BE641209A - - Google Patents

Description

       

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 EMI1.1
 

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 EMI2.1
 

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 EMI3.1
 process for the preparation of a new antibacterial agent

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 EMI4.1
 The * invention in relation * to a new and useful substance called notomyeia, thus * * # *,
 EMI4.2
 a process for its production; it concerns more by-
 EMI4.3
 particularly a process to produce it by t'f # .na-
 EMI4.4
 tion and methods for recovering and purifying it *
 EMI4.5
 The invention is aimed at this acid antibacterial aaent and its alkaline 0410 in dilute aolutioa, we form crude ooncentr44, .ou. purified solid product form and 10 = 0 cri $ tal11n. pure. Natamyaine is effective in inhibiting the growth of Gram-positive bacteria ...

   The notomy-
 EMI4.6
 cine is not torque and it manifests a therapeutic effect.
 EMI4.7
 can in mice Infected with Ga # - positive bacteria. Notomyoine is also useful in curing # in human $ the infections caused by det
 EMI4.8
 Gram-positive bacteria, eg pneumonia. The
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 natomoina has also been referred to as the antibiotic Du 620.



  The Applicant now provides, ocaforméoeat to the present invention, an effective antibiotic substance.
 EMI4.10
 cient to inhibit the growth of Gram-
 EMI4.11
 positive, selected from the group, ooaetituee by a sustained acid stance, notoaycine, which is easily soluble in acetone, d10xane and alkaline water, moderately
 EMI4.12
 soluble in ethanol, butanol, ethyl acetate,
 EMI4.13
 butyl acetate and mithyl l80but11 oétoae, less soluble in beneône, methanol and ohloroferme and
 EMI4.14
 insoluble in carbon tetrachloride petroleum ether and acidulated water, which gives reactions of
 EMI4.15
 Fehling and ldaliaah positive, discolors bromine and gives negative reactions with nlnh74rine, Tollens and anthrone,

   and which in purified form melts at 222-224 "z un; lPh! 7 of -134 * (0 - 1.0i acetone),

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   exhibits an ultra violet absorption spectrum in
 EMI5.1
 'ethanol having maxima. 275 millimicrons (2 | Ja 595) and at 555 millimierons (1 ex si 498) in Hal If / 10 having maxims at 275 taillimic = no (n 1 * ffl - 287) and t '4' = il- limicrons (1 Ja 277), and in Naon N / 10 having a maxi- aum at 280 willioiorons (m 3 '- 766), has a neutralization equivalent of 548 and whose mean elemental analysis is as follows 0 w 59 # 1% î H. 5155%;

   N m 5tgo% and 0 (by difference) * 29.65%; and when we pellet it in
 EMI5.2
 potassium bromide exhibits caraotdviatic absorption in the infrared region of the spectrum; as shown in Figure 1; and mixes them with said acidic substance, referring to the drawings
Figure 1 is a plot of the infrared absorption spectrum of notomyoine, free acid, when pelletized in potassium bromide.



   Figure 2 this a curve of the spectrum of absorp-
 EMI5.3
 tion in the ultraviolet of notomyoine in solution in ethanol, 0.1 N dbna H01 and in 0.1 N NaOH.



   The Applicant further provides, in accordance with the present invention $ the method of producing a
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 antibiotic substance called notomyoine, which consists in cultivating a layer of tr, omy "itbr, n8ft in an aqueuna carbohydrate solution containing a nitrogenous nutrient, in an aerobic immersed medium,
 EMI5.5
 until 1ptte activity towards Fat-positive bacteria is imparted to this solution, and notomyoine is recovered from this solution.



     The organism producing the antibiotic of the pre-
 EMI5.6
 This invention is isolated from a soil sample collected in the island of Richirit Hokkaidot Japan, and it con-

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 EMI6.1
 identifies a new species called tretoilo's ish1, n! Lot of the genus Strectomyces. A culture of the living organism, to which has given the laboratory designations 404 y 3 and A 979 ;, has been deposited in the American Type culture Collection, in Washington, D # 0.9 Biais-Unie 41 AM4- rique, and has been added to its permanent collection of micro- ', Organisms $ OR the designation of A.Iri.0.0j, 14812.



  The characteristics of the mouth representativet. 404 Y 3, from 8. rishirenais. are the following! PAHA0T8RIOTIQUBB. PB OUX / PUft $.



