP> na- <SEP> white <SEP> rosé
<tb> created
<tb> Pigment <SEP> soluble <SEP> none <SEP> havana
<tb>
<tb> Agar-agar <SEP> at <SEP> Growth <SEP> good <SEP> good
<tb> D-glucose <SEP> Aerial <SEP> none <SEP> gray-blue,
<tb>.

   <SEP> white
<tb> Pigment <SEP> soluble <SEP> none <SEP> havana
<tb>
<tb> Agar-agar <SEP> tyro- <SEP> Growth <SEP> fairly <SEP> good <SEP> to <SEP> good
<tb> sine <SEP> Waksman <SEP> good
<tb> Air <SEP> none <SEP> none
<tb> Pigment <SEP> none <SEP> none
<tb>
<tb> Agar-agar <SEP> B <SEP> Growth <SEP> none <SEP> none
<tb> starch <SEP> Waksman
<tb>
<tb> Conversion <SEP> from
<tb> nitrate <SEP> en
<tb> nitrite <SEP> negative <SEP> negative
<tb>
 
Although the known Streptomyces caelestis sporulates more readily on organic media than strain M-188, this appears to be a strain-to-strain difference.



  The two organisms have been shown to produce separate and distinct antimicrobial agents.



   A complete taxonomic study of Streptomyces caelestis strain M-188 on various media is given below; co-color indications given in parentheses refer to the "Color Harmony Manual", 3rd Edition, by Jacobsen, Granville and Foss, 1948, Container Corporation of America.

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   - Medium No. 1 - Tryptone, glucose, agar-agar with beef extract -
Growth on this medium is rapid and abundant.



  The darkest color of the substrate on the 6th day is mustard brown (2 pl). By the 11th day, the lightest substrate is golden brown, tobacco brown (3 µl), the darker areas and the centers of densely populated colonies are clove colored (3 µl). On the 18th day, these areas are respectively camel, maple sugar, tan (3 le) and clove, dark brown (3 pl).



   Aerial mycelium is abundant, covering the surface of colonies by 2-3 mm by day 3 and in the central portion of other larger colonies. It is pearly white (b) to light gray (c), unchanged after 26 days of incubation. Microscopic examination of the aerial mycelium on day 26 does not show any spores.



   Isolated colonies have convex centers, with aerial mycelium confined to 1-2 mm in the center. After 6 and 11 days, a single diametral wrinkle or crack appears in the central raised portion. As growth progresses the colonies spread out flat with a surface growth of 2-4 mm, the remainder spreading densely below the agar surface to a total diameter of 7- 7.5 mm on the 18th day. The aerial mycelium remains confined to the initial zones noted on the 3rd day.



   Soluble pigment brownish on the 3rd day, medium clove (3 nl) on the 6th day becoming chocolate, dark brown (4 nl) on the 18th day.



  - Medium No. 2 - Peptone, glucose, sodium chloride, beef extract agar-agar -
Very fast and abundant growth - mustard substrate, old gold (2 on) 6th day - darker portions 11th day

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 amber, topaz (3 pe) - golden brown (3 Pg) 18th day, with new growth mustard, old gold (2 le) in expanding colonies.



   Scattered to moderate aerial mycelium only in dense growing areas, pearly (2 ba), day 6, turning yellow (1 ba) day 11. Microscopic examination does not show spores on the 27th day.



   The isolated colonies, on the 6th day, show domed centers, wrinkled radially. On the 18th day, a few radial indentations go up to 1/2 mm from the edge of the colonies.



  Isolated colonies of 2.25 mm on the 3rd day reach 8-11 mm on the 18th day. Viewed from above, the surface of isolated colonies is light mustard (2 le) except the centers which are cream colored (1 1/2 ca).



   A very light brownish soluble pigment, 3-11 days, becomes diffuse light greenish brown by the 18th day.



  - Medium # 3 - Nutrient Agar -
Excellent growth. Moderate to abundant aerial mycelium covering colonies of 2.0 mm on day 3 but not increasing as growth expands to 4-6 mm on day 11. The aerial mycelium is pearly white (b) to nacre (2 ba) or ivory (2 cb) as incubation progresses.



  Spores are not observed on microscopic examination on days 11, 18 and 27.



   Well isolated colonies 6th to 11th day, 4-6 mm in diameter, with radial wrinkles and a single more marked diametric fissure, confined mainly to a raised central portion of 2 mm bearing aerial mycelium.



  The rest of the growth extends flat on the agar surface to a well-defined edge. Growth does not penetrate the agar beyond the limits of surface growth.

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   Darker substrate, clove, dark brown (3 ft) to dark brown, coffee, sepia, seal (3 pn) on days 6 to 11, becoming cloves (3 nl) on day 18.



  Substrate lighter in portions without aerial mycelium, light brown, cinnamon (3 1g) on the 11th day, passing to yellow maple (3 ng) on the 18th day.



   Dark brown soluble pigment, clove (3 pl), day 6, dark brown (4 pl) day 11 and diffuse mustard brown (2 pl) day 18.



  - Medium n * 4 - Glucose agar -
Growth on this medium is rapid and abundant.



  No aerial mycelium formed within 27 days of incubation, and no soluble pigment was observed.



   Smooth dull colonies of 2.0 mm on day 3 flatten out flat on day 6 at 4-4.5 mm, with symmetrical radial fissures or indentations wrinkling the raised central portion. As growth progresses, it does not spread below the agar surface as in other organic media containing less glucose.



   Substrate color in dark colony centers is mustard, old gold (2 le) by day 6, no change thereafter. Lighter substrate of new extension growth, bamboo, buff (2 gc).



  - Medium n 5 - Agar-agar and oatmeal Carvajal-
No growth occurs on this medium within 27 days of incubation.



  - Medium n 6 - Czapek's solution, dextrose, agar-agar -
Growth is slow to start with pinpoint colonies on day 3, progressing to 7.0 mm.



  Isolated colonies on the 18th day. Scattered aerial mycelium of yellow tint (1 ba) on the 6th day observed in the center of 1-1.5 mm colonies. On the lth day, an aerial mycelium

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 pearly (2 bc) is placed in a scattered ring around a central non-aerial portion. From above, this non-aerial center is mustard, old gold (2 le). On the 11th day, the substrate of the band and colony centers is parchment colored (1 1/2 db). On the 18th day, the growth of the clonies on the surface is about 3.0 mm, the rest spreading below the surface of the agar-agar up to 7.0 mm.



   No soluble pigment is formed in 27 days. On microscopic examination, no spores are found after 27 days.



  - Medium No. 6c - Czapek's solution, corn starch, agar- agar -
On the 3rd day, moderate colorless growth in bands.



  Isolated colonies fluffy, 1.5-2.0 mm. No hydrolysis is noted. On the 6th day, the growth remains colorless.