  C a growth 6 '- soluble pigment A a Aerial mycelium B a biochemical property 1. Czapek agar moderate, yellowish gray. to light brownish gray, or ivory yellow At poor, powdery, white S: none
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 2o u of Oeapek at moderate cg, yellowish gray or gyyrool light brownish gray to pale yellow with slightly pronounced brown A; mediocre, powdery, white S: none or light yellowish brown not very pronounced
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 3. Asar with glycerol and nice moderate bone, from yellowish gray to clear ammonium to yellowish white
A: scarce, powdery, light yellowish gray to light gray.



   S: nothing
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 4.Ear to glucose and ot poor, thin, shiny, asparasin Light yellowish gray

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A: none
S: none 5. Agar @ 1 .starch C: good, with pale yellowish cries. ivory yellow
A: moderate, powdery, not spreading, from washed brown to light brown with gray
S: nothing
B: hydrolysis is moderately strong 6. Nutrient agar C: moderate, burnt umber to brown
A: mediocre, powdery, white.



   S: dark brown 7. Bennett's Agar C: moderate, from greyish olive brown to greyish brown
A: moderate, from white to light brown to brownish gray
S: yellow brown, S: flour agar C: good, yellow pays with oat brown and boyton wash
A: moderate, white to gray with faded brown
So pale yellow with faded brown
9. C apple cap: good, shiny, earthen surface wrinkled
A: none
S: the plug turns brownish black 10.

   Pitting on gelatin C: brownish black colony on the surface
A: none S: dark brown
B: negative liquefaction

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 11 Sting on gelatin at C: brownish black colony with tyrosine yeast surface
A: none
S: dark brown
B: negative liquefaction 12, Milk Ci formation of a brown ring * tre
A: none
So faded brown
B: undigested 13. Nitrate solution Ci white granular mass on the surface
B: reduction to positive nitrite.



  14. Training media C: aediooric, thin, grayish melanin black
A: rare, blano
S: brownish gray to greyish brown
B: melanin positive, 15. C dextrose agar: good, shiny, dark brownish potatoes
AT:

   moderate, white to pale pink to pale brown with washed hair
S: shout or shout hot.

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 EMI9.1
 muotillsàej ± n 9, .o 2.0mge of oeyipono
 EMI9.2
 XIVVI-000 ++ Inulin t "t 'Arabiaose ++ salloine Olucont ++ Manaitol Qalaotone ++ Sorbitol Fructose -' ')' 0 <llobia Scrbose Rh & cmc'Nt r + sucrose + <- boa citrate = 1; aai ** 8uoo , uxtr de sodlua -t- Laatoee - * "'- lucaitol + # Raffinons + * Witness
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 ++ m good growth + fair growth t * questionable growth
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 - M no growth
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 paraatg'8 molq1Qu. The morphological properties of the strain were observed on starch agar and on Bennett's agar.



  Microscopic examination of the aerial mycelium reveals entangled and branched hyphae, sometimes in tufts, and aporophorea which produce sinuous spirals. On examination under a microscope the spores are found to have a
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 elliptical to oval shape and smooth surface *
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 3Gratomvcer 41a Y 3 has an aerial mycelium
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 brownish gray ot it produces a brown or dark brown pigment on organic media. The liquefaction of gelatin is negative both as a prick on the gelatin and as a prick
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 on Sdlatine with tyrosine and yeast, and it shines
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 is not digested * Nitrite forms from nitrate and starch is hydrolyzed.

   Strain 404 Y 3 resembles
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 di, rnta.nclrō. to 8. sriaooruber, to 8. r al1voohro,:, m ('} fenop, to S. nUl "!: 1Ut., b to U. r; rifJoochroItIO [Emo .1 to t3. ha-

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 EMI10.1
 waiiensis and 3. nasanish & some points of view, such as the formation of npiralou, the color of the aerial mycelium and the pigment m61anotde, but it differed from it in certain cultural and physiological properties, as indicated below. didd'tgS :, According to Waksman and Ourt1s, the aporophorea are straight and according to Jonsen, it forms sin-
 EMI10.2
 strorsua. The color of the aerial mycelium is indicated to vary from white to ash gray on Osapek's act and is
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 gray on Q8ar with glucose and asporagin The bite quifaction in gdiatin is quite rapid.