  Isolated colonies 3.0-3.5 mm. Partial hydrolysis of a 2.0 mm band, doubtful, more or less dense growth. on the 11th day, dubious hydrolysis, cloudy halo around the growth with a small zone of partial clarification beyond. On the 18th day, numerous colonies showing abundant green to green-blue aerial mycelium - spores - isolated colonies spreading densely in the agar-agar 2.0 mm beyond the superficial growth. Typical measurement: diameter of superficial growth 9.0 mm, below agar-agar up to 11.0 mm, cloudy halo up to 15 mm, and weak to moderate hydrolysis up to 22.0 mm.



  Clear area appreciated in comparison to the control portion of the agar agar, not in comparison to the cloudy interior halo.



  - Medium No. 7 - Dextrose, asparagine, agar-agr -
Growth is moderate to good on this medium.



  Substrate very strongly pigmented on the 6th day, parchment

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 (1 1/2 db). This color remains the darkest taken by central portions of isolated colonies. New growth exhibits a yellow (1 ba) or colorless substrate as one approaches the edge of the expanding growth. Moderate aerial mycelium, yellow (ba) to pearly white (b). There is no soluble pigment or spores after 27 days of incubation.



  - Medium No. 8 - Calcium malate and agar-agar -
Growth is excellent and the patient's digestion is good or marked. On the 3rd day, the colonies, all isolated even in a very dense band, are composed of flat, lamellar, non-aerial mycelium of 2 mm, estimated at <95% of the growth. The rest of the smaller colonies have aerial mycelium. On the 6th day, a slight clarification of the malate is noted. All colonies now have yellow aerial mycelium (1 ba) apart from a few almond green spots (23 ig). It was not noted whether colonies with green aerial mycelium were those that first appeared with aerial mycelium on day 3. By day 11, digestion is marked at 5-6 m from the edge of the strip and the previously green aerial mycelium is now dark gray to slate.

   On the 18th day the mass of the aerial mycelium is pearly white (b) to light ivory, eggshell (2 ca), and on the 27th day there are abundant new colonies with green aerial mycelium in the strip.



   Microscopic examination of the green aerial mycelium: profuse sporula- tion.



  - Medium # 9 - L-tyrosine agar slant cultures
No soluble pigment is formed by the many isolated colonies which grow slowly at first but well as incubation progresses, over 27 days.

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   - Medium n 10a-agar-agar n 1 with corn starch -
3rd day: no visible growth
6th day: a colony 11th day: a colony
18th day: colony of 9.0 mm with 5 radial indentations. Hydrolysis zone 13.0 mm in diameter.



     27th day: colony of 19.0 mm, hydrolysis 34 mm.



   - Medium No. 11 - Tryptose, agar-agar and blood -
Abundant non-aerial growth, dull, dry, no hymolysis in 18 days of incubation. Within 3 days a strong dark almost black colouration of the surrounding environment occurs in the vicinity of the growth. One can find intact red blood cells for example up close.



   - Medium No. 13 - Gelatin buffer -
Growth is not visible by day 3 but is good by day 6 with sparse aerial mycelium, pearly white (b) and moderate to abundant soluble pigment, ebony color, teak (3 in) below the surface film .



   On the 18th day, the film is coarse and the soluble pigment dark brown. Gelatin is not liquefied after 27 days of incubation.



  - Middle n 14- Potato wedge -
Growth is good to excellent after a slow start. On the 6th day, the majority is formed of numerous dry, crowded colonies without aerial mycelium. A rare aerial mycelium is present on a thin upper portion of the slant culture. The colonies are wrinkled on the 11th day by numerous cracks or fine radial indentations. Non-aerial growth adobe brown, cinnamon, light brown (3 µg) by day 6, becoming golden brown, tobacco brown (3 µl) to dark brown, cloves (3 µl) by day 11.

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   The rare aerial mycelium at the top of the culture is ivory (2 db). The potato is only moderately dark after 27 days of incubation.



   - Medium n T5 - Glucose broth -
With the inoculum used, all growth on this medium is formed as separate colonies, placed under the surface, clinging to the sides and bottom of the tube. On the 11th day, the colonies on the sides present heavy and light portions radiating from a dense center, typical of radial wrinkles on favorable solid media. All the growth at the bottom of the tube on the 18th day. No film formation, therefore no aerial mycelium or trace of substrate pigment. The growth is colorless. No soluble pigment is observed.



  - Medium No. 16 - Czapek's solution with dextrose -
No growth occurs within 27 days of incubation.



  - Medium No. 17 - Sunflower milk -
The growth is formed by a partial film.



  On the 11th day, it is noted in the form of two large wrinkled colonies. The tube is shaken carefully to distribute the growth. On the 18th day, no visible change occurred. After 27 days, there is no coagulation or peptonization. On the 27th day, the final pH is 5.7.



  - Medium No. 18 - Yeast extract, maltose and agar-agar -
Abundant and rapid growth in this environment. Isolated non-aerial colonies. Aerial mycelium moderate in areas of dense growth, pearly white (b). No spores were observed on microscopic examination of the aerial mycelium on days 11, 18 and 27.



   Color of the substrate bamboo, buff (2 gc) on the 6th day becoming honey, light gold (2 lc) on the 11th day and brown

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 - approximately golden (3 pg) on the 11th day. Colonies well isolated, non-aerial, 4-5 mm by 6th day, convex with numerous cracks or symmetrical radial wrinkles. They reach 8.0 mm on the 11th day with the outer portion spreading out flat. Raised central portion radially fissured, converging to a small central depression. Growth at the edges of the colonies does not penetrate the agar beyond the surface growth. Soluble pigment is not observed.



   - Medium n 19 - Tomato puree, oatmeal and agar-agar. -
Rapid and abundant growth. Pearly white aerial mycelium (b) abundant in areas of dense growth in
3rd day. Isolated colonies typical of those described for other organic media. By day 18, aerial mycelium appears on isolated colonies, in spots or in a thin ring around a raised non-aerial center, radially wrinkled and rounded. In areas of dense growth, an aerial mycelium appears in the center of the colonies in the most populated portions. No change in the color of the surrounding medium is observed. The aerial mycelium does not turn green during 27 days of incubation. Microscopic examination of the aerial mycelium after 27 days did not show any spores.

   Maximum size of the colonies isolated on the 27th day: 21.0 mm.



  - Spore morphology - Pridham base medium and
Gottlieb with dextrin as carbon source -
Spores measuring about 0.8 x 1.0 microns grow into individual chains which are sinuous, curls, coils and primitive spirals. We do not observe real spirals. The channels appear

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 Usually grow as tufts at the ends of mycelial filaments or short branches of mycelium, but occasionally as individual chains starting from a primary branch. The tufts are produced by several chains formed by tight branching and subbranching of the mycelium rather than a brush of a single origin.



   Electron micrographs show that the mature spores are covered with numerous blunt ridges and spines. The young spores are relatively smooth.



  Some of these appear binucleated. The ovoid to bascillary morphology described by the measurements given above is confirmed by electron micrographs.



   As stated above, the present invention further comprises a method for culturing Streptomyces caelestis, strain M-188, under controlled conditions which include a temperature of 24-32 C, in submerged fermentation with appropriate agitation and aeration, on media consisting of a source of carbohydrates such as glycerol, starch, dextrin or sugars, glucose, lactose or sucrose, or mixtures of these sources; a source of organic nitrogen such as soybean oil, corn maceration liquor or peptone, a source of growth factors such as yeast, soluble distillery products or molasses;

   mineral salts such as sodium chloride, an insoluble buffer to prevent acid build-up, for example calcium carbonate, and a non-toxic defoamer such as vegetable oil. When the growth of the organism has given a satisfactory amount of antimicrobial substance, as indicated by the titration

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 using Bacillus subtilis, the entire culture is processed to isolate the desired antimicrobial product. The amount of active substance present in the liquid phase depends on the pH because the antimicrobial product is a weak base which is poorly soluble in water. Also, antibacterial assays performed on the filtered or centrifuged fermentation liquor cannot measure all of the activity produced. It is necessary to treat the entire culture with acid before performing a titration.

   Most of the antimicrobial product, in a good fermentation, will end up in the solids. Also, it is more efficient to extract the antimicrobial substance from the whole culture and not from the filtrate or the liquid portion. A solvent extraction process is applied taking advantage of the low solubility of the substance in water in alkaline medium and the high solubility of the substance in water in acidic medium. The operations are described more fully in the examples. A particular subatance obtained in this way has remarkable and valuable properties. It has characteristics which distinguish it from antimicrobial substances known and previously described.



   To obtain an inoculum suitable for use in shaker flasks, the vegetation obtained with cultures slanted on tryptone agar can be used. This medium can also be used to maintain by transfer from one culture to another suitable viable cultures which yield the antimicrobial product.



  However, in general practice it has been found that a safer method has been to maintain the culture of M = 188 in soil or with freeze drying. Growth on slant cultures is used to inoculate

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 shaker flasks which can in turn be used to inoculate laboratory scale fermentation tanks. Another method is to use the shaker flasks to inoculate suitable metal containers, preferably aluminum, containing a suitable medium which serves to inoculate pilot plant scale fermentation vessels or well the seed tanks of fermentation tanks on a commercial scale.

   In general, the growth of the organism in fermentation tanks varying from 23 liters to 38,000 liters reaches its maximum in 3 to 4 days, although for the purpose of obtaining an inocula the incubation may be of 24 hours only. Aerobic conditions are maintained in the fermentation tanks by pushing sterile air to the bottom of the tank using a dispersing device.



  The flow of air discharged into the culture medium depends more or less on the size and shape of the fermentation tank. An aeration rate of 0.8-1 volume of air per volume of culture per minute has been found to be satisfactory. If the culture medium foams inconveniently during fermentation, sufficient non-toxic anti-foam vegetable oils can be added to suppress the foam. Throughout the fermentation, the culture medium is stirred. However, in a phase of inoculum preparation where the yield of antimicrobial agent in the inoculum itself is not of critical importance, sufficient agitation is achieved by bubbling air. through the liquid.

   On the other hand, when the yield of antimicrobial agent is high, the agitation is carried out by means of agitating devices which form part of the fermentation apparatus. The degree of agitation

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 depends on the structure of the fermentation vessels of various sizes as it / well known that pilot plant scale and commercial scale fermentation vessels are designed for general use and not for a specific fermentation process. The body
Streptomyces caelestis strain M-188 can produce the desired antimicrobial agent in satisfactory amounts in various culture media over a temperature range of at least 24-32 C and it is not necessary to maintain a level. exact aeration or a precise degree of mechanical agitation.



   The following examples illustrate the formation, recovery, concentration, purification and identification of the antimicrobial agent M-188 and its acid addition salts. These examples are merely illustrative and should not be construed as limiting.



   Example 1 Manufacture in 23 liter fermentation tanks with molasses / peptone medium
In a 500 cm3 Erlenmeyer flask, 150 cm3 of a seed medium containing the following ingredients are placed at the concentrations indicated:
 EMI16.1
 
<tb> grams <SEP> per <SEP> liter
<tb>
<tb> glucose <SEP> monohydrate <SEP> 15
<tb>
<tb> flour <SEP> from <SEP> flakes <SEP> from <SEP> soybean <SEP> (soybean <SEP> finely ground <SEP> <SEP> and <SEP> defatted) <SEP> 15
<tb>
<tb> <SEP> sodium <SEP> chloride <SEP> 5
<tb>
   tap water :

   to make a liter
The vial and its contents are sterilized by autoclaving for 25-30 minutes at a temperature of 120 C. After cooling, the vial is inoculated with

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 a piece of the surface of a tryptone agar culture in which Streptomyces caelestis strain M-188 has been grown for at least 6 days. The inoculated vial is shaken at 28 ° C. on a rotary shaker which has a stroke of 57 mm and operates at about 230 rpm for 48 hours.



  The above culture is used to inoculate additional vials prepared and sterilized as above. Each flask is inoculated with approximately 3 cc of the 48 hour culture. Seed flasks are incubated and shaken as above for 48 hours.



   In a small fermentation tank with a capacity of 23 liters, we put 12 liters of a medium comprising: g / 1 glucose monohydrate 10 molasses 20 peptone 5 antifoam oil 5 tap water! to make a liter.



   The fermentation vessel and its contents are sterilized by autoclaving for 75 minutes at 121 ° C.



  After cooling, the fermentation vessel is aseptically inoculated with the contents of three of the second pass seed culture flasks described above. The culture is carried out in the tank at 24 ° C. for 4 days and during this time the broth is mechanically stirred and sterile air is blown into the bottom of the tank at a rate of approximately 0.8 volume of air per volume of broth per minute. During fermentation the pH drops from about 6.0 to about 5.0 and then rises to about 5.5. Maximum biological activity is reached after about 4 days. The presence of the antimicrobial product in the liquor is indicated by

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 a 24 mm zone of inhibition on a tray seeded with Bacillus subtilis.



   Example 2
Manufacture in 23 liter downstream fermentation tanks a medium formed from soybean flour and soluble dried distillery products.



   In a fermentation tank with a capacity of 23 liters are placed 12 liters of a fermentation medium which has the following composition: g / 1 glucose monohydrate 15 soybean flour 12 sodium chloride 5 soluble dried distillers products, obtained from from the fermentation of molasses 5 glycerol 2.5 calcium carbonate 1 tap water: to make 1 liter
About 300 cc of transesterified vegetable oil are added to the vessel and the vessel and its contents are autoclaved at 121 ° C. for 75 minutes. After cooling, the fermentation vessel is aseptically inoculated with three flasks of second pass seed culture of the type described in Example 1.

   The culture is carried out for 4 days at 28 C; during this time, it is mechanically stirred and sterile air is blown to the bottom of the tank at a rate of about 10 l / min. During this growth time, the pH of the broth slowly rises from about 6.2 to about 7.4.



  Antibacterial activity, as measured by a tray test with Bacillus subtilis, reaches a

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 maximum after about 3 to 4 days. A 25 mm inhibition zone is obtained. After 4 days, the culture liquid is removed from the tank and filtered with suction using filter aid. Due to the low solubility of the antibacterial substance in water, especially at neutral or slightly alkaline pHs, both the cake and the liquid phase must be treated to recover the desired material, as described in Example 9. .