  8 * ¯.iip; riseoru'bert It is stated that the spores have a cylindrical shape. The color of the development is reddish orange on Ozapek's agar and adorned reddish olal to reddish purple on starch agar. Differences in the use of carbon sources are also found.
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 such as sucrose, raff.nvdd, citrate and 8uo01- matte S "oli .."! 9ch.t'Qrnor: en.



   Differences are found in developmental color, soluble pigment in glucose and asparagine agar, and in the digestion of gelatin and milk.
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  S. jaureus Developmental color is dark brown on Ozapek's agar and gold -. AnsÓ light on 3.4aaar on glucose and asparagine. The gelatin liquefies quickly and then slows down later.
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 grieQOahrçloe: lJne The color of the aerial mycelium ranges from white to gray

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 EMI11.1
 clear neutral. The liquefaction of gelatin is .04 '' ..



  Milk did not coagulate. Differences were also found in the use of carbon sources * such as rhamiose, inulin, salicin, citrate and auccinates. Hawaiians ta The color of development on glucose aga * and Aspuraaine is light brown, and aerial myoeus ranges from blano to gray. Ditrivenota are also found in
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 The use of carbon sources such as sorbitol and
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 the l3uoo: l.nate. ti's., n2, eiiniuh.à The color of development on igar & starch is cranium with rouseutte violet and the soluble pigment on starch aga est- rosy weak: *, Gelatin this mode- retains liquefied milk is coagulated and poptonjui. Differences are observed in the use of carbon sources, such as acarOBe and sorbitol.



  In order to choose Bites with high productivity, we proceed to irradiation of pure ultraviolet light in the primitive strain and we choose mutant colonizers. One studies uycolofjiquamont among these layers cer tuina dpjj-lua strong producers, but one does not observe any
 EMI11.4
 notable difference except that we get something. touches of white aerial mycelium.
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  In view of the above-mentioned characteristics of this strain, it was decided that Utroptomyces 404 Y 3 eon "caused a new outbreak and it was designated 8treptomyees Ziahirîenais nov. Sp.



  The species UtreptoNyeea described here-% dot includes all strains of streptomyoea which form

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   , notomyoine and which cannot be clearly
 EMI12.1
 , 41 * f ereneées of the layer Ni 404 Y 3 and its aoufaulturea,;

  including mutants and variant $$ The properties of netomycin are deopiton ioi-inûrae and, although these properties are known, it is easy to differentiate notomyoine-producing strains from others.
 EMI12.2
 The., Of3-tjoi, vitncigt cultivated under suitable conditions * product of notomyointo A fermentation broth containing notomwine is prepared by inoculating sports or myoelia of the organism producing notomyoine in a medium. appropriate, and then growing under aerobic conditions. It is possible, to produce notomyoine,

   to grow on solid medium, but for large quantity production, liquid medium is preferable. The temperature of the culture can be varied over a wide area, between
 EMI12.3
 20 and 550 0, in which the organism can and grow, but a temperature of 26 to 300 0 and the pH being substantially neutral are preferred.

   In the submerged aerobic fermentation of the body to produce notoayoine, the medium contains as a carbon source a commercial clyeeride oil or a carbohydrate such as
 EMI12.4
 ClyC6rol Glucose, maltoses, sucrose, lactose, dextrin, starch, etc., in the pure state or in the raw state, and as a source of nitrogen an organic substance such as soybean flour, produces them distillery solubles, the.
 EMI12.5
 peanut flour, Craines flour:

   cotton wool, meat extract, peptone, fish meal, yeast extract, mate maceration water, etc. and, if desired, mineral sources of nitrogen such as nitrates
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 and ammonium salts, and mineral soils such as chlo.

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 rare sodium, potassium chloride and sulphate
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 potassium, as well as s-buffering agents like calcium carbonate or phosphates and traces of heavy metal salts;

   these constituent elements of the medium include those listed in Canadian patent 513,324 and in the British patents
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 N 730,341 du and 736,325 du and in the patents of the United States of America J. 2,691,618 du, 2.6S.t7,8 du, 2,653,899 du, 2,586,762 du t 2,516 * 080 du, 2,843,892 du 2,609,329 of, and 2,709,672 of. His culture
 EMI13.3
 aerated submerged, an anti-munse agent such as liquid paraffin, fatty oils or a silicone is used.