   Example 3 Manufacture in 23 liter fermentation tanks on a medium formed from soybean meal and dried distillery soluble product, with various compounds used as carbon source.



   The same medium as in Example 2 was used for a series of 23 liter fermentation tanks with the exception of the carbohydrate source. Two tanks are prepared, each of which contains 12 liters of medium, with each of the following carbohydrates: glucose monohydro, sucrose, lactose, starch, dextrin and glycerol.



  These 12 cuvettes are prepared as described in Example 2, each set of two cuvettes containing 17.5 g / l of the desired carbon hydrate in place of the 15 g of monohydric glucose and the 2 cuvettes. , 5 g of glycerol. For each vat, the fermentation process is carried out exactly as indicated in example 2.



   The antimicrobial product is prepared with the six culture media. The antibacterial activity, measured by a plate titration with Bacillus subtilis, indicates that no medium is clearly superior to the others. The six media give zones of inhibition varying from 24 to 27 mm on the last two days of the 4-day fermentation.

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   On completion of the 4 day fermentation, the entire contents of 11 of the 12 tanks are combined and suction filtered using filter aid.



   Due to the low solubility of the antibacterial substance in water, especially in a slightly alkaline medium, both the cake and the liquid phase must be treated to recover the active antimicrobial material, as described in Example. 9.



   Example 4
Manufactured in 23-liter tanks with a medium formed from soy flour and maceration-corn liquor.



   12 liters of a fermentation medium of the following composition are placed in a fermentation tank with a capacity of 23 liters: g / 1 glucose monohydrate 30 soybean flour 20 corn maceration liquor (wet weight) 10 sodium chloride 5 calcium carbonate 2 trnsesterified vegetable oil 5 pH adjusted to 6.6-6.8 using Na2CO3
The tank and its contents are autoclaved at 121 ° C. for 75 minutes. After cooling, the fermentation vessel is aseptically inoculated with three flasks of second pass seed culture, as described in Example 1.

   The organism is cultured for 4 days at 28 ° C. during this time the culture is shaken mechanically and sterilized air is blown into the bottom of each tank at a rate of about 10 l / one.



  During the growth time, the pH of the medium rises

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 slowly to around 7.6 when harvested after an initial drop to 6.2-6.4. Antibacterial activity as measured by tray titration with Bacillus subtllia reaches its maximum on the 3rd or 4th day of fermentation. Since there is no apparent drop in yield once the maximum is reached, harvesting is done on the 4th day to ensure maximum production. On the titration tray with Bacillus subtilis, inhibition zones of 26 mm are obtained.



   Example 5 Manufacture in 190 liter tanks with a medium of soy flour, corn maceration liquor and yeast.



   The organism Streptomyces caelestis strain M-188 is cultivated in slant culture on tryptone agar for 6 days at 28 C. The vegetation of a culture is suspended in a few cubic centimeters of water and inoculated. two 500 cm3 Erlenmeyer flasks each containing 150 cm3 of the following inoculating medium: g / 1 glucose monohydrate 15 soy flake flour 15 sodium chloride 5 calcium carbonate 1
The vials containing 150 cc of this medium are sterilized by autoclaving them for 25-30 minutes at 120 C. After cooling, the vials are inoculated with the vegetation taken from the above agar cultures. . The inoculated vials were shaken for 48 hours at 28 ° C. on a rotary shaker which had an eccentricity of 57 mm.

   With all the contents of these vials, an aerated metal cylinder with a capacity

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 approximately 12 liters containing 10 liters of the following medium: g / 1 soybean flour 10 glucose monohydrate 10 calcium carbonate 1 transesterified vegetable oil 5
The container and its contents are pre-sterilized at 120 C for 80 minutes and cooled to 28 C.



   The aerated cylinders are incubated for 48 hours at 28 ° C. Air is bubbled through the culture medium, through a tube placed at the bottom, at a rate of approximately 10 l / min. The entire contents of the carboy are then used to inoculate a fermentation tank with a capacity of 190 liters containing 115 liters of the following medium which has previously been sterilized at 124 ° C for 1 hour and cooled to 32 C: g / 1 starch 10 soy flour 20 corn maceration liquor (wet weight) 10 whole dried brewer's yeast 5 calcium carbonate 3 transesterified vegetable oil 30
The inoculated medium from the fermentation tank is kept under vigorous stirring at a temperature of 32 ° C. for 4 days while aerating at a rate of one volume per volume of medium per minute.

   At the end of fermentation, growth is often so strong that it is necessary to dilute the culture with water to remove it from the fermentation tank.



   Untreated culture assays have little or no value in determining culture activity

 <Desc / Clms Page number 23>

 during harvest given the insolubility of the antimicrobial product. When the cultures are treated in the following way, the titrations become more useful. We adjust
50 cm3 of complete culture at pH 2.0 with hydrochloric acid and then this acidified culture is placed on the rotary sewer for 30 minutes. Then vacuum filtered through three layers of filter paper. After adjustment to pH 4.0 with sodium hydroxide, the filtrate is ready for titration. The titer of the fermentation described in this example is 225 units / cm3.



   Example 6 Manufacture in 1900 liter fermentation tanks on a medium of soy flour, corn maceration liquor and yeast.



   An inoculum for the density vessel is prepared by the aerated cylinder method described in Example 5. The entire contents of one of the metal culture cylinders are used to inoculate a fermentation vessel. seeding with a capacity of 190 liters containing 115 liters of the following medium which was sterilized at 124 C for 1 hour and cooled to 32 C. g / 1 starch 10 soybean meal 20 corn maceration liquor ( wet weight) 10 whole dried brewer's yeast 5 calcium carbonnate 3 transesterified vegetable oil 30 pH to 6.7 using Na2CO3
The seed tank is maintained at 32 ° C. for 24 hours with vigorous mechanical agitation and aerating at the rate of one volume of air per volume of culture medium.

 <Desc / Clms Page number 24>

 per minute.

   At the end of these 24 hours, the entire contents of the fermentation tank are used to inoculate 1150 liters of the same medium in a fermentation tank with a capacity of 1900 liters, The inoculated medium of the fermentation tank is kept under a vigorous mechanical stirring at a temperature of 32 C for 4 days.



  Sterile air is introduced for aeration of the culture medium at a rate of 1 volume per volume of medium per minute. At the end of the fermentation cycle, the culture is diluted with enough water so that it is sufficiently fluid and can be pumped out of the tank, allowing the recovery operation to begin.



  Prior to diluting the culture, a sample was taken for titration and processed as described in Example 5. The titer of the material in this 1150 liter fermentation was 375 units / cm3.



   Example 7 Manufacture in 36,000 liter fermentation tanks on a medium of soy flour, corn maceration liquor and yeast.