    More than one kind of carbon source can be used for the production of notomyoine. Cultivation is generally continued until at least several hundred mcg / ml of notomyoine have accumulated in the medium. In some cases the pH of the broth decreases initially and then rises slightly.



   The following examples are intended. , illustrate this invention, but not limit it.



     EXAMPLE 1 The fermentation conditions suitable for the production of the antibiotic referred to as noto are studied.
 EMI13.4
 myosin and the following composition of Medium. is useful in shake culture 1.5% soluble starch, 2% cottonseed endosporm meal (Fharaamedia), 0.1% K2HPOlf .. 0.1z laOl, 0.05z5 * % 6 (> v 7 8201 0.05% Oa012 '0.001 i ZnS04t? H20 and 0.% yeast extract.

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   Notomycin is digested within 3-5 days of fermentation with adequate aeration and agitation, and activity is found in both the broth filtrate and in the mycelium. We dose notomyoine against
 EMI14.1
 taphylococcuB aurus IDA 209 P by the paper disc or cylindrical plate method.
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 example Jo The submerged aerobic fermentation of S. ri8hirie & - 'aie (strain 404 Y 3) in shaken flasks, in a medium containing 6% starch (Staclipae J), 3% brewer's yeast freed of its bitter ingredients and 1% of Oa003 'gives 300 & 400 raog / m3, of notomyoine.



  EXE! .Fi) LI 3 The submerged aerobic fermentation of, 8 "," iahir, i ": sis (strain 404 Y 3) in shaken flasks, in medium containing 6% starch (Staclipae J) 3% flour of-
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 cottonseed sperm (Miarmamedia), 215% soluble distillery product and 1 tt of OAOO gives 150 to 200 mcg / ml of notomyoine.



  . & JXE1 \ ÍPLE 4 The submerged aerobic fermentation of, r1ship1n1J! (strain lez Y 3) in shaken flasks, in a medium containing 1.0% soybean meal, 2% cottonseed endosporma flour (Pharmamedia), 2.0% soluble starch, 0.5 % glycerin, 0.2% yeast extracts, 0.2%
 EMI14.4
 K2 "PO4 '0.1% MgS04' 1.0% OaOO, and 0.05% defoamer silicone, gives pU?, 0 of # ¯¯¯¯¯¯ .... rtaGuya. ao of the following rauniôre!

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 EMI15.1
 Duration Iili z
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<tb> 5 <SEP> (days <SEP> 7.8 <SEP> 70
<tb>
<tb> 6 <SEP> Days <SEP> 8,1 <SEP> 80
<tb>
 
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 7joura 8t3 120
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 mM? .mm pnltituo.nts d1fmW!. \ 1 '/ Soybean marinates 2.0% Glycerin 1.5% Yeast extract 0.2%
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 Dipotassium Phosphate 0.1.



  0.1% sodium chloride
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 Calcium chloride 0.05% Magnesium sulphate 0.09% Zinc sulphate 0.001% pH 7.0
 EMI15.7
 100 ml of a culture containing the above components are sterilized in a 500 ml flask, this medium is inoculated with a seed culture of the sri2top to rit3hirioU strain 404 Y 3 and cultured at 27 t 10 0 for 6 days by shaking it, whereupon the formation of nototayoin in the fermentation broth reaches 150 mes / mi.

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 EMI16.1
 



  9 = 110LIC - - 9.ens1ii tum} tar du uIpilioM Cottonseed endoapcrme flour (Pharaaaodia) 2 0 S Soluble starch 1.5% Yeast extract 0.2%
 EMI16.2
 Dipotagoic phosphate 0.1% Sodium chloride 0.1% Calcium chloride 0.05% Magnesium sulphate 0.05% Zinc sulphate 0.001%. pH 7.0
100 ml of a culture medium containing the above components were sterilized in a flask with a volume of 500 ml, inoculated with a culture of on-
 EMI16.3
 seed of 3treptom, yceg i'Jiah1tqnu11 404 Y -30 (which is a mutant strain of 404 Y 3 obtained by irradiation
 EMI16.4
 with ultra-violet rays),

   and cultivate it at 27 t 10 0 for 9 days by shaking it, whereupon the production of notomycin in the fermentation broth reaches 400 mog / ml,
 EMI16.5
 30 liters of a culture medium containing the same components as in Example 6 are placed in a stainless steel tank with a volume of 50 liters, inoculated with a seed culture of LtÙ2m.Ug2 rishiriansis and it was grown at 28 × 10 0 for 1 hour, whereupon the production of notomyoine in the fermentation broth reached 120 cog / ml.

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 EMI17.1
 



  5 liters of fermentation broth are filtered
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 PU 8.0, adjusted to 6to 1 * pHfiltrate and extracted
 EMI17.3
 with 2 liters of methyl isobutyl oetone. We extract the
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 Cake c'11.n with JK> 0 al acetone with vigorous stirring, and the all drained extract is evaporated to remove the solvent. The resulting aqueous concentrate is extracted at pH 6 with koO ml of methylioobutyl ketonoi. The two ketone extracts are mixed, washed with 500 ml of water,
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 then we run the activity donates 1000 liters of water
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 cold whose pH has been adjusted to 10.0 with hydroxide
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 sodium. The pH of the aqueous extract was adjusted to 6.0 and
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 we extract it again with OO ml of ethyl acetate which we empty to 30 ml.

   Zn adding 150 ml of ether
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 petroleum in concentrate, a light brown amorphous precipitate appears} it is collected, washed with small
 EMI17.10
 amount. of methanol to remove colordes impurities and it is dried, which gives 750 mg of notoayaine under
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 form of a powder.
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  400 mg of the powder de'notoayoine thus obtained are dissolved in 100 ml of ethyl acetate and adsorbed on a column of a0 g of alumina previously treated with sulfuric acid. The column is washed with 200 μl. ethyl acetate and eluted with methanol. We collect '*
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 the eluate in fractions of 10 ml, the fractions are mixed
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 active and they are concentrated uouo empty up to o1oQ1t6. We reCtiStUllISe notomyoine twice in methanol
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 solid of pale yellow color thus obtained, which gives
 EMI17.16
 70 g of crystalline uotomycin blfJnùhJ, which shears. 218-210 * 0;
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 by decomposing.
 EMI17.18
 



  The notouyoine was further purified by the Oraig technique of distribution b counter-ouut using a

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 solvent system of 5 parts (by volume) of carbon tetrachloride, one part of chloroform, five parts of methanol and one part of water. After 50 transfers, the maximum concentration is found in tube N 26, by the biological test curve, by the ultra-violet test curve and by the weight curve; this * curves agree with the theoretical curve.

   By freeze-drying 10 tubes around this maximum, pure notoayoine is obtained.



   Notomyoine was also purified by recrystallization from aqueous acetone followed by absolute methanol to give a substance melting at 220 0 on decomposition.



   Notomyoine was also purified by alumina chromatography, in which 39.7 g (activity 500 mcg / mg) was dissolved in ethyl acetate and adsorbed on 2 g of Al2O3 (pH 4, 7, treated with H2SO4).



  After washing the column with ethyl acetate, it is eluted successively with methanol and then with acidic methanol. The active fractions were mixed, neutralized, concentrated in vacuo and then lyophilized to give 12.4 mg as a pale yellowish powder (activity 700 mcg / mg);
480 mg of notomycin are treated with a small amount of activated charcoal in scetonic solution, then recrystallized from hot ethanol, which gives 230 mg of crystalline notomyoine, which is dried under vacuum for 3 hours at 50 ° C. .

   Melting point 222-224 0 (with deoomposition); pKa 6.32 in 75% acetone. Equivalent to titrations 526.5 Analysis. Calculated for C26H26N2O10: C - 59.35 H - 4.98 N - 5.32 Found: C - 59.1 H - 5.35 N - 5.90

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 Molecular weight determination by
 EMI19.1
 the ovysooopic method in dioxane gives a value of 500 20. te specific rotatory power $ & pha7 | is -134 $ 40 (0 - 1., acetone), in C., ara1.oA with -69.6 'for novobicein under the same conditions. Notomyoine gave a negative reaction (brown) with anthrone, while novobicelne gave with anbron. a positive (green) reaction.
 EMI19.2
 



  A solution of 100 mg of aotoayoine in 120 ml of absolute ethanol is stirred with 0125 g of Adam's catalyst, under atmospheric pressure of hydrogen, pen- '. including 30 minutes at room temperature. Almost no hydrogen absorption is observed. After separating the catalyst, 35 ml of water are added to the filtrate with stirring.
 EMI19.3
 energetically. Adding to the 100 ml solution of normal hydrochloric acid precipitates a white solid product; we collect it, we dry it and we find that it weighed Sa, 7 mg, and that it is the original starting product,
 EMI19.4
 that is to say, noto01n., as confirmed by the 6bvoma- tographis on paper.