   An inoculum for the inoculation fermentation vessel is prepared by the aerated cylinder method described in Example 5. The entire contents of one of the metal culture cylinders are used to inoculate a fermentation vessel. seed fermentation with a capacity of 3150 liters containing 1285 liters of the following medium, which was sterilized at 124 C for 1 hour and cooled to 32 C: g / 1 starch 10 soy flour 20 corn maceration liquor (wet weight) 10

 <Desc / Clms Page number 25>

 three times with chloroform using 1.5 liters of chloroform for each extraction. After each extraction, the mixture is allowed to stand until the phases separate and the chloroform phase is withdrawn.

   The three portions of chloroform are combined and passed through a column packed with excess adsorbent formed from synthetic magnesium silicate. After washing the column with fresh chloroform, the desired substance is eluted by passing a mixture of 10% ethanol and 90% chloroform through the column. By performing a tray titration with Bacillus subtilis, it is seen that 30% of the antimicrobial activity is derived from the 936 mg of solids which is obtained by evaporating the solution of ethanol and chloroform.



   Growth of Staphylococcus aureus in a broth is hindered by 2 # g / cm3 of the above material.



  When dissolved in acidified methanol or in 0.1n aqueous hydrochloric acid, its ultraviolet absorption spectrum shows a maximum at 279 millimicrons, of magnitude E 1% cm = 240.



   Example 9 Recovery of antimicrobial agent from fermentation liquor and filter cake.



   The fermentation liquor from 11 23-liter tanks obtained essentially as in Example 3 is combined with six different carbohydrates, filtered with suction using a filter aid to obtain 60 liters of clear filtrate. . The filtrate is extracted three times with chloroform, using for each extraction a volume of chloroform equal to about 1/10 of the volume of the aqueous phase. After each extraction, the phases are allowed to separate, we

 <Desc / Clms Page number 26>

 whole dried brewer's yeast 5 calcium carhonate 3 transesterified vegetable oil 10 pH to 6.7 using Na2CO3
The seeding fermentation vessel was maintained at 32 ° C. for 28 hours with vigorous mechanical agitation and aeration at the rate of one volume of air per volume of culture medium per minute.

   At the end of these 28 hours, the entire contents of the fermentation tank are used to inoculate 22,000 liters of the same medium into a fermentation tank with a capacity of 38,000 liters. The inoculated medium is kept from the fermentation tank under vigorous mechanical stirring at a temperature of 32 ° C. for 4 days. Sterile air is introduced to aerate the culture medium at a rate of one volume per volume of medium per minute. At the end of the fermentation cycle, the culture is diluted with enough water so that it is sufficiently fluid and can be pumped out of the tank to begin the recovery operation.

   Before diluting the culture, a sample is taken for titration and processed as described in Example 5.



  The titer of the material in this 22,000 liter fermentation is 360 units / cubic.



   Example 8 Recovery of the antimicrobial agent obtained from a 12 liter fermentation, by a chloroform extraction process. - The fermented liquor taken from a 23 liter fermentation tank and prepared essentially as in Example 1 is stirred with a filter aid and separated from the solids by suction filtration to obtain 7.6 liters of clear filtrate . We extract this to

 <Desc / Clms Page number 27>

 retained the chloroform phase and if there is an emulsion layer it is centrifuged to recover the chloroform.



   The combined extracts contain more than 80% of the antimicrobial activity of the filtrate. The cake was extracted twice with 10 liter portions of 50% aqueous acetone to recover most of the antimicrobial activity.



   In the same way, the whole fermentation liquor from six 23 liter tanks and obtained as in Example 2 is filtered to obtain 42 liters of clear filtrate which is extracted three times with chloroform as described above. The cake is extracted twice with 10 liter portions of 50% aqueous acetone, as described above.



   The chloroform extracts of the filtrates are combined and evaporated in vacuo to dryness. The dried residue water is suspended in 50 cm3 / and the extract is repeatedly extracted with 50 cm3 portions of ethyl ether. The ether extracts were combined and evaporated to give 4.37 g of solids.



   The aqueous acetone extracts of the cake are combined and concentrated to 14 liters to remove acetone. The aqueous solution is extracted three times with 3 or 4 liters of chloroform. The extracts are combined and evaporated until a dry powder is obtained which is resuspended in 50 cm3 of water, then extracted several times with portions of 50 cm3 of ether.

   Ether extracts give
6.90 g of solids. -
Since the titrations carried out on the two dried preparations show that they have similar activities, 200-250 units / mg, and that the chromatograms

 <Desc / Clms Page number 28>

 mes on paper and the toxicities are similar, the two materials are combined for subsequent purification. Each preparation was dissolved in methanol, the solutions mixed and dried to obtain 10 g of solids.



   4 g of this aggregate material is extracted with several portions of water acidified at most to pH 2.0.



   The extracts are combined with acidified water, ie 180 cm 3, they are extracted with ether and the ether phase is discarded.



   The aqueous phase is adjusted to a slightly alkaline pH and extracted with ether. The extract is evaporated to obtain 2.0 g of dry residue assaying 320 units / mg.



   Example 10 Recovery of antimicrobial agent from 115 liter fermentations by a chloroform extraction process.



   The obtained whole fermented cultures were combined in four 190 liter tanks in the same way as in Example 5, to obtain 435 liters. The overall culture was adjusted to pH 2.0 with 10% hydrochloric acid and stirred for 30 minutes. The material was filtered to give 390 liters of filtrate. The filtrate is adjusted to pH 4.0 with 10% sodium hydroxide solution. Three extractions are carried out with 1/10 volume of chloroform each time. The two liquid phases are separated in a centrifuge. The extract is concentrated under reduced pressure to 2200 cm3. 3 volumes of pentane are added to the chloroform concentrate and the mixture is extracted three times with 1/8 volume of 5% aqueous acetic acid. The solvent layer is discarded.

   The acidic aqueous layer is concentrated under reduced pressure to about 1/8 of its original volume. The impurities are precipitated by stirring and slowly adjusting the pH to 5.3.

 <Desc / Clms Page number 29>

 using a 10% sodium hydroxide solution. A gummy residue forms, is filtered off and discarded. The pH of the clarified solution is adjusted to 11 with 10% sodium hydroxide to precipitate the antimicrobial agent; this is collected by filtration and washed thoroughly with distilled water adjusted to pH 9.0. The powder is dried under vacuum at 45 ° C. 75 g of antimicrobial agent M-188 are recovered which has a titer of 300 unita / ng.



   Example 11 Recovery of the antimicrobial agent from 115 liter fermentations by extraction with amyl acetate at pH 8.4.



   The fermented whole cultures obtained are combined in 190 liter tanks, essentially as described in Example 5, to obtain 225 liters. The overall culture is adjusted to pH 8.4 using a 10% sodium hydroxide solution. The adjusted culture is extracted with 1/5 volume of amyl acetate. After separation of the allyl acetate layer, it is extracted with about 1/2 volume of distilled water, maintained at pH 2.5 with sulfuric acid. The aqueous phase is separated and adjusted to pH 5.5 using 10% sodium hydroxide. This solution which contains the antimicrobial product is concentrated up to 3 liters under reduced pressure. The pH of the concentrate is adjusted to 8.5-9.0 with sodium hydroxide, which precipitates the antimicrobial product.

   The precipitate is removed by filtration and washed thoroughly with distilled water adjusted to pH 9.0. The precipitate was dried in vacuo at 45 ° C. 26 g of antimicrobial agent M-188 was recovered which had a titer of 435 units / mg.

 <Desc / Clms Page number 30>

 



   Example 12 Recovery of antimicrobial agent from a 115 liter fermentation by acid treatment prior to extraction with amyl acetate at alkaline pH.



   78 liters of whole fermented culture obtained in a 190 liter tank were taken essentially as described in Example 5, and the pH was adjusted to 2.0 with sulfuric acid. After maintaining the culture at pH 2.0 and shaking it for 3 hours, it is filtered to remove the mycelium. The filtrate is adjusted to pH 8.3 with a 10% sodium hydroxide solution. 1/6 volume of amyl acetate is added and the mixture is stirred for 30 minutes. The two phases are separated in a centrifuge. The aqueous phase is discarded which exhibits virtually no antimicrobial activity. The amyl acetate is extracted with about 1/40 volume of water maintained at pH 2.5 with sulfuric acid and the particles are separated.
 EMI30.1
 #: aass. I adju8te alôr8 the aqueous solution to the pti 9.5 using sodium hydroxide while stirring vigorously.

   The slurry is filtered and the cake washed thoroughly with distilled water at pH 9.0 approximately. The solids were dried in vacuo at 45 ° C. to give 10.5 g of product titrating 474 units / mg.



   Example 13 Recovery of the antimicrobial agent from a 1150 liter fermentation by extraction with amyl acetate at pH 8.4.



   The fermented whole cultures obtained are combined in four 1900 liter tanks, essentially as described in Example 6, to obtain 6200 liters of fermentation liquor. The total volume of the culture includes the water necessary for the dilution allowing the medium to be withdrawn from the tank by pumping. We adjust the ass-

 <Desc / Clms Page number 31>

 Overall ture to pH 8.4 using a 10% sodium hydroxide solution and extraction is carried out with 1/5 volume of amyl acetate.



   The phases are separated and the aqueous phase is discarded. The amyl acetate phase is extracted with about 1/40 volume of distilled water maintained at pH 2.5 with sulfuric acid. The aqueous solution is separated from the antimicrobial product and adjusted to pH 9.5, which precipitates the antimicrobial product. The precipitate is filtered off and washed well with distilled water adjusted to about pH 9.0. The precipitate was dried in air at 50 ° C. The antimicrobial product obtained weighed 563 g and had a titer of 325 units / mg.



   Example 14 Recovery of the antimicrobial agent from a 22,000 liter * fermentation by acid treatment before extraction with amyl acetate at alkaline pH.



   A fermented whole culture obtained as described in Example 7 was adjusted to pH 2.0 with sulfuric acid, and stirred for 3 hours to remove the antiaicrobial product from the mycelium. The culture is filtered and the pH of the filtrate is adjusted to 8.4 using 10% sodium hydroxide. At this stage, 26,190 liters of filtrate are obtained.



  This filtrate is extracted with 1/8 volume of amyl acetate in a liquid / liquid extractor at a rate of 114 liters per minute. The amyl acetate solution was extracted with 1/40 volume of water maintained at pH 2.0 using sulfuric acid. This aqueous extract is adjusted to pH 9.5 to precipitate the antimicrobial product. The product is filtered off and washed thoroughly with distilled water adjusted to approximately pH 9.0. The precipitate is dried in air at 50 ° C. The antimicrobial product obtained weighs 7.38 kg and has a titer of 482 units / mg.

 <Desc / Clms Page number 32>

 



   Example 15 Further purification of the antimicrobial agent by chromatography on magnesium silicate.



   17 g of the antimicrobial product titrating 395 units / mg (E1% 1 cm point 278 m @ 236) obtained by the process of example 11 are dissolved in 100 cm3 of chloroform. This solution is applied to a column containing 500 g. of magnesium silicate as adsorbent and development is started with 1% ethanol in chloroform.

   Fractions of 15cm3 are collected and classified as follows according to the ultraviolet peaks:
 EMI32.1
 
<tb> Fraction, <SEP> cm3 <SEP> Solution <SEP> of <SEP> Absorption <SEP> Title <SEP> in <SEP> Weight <SEP> of <SEP> the
<tb>
 
 EMI32.2
 development of UV. units / my preparation.g
 EMI32.3
 
<tb> Q5 <SEP> cm <SEP>
<tb>
<tb>
<tb>
<tb>
<tb> 1-5 <SEP> ml <SEP> 1% <SEP> ethanol
<tb>
<tb>
<tb>
<tb> in <SEP> the <SEP> chloro-
<tb>
<tb>
<tb> form <SEP> 196 <SEP> 382 <SEP> 0.302
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 60-75 <SEP> ml <SEP> "<SEP> 217 <SEP> 470 <SEP> 3.1
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 76-90 <SEP> ml <SEP> "<SEP> 258 <SEP> 532 <SEP> 2.6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 91-105 <SEP> ml <SEP> "<SEP> 254 <SEP> 550 <SEP> 2.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 106-122 <SEP> ml <SEP> "<SEP> 254 <SEP> 550 <SEP> 1,

  3
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 122-132 <SEP> ml <SEP> "<SEP> 212 <SEP> 430 <SEP> 0.60
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> product <SEP> of elu- <SEP> ethanol <SEP> at <SEP> 100%
<tb>
<tb>
<tb> tion <SEP> of <SEP> the <SEP> co-
<tb>
<tb>
<tb>
<tb> lonne, <SEP> low <SEP> 234 <SEP> 380 <SEP> 2.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> product <SEP> of elu-
<tb>
<tb>
<tb>
<tb> tion <SEP> of <SEP> the <SEP> co-
<tb>
<tb>
<tb> length <SEP> (*) <SEP> top <SEP> "<SEP> 189 <SEP> 269 <SEP> 0.96
<tb>
   (*) The pigmented impurities are largely retained in the upper portion of the column.



   Example 16 Further purification of the antimicrobial agent on a column of magnesium silicate.



   60 g of the antimicrobial product containing 395 units / mg are dissolved in 200 cm3 of chloroform (E1% cm point at 278 m
236) obtained by the process of Example 11, and it is applied

 <Desc / Clms Page number 33>

 to a column of 120 g of magnesium silicate. The column is washed with 1% methanol in chloroform and the following two fractions are collected:
 EMI33.1
 
<tb> Fraction <SEP> E1% 1 <SEP> cm <SEP> to <SEP> 280 <SEP> m @ <SEP> Title <SEP> in <SEP> units / mg <SEP> Weight <SEP> of < SEP> the <SEP> frac,
<tb>
<tb>
<tb> 1 <SEP> cm <SEP> @ <SEP> tion, <SEP> g
<tb>
<tb>
<tb> A <SEP> 237 <SEP> 560 <SEP> 43.6
<tb>
<tb>
<tb>
<tb> B <SEP> 225 <SEP> 450 <SEP> 2.2
<tb>
 
Example 17 Formation of M-188 hydrochloride
A sample of the free base obtained in Example 12 is dissolved in dry diethyl ether.

   Hydrochloric acid gas is bubbled through ether and the hydrochloride precipitates.



   In order to be able to evaluate the activity of various preparations of antimicrobial agent M-188, an agar-tray titration with Bacillus subtilis ATGC-10707 as the test organism was used, and a preparation of M-188 containing 320 units / mg. The titration method is based on that of DC Grove and WA Randall, "Assay Methods of Antibiotics and a Laboratory Manual", edited by Medical Encyclopedia, New York 1955, pages 34, 35 and 38. Agar medium is used. yeast extract agar for streptomycin titrations, 10 cm3 per tray. The test solution is diluted in 1% phosphate buffer, pH 6.0 and added to the steel trays which are incubated at 37 ° C for 16-17 hours.



  The interval of the titration curve is 2, 4, 6, 8 and 16 units / cm3, the figure of 4 units / cm3 serving as the reference standard. A 2 g sample prepared according to Example 9 is arbitrarily assigned a titer of 320 units / mg. All the titers indicated in the preceding examples are obtained on this basis. From this point of view, samples of M-188 which assay 600 units / mg are very purified and approach the maximum titre.



   After cultivating the organism for about 4 days

 <Desc / Clms Page number 34>

 and having reached an appropriate concentration of the antimicrobial product in the fermentation liquor, the product can be separated from the cultures by several methods. Since the product is almost insoluble in neutral or alkaline water, most of it is in the solid phase. If desired, the culture can be separated by filtration and the liquid treated as described below, while the solid phase is extracted with an organic solvent such as acetone or an organic solvent. alcohol in which the base is soluble. Alternatively, the fermentation liquor can be acidified and stirred until the antimicrobial substance is dissolved.

   The culture is then filtered and the liquid phase is treated to recover the desired product.



   In order to recover the M-188 from an aqueous solution obtained as above, it is advantageous to make the solution alkaline and to extract the active substance with a solvent immiscible with water. A variety of solvents are useful for the extraction, but chloroform or ethyl or amyl acetate is preferred. To recover M-188 from a solution in an organic solvent, the solution can be evaporated to dryness but it is advantageous to extract the antimicrobial substance into a dilute aqueous acid using enough acid to neutralize the free base. contained in the organic solvent and dissolve it in a relatively small volume of water, about 1% of the initial volume of the culture. When an alkali such as sodium hydroxide solution is added to the solution thus obtained, the desired substance precipitates in the form of the free base.

   The free base can still be purified by chromatography although it is impossible to obtain a completely homogeneous substance thereby. By passing a solution of base M-188 in chloroform over a column of magnesium silicate and eluting the active substance with chloroform containing about 1% ethanol, a fractionation of the material is obtained. 'we can follow by observing

 <Desc / Clms Page number 35>

 optical density to 280 millimicrons.



   Streptomyces caelestis strain M-188 produces a mixture of antibiotic substances. The best preparations contain more than one biologically active material, as shown by the chromatograms on paper. Two of the biologically active substances which are present as impurities in the best preparations of M-188 antimicrobial agent were obtained as purified preparations and their antibacterial properties were found to be essentially the same as those of the main constituent. Thus, it should be understood that the antimicrobial agent described herein does not have to be 100% pure to be 100% active.



   The purified antimicrobial agent M-188 is an amorphous white powder which is soluble in water at 25 ° C. at only 2.5 mg / cm3. It is very soluble in most organic solvents such as methanol, ethanol, chloroform, ethyl acetate and benzene. It is insoluble in saturated hydrocarbons. It is easily soluble in dilute aqueous mineral acids. Electrometric titration in 50% aqueous ethanol indicates a titratable group in which pKa - 6.7. The ultraviolet absorption spectrum of the free base in chloroform shows a maximum at 279 millimicrons, E1% = 280 and a minimum at 232 millimicrons, E1% = 29.

   The infrared absorption spectrum of purified M-188 is shown in the accompanying drawing.



  When determining an infrared spectrum of the antimicrobial agent M-188 as free base using a 7% solution in chloroform using a double beam spectrophotometer, the bands are seen absorption following:

 <Desc / Clms Page number 36>

 
 EMI36.1
 
<tb> Wave length <SEP>, <SEP> microns <SEP> Frequency, <SEP> cm-1 <SEP> Intensity
<tb>
<tb>
<tb>
<tb> 2.88 <SEP> 3472 <SEP> M
<tb>
<tb>
<tb>
<tb> 3.41 <SEP> 2933 <SEP> S
<tb>
<tb>
<tb>
<tb>
<tb> 5.79 <SEP> 1727 <SEP> S
<tb>
<tb>
<tb>
<tb> 5.94 <SEP> 1684 <SEP> W
<tb>
<tb>
<tb>
<tb>
<tb> 6.12 <SEP> 1634 <SEP> W
<tb>
<tb>
<tb>
<tb> 6.26 <SEP> 1597 <SEP> s
<tb>
<tb>
<tb>
<tb>
<tb> 6.89 <SEP> 1451 <SEP> M
<tb>
<tb>
<tb>
<tb>
<tb> 7.11 <SEP> 1406 <SEP> w
<tb>
<tb>
<tb>
<tb> 7.36 <SEP> 1359 <SEP> w
<tb>
<tb>
<tb>
<tb> 7,

  46 <SEP> 1340 <SEP> w
<tb>
<tb>
<tb>
<tb>
<tb> 7.73 <SEP> 1294
<tb>
<tb>
<tb>
<tb>
<tb> 7.85 <SEP> 1274 <SEP> w
<tb>
<tb>
<tb>
<tb>
<tb> 8.00 <SEP> 1250 <SEP> W
<tb>
<tb>
<tb>
<tb> 8.60 <SEP> 1163 <SEP> s
<tb>
<tb>
<tb>
<tb>
<tb> 8.95 <SEP> 1117 <SEP> M
<tb>
<tb>
<tb>
<tb> 9.25 <SEP> 1081
<tb>
<tb>
<tb>
<tb>
<tb> 9.52 <SEP> 1050
<tb>
<tb>
<tb>
<tb> 9.75 <SEP> 1026 <SEP> M
<tb>
<tb>
<tb>
<tb>
<tb> 9.85 <SEP> 1015 <SEP> M
<tb>
<tb>
<tb>
<tb> 9.98 <SEP> 1002 <SEP> M
<tb>
<tb>
<tb>
<tb>
<tb> 10.22 <SEP> 978
<tb>
<tb>
<tb>
<tb> 10.9 <SEP> 917 <SEP> w
<tb>
<tb>
<tb>
<tb>
<tb> Il, 12 <SEP> 899 <SEP> W
<tb>
<tb>
<tb>
<tb> 11.53 <SEP> 868 <SEP> w
<tb>
<tb>
<tb>
<tb>
<tb> 11.94 <SEP> 838 <SEP> @ <SEP> M
<tb>
 S - strong; M @ medium; W - weak.



   The specific rotation of a 1% solution of the free base in chloroform is [[alpha]] 25D = -40.



   Elemental analysis of a representative sample of the free base M-188 shows that it contains 60.37% carbon, 8.21% hydrogen, 1.91% nitrogen and 29.51% oxygen by difference, which corresponds to an empirical formula C38H61NO14

 <Desc / Clms Page number 37>

 which theoretically gives 60.38% carbon, 8.13% hydrogen, 1.85% nitrogen and 29.63% oxygen. Thus, the calculated molecular weight is 756.



   Acid addition salts of M-188 antimicrobial agent can be obtained by treating a solution of the free base in water or in an organic solvent such as methanol, ethyl acetate or chloride. methylene, by an equivalent amount of an aqueous solution of an acid such as hydrochloric acid, oxalic acid, salicylic acid or citric acid and evaporating the solution to dryness. Alternatively, a solution of M-188 antimicrobial agent in an organic solvent can be treated with a selected acid or a solution thereof and the corresponding acid addition salt precipitated directly from the solution. Examples of such salts are the hydrochloride, oxalate, salicylate, citrate, benzoate and sulfate. For therapeutic uses, the salt chosen must obviously be non-toxic.



   The invention is not limited to the preparation of the antimicrobial agent M-188 using the described strain of Streptomyces caelestia. It is understood that the fermentation process of the present invention can also relate to other strains of Streptomyces caelestis producing M-188 which are obtained by exposing the indicated organism to modifying agents such as X-rays. , ultraviolet rays and chemicals such as chlorethazine.



   The antimicrobial agent M-188 and its acid addition salts are characterized by a broad antibacterial spectrum.



  The activity of this agent against typical organisms is indicated by the following table:

 <Desc / Clms Page number 38>

 
 EMI38.1
 
<tb> Antimicrobial <SEP> spectrum
<tb>
<tb> Minimum <SEP> concentration
<tb> Inhibition <SEP> organism <SEP> after <SEP> 48
<tb> hours. <SEP> g / cm3
<tb>
 
 EMI38.2
 Staphylococcus aurs' * 209-P 0.5
 EMI38.3
 
<tb> Staphylococcus <SEP> aureus <SEP> Treaster <SEP> 0.5
<tb>
<tb>
<tb>
<tb> Staphylococcus <SEP> aureus <SEP> Wise <SEP> 391 <SEP> 1.0
<tb>
<tb>
<tb>
<tb>
<tb> Staphylococcus <SEP> albus <SEP> 0.75
<tb>
<tb>
<tb>
<tb>
<tb> Streptococcus <SEP> pyogenic <SEP> 0.25
<tb>
<tb>
<tb>
<tb>
<tb> Streptococcus <SEP> faecalis <SEP> 2,0
<tb>
<tb>
<tb>
<tb>
<tb> Streptococcus <SEP> agalaciae <SEP> 0.13
<tb>
<tb>
<tb>
<tb>
<tb> Streptococcus <SEP> dysgalactlae <SEP> 0,

  25
<tb>
<tb>
<tb>
<tb>
<tb> Enterococcus <SEP> 89 <SEP> 2,0 <SEP> @
<tb>
<tb>
<tb>
<tb>
<tb> Enterococcus <SEP> 93 <SEP> 2.0
<tb>
<tb>
<tb>
<tb>
<tb> Enterococcus <SEP> Blaschke <SEP> 1.0
<tb>
<tb>
<tb>
<tb>
<tb> Clostridium <SEP> sporogenes <SEP> 48.0
<tb>
<tb>
<tb>
<tb>
<tb> Cloatridium <SEP> perfringens <SEP> 64.0
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Corynebacterium <SEP> species <SEP> 0.25
<tb>
<tb>
<tb>
<tb> Sarcina <SEP> lutea <SEP> 0.06
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Diplococcus <SEP> pneumoniae <SEP> il, 25
<tb>
<tb>
<tb>
<tb>
<tb> Lactobacillus <SEP> casei <SEP> 0,

  5
<tb>
<tb>
<tb>
<tb>
<tb> Salmonella <SEP> enteritidis <SEP>> 256
<tb>
<tb>
<tb>
<tb>
<tb> Aerobacter <SEP> aerogenes <SEP>> 256
<tb>
<tb>
<tb>
<tb>
<tb> Escherichla <SEP> coli <SEP> 128
<tb>
<tb>
<tb>
<tb>
<tb> Klebsiella <SEP> pneumonia <SEP> 4
<tb>
<tb>
<tb>
<tb>
<tb> Pasteurella <SEP> multocida <SEP> ATCC <SEP> 10544 <SEP> 4
<tb>
<tb>
<tb>
<tb>
<tb> Pasteurella <SEP> multocida <SEP> (hen)

   <SEP> 4
<tb>
<tb>
<tb>
<tb>
<tb> Shigella <SEP> sonnei <SEP> 95
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Neisseria <SEP> catarrhalls <SEP> 2
<tb>
<tb>
<tb>
<tb>
<tb> Actinomyces <SEP> bovis <SEP> 5
<tb>
<tb>
<tb>
<tb>
<tb> Aspergillus <SEP> niger <SEP>> 512
<tb>
<tb>
<tb>
<tb>
<tb> Chaetomlum <SEP> globosum <SEP>> 512
<tb>
<tb>
<tb>
<tb>
<tb> Saccharomyces <SEP> cerevisiae <SEP> <<SEP> 32
<tb>
<tb>
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<tb> Candida <SEP> albicans <SEP>> 512
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The antimicrobial agent M-188 has been found to be extremely useful in the control of turkey sinusitis and coccidiosis. In representative operations :), a good result against infectious sinusitis in turkeys has been obtained by injecting directly into the sinuses of infected turkeys 25-50 mg of antimicrobial agent M-188.

   Likewise, it has been found that a good result against coccidiosis is obtained by incorporating the antimicrobial agent M-188 into the feed of the hens at a rate of 0.05-0.10% in. weight on the total diet given to hens severely affected by coccidiosis due to the presence of the protozoan Elmeria tenella.


    

Claims (1)

ABSTRACT The subject of the invention is: I - A process for preparing an antibiotic substance called “antibacterial agent M-188.”, A process characterized by the following points, considered individually or in various combinations: 1) a strain of Streptomyces caelestis producing antimierobial agent M-188 is cultivated in an aerobic medium, in a medium containing assimilable sources of cabone hydrate. nitrogen and inorganic saltsa until appreciable antimicrobial activity is obtained; 2) an aerobic submerged culture is carried out; 3) the strain is Streptomyces caelestis NRRL 2821; 4) the broth is extracted, at a pH of between 3 and 10 approximately, with a polar organic solvent immiscible with water; 5) the culture medium is maintained between 20 and 32 C approximately and the organism is cultivated for 2 to 6 days. II - The product obtained by the following process I, in the form of the free base or of acid addition salts.