   Under the same conditions, 1,000 of novobiooine give 946 mg of dihydronovoblocinst as
 EMI19.5
 This is confirmed by the ohrOma'co! raph1Q.ue analysis on paper, in accordance with the OF.J. Wolf et al., Antibiotics Annual, 1956-1957 # page 1035).



  Notomyol gives positive Pahling and Moliboh reactions and clogs bromine. The reaction with ninhydrino # the reaction of (Pollens and the reaction with anthrone are negative.



   The paper strip chromatography of the
 EMI19.6
 notonyoine gives the following results!

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 EMI20.1
 Moist M lutanol Vnlaur Solvent System 0.15 Ohlorurw 9S Aqueous Solution
 EMI20.2
 
<tb> ammonium <SEP> 0.03
<tb>
<tb> Phenol <SEP> to <SEP> 75 <SEP>% <SEP> 0.95
<tb>
<tb> Acetone <SEP> to <SEP> 50 <SEP>% <SEP>. <SEP> 0.75
<tb>
<tb> Butanol-methanol-water <SEP> (4: 1: 2) <SEP> +
<tb>
 
 EMI20.3
 2% methyl orange ## 0.45 Butanol-methanol-water ($ 4 192) 0.40 Benz6ne.-methanol (4il) oto5
 EMI20.4
 
<tb> Distilled <SEP> water <SEP>. <SEP> 0.05
<tb>
 
 EMI20.5
 Butanol-water (4:

  1) + 0.25% p-tolueneaultonic acid 0.30 ButaNol-water-aoetic acid (21111) ot5o Butanol-water (4tl) + 2% pipdridine 0.25
 EMI20.6
 Notomyoine dissolves ta1lam.nt dani 1 '& 0'- tonot dloxanno and alkaline water, they have aoderately soluble in 1 $ 6 ethanolt butanol, ethyl acetate butyl acetate and methyl ketonal i8obu1 it this minus 80Juble in bonsènet, môthamol, and chloroform, and it is insoluble in carbon tetrachloride, petroleum ether, and acidulated water.
 EMI20.7
 As shown in Figure 2, notoayoin exhibits an absorption spectrum in the ultra-violet comprising the following maxima
 EMI20.8
 Maxima in ethanol zon5 mil11m1cronl - (3 | Jft m 595) 335 ml111m1orons (1 i JR * 498) Maxiaa in HOI 1;

  / 10 275 millimiorone (1 t: a. 287) 345 milllmiorona (1: .. 277) Maximum in NAOU H / 10 280 mil11miorona (0 c "766)

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As shown in Figure 1, the notomyoine, pu * -
 EMI21.1
 used in bromide d. p.taS / sima, presents l1U1X1111.1. absorption characteristics in the infrared at the following wavelengths, expressed in microns:
 EMI21.2
 '2.99; 3.36 ('paul.mont). 3145; 5.88 (shoulder); 3.92; 6110; 6.24i 6 $ 451 6 ,; 6 #? 17.2; . -7.63; 7s85l 8.9} 9t2 $ 9.6; 10 # 05; 10.3! 10.6; 11b> i 12 # 251 12.65; 1> #I; 13.39.



  Thus notomyoine and novobiooint diffe "easily, for example by the melting point, by elementary analysis, by the specific rotatory power,
 EMI21.3
 by absorption 1.ur.piQtre8 in the ultraviolet and in the infra-red, by the reaction with anthrone and by their behavior in chromatceraphit on paper bunde.



  Notomyoine is an acidic organic compound which is readily soluble in alkaline solutions, such as aqueous solutions of alkali metal hydroxides and
 EMI21.4
 alkaline-earthy. For example, a solution of nataayoisre in an aqueous solution of sodium hydroxide or calcium
 EMI21.5
 oium forms respectively I alone of sodium or calcium, which can if desired be isolated, for example by lyophilization.

   Salts are formed in an analogous manner with organic bases; the addition of streptomycin or
 EMI21.6
 from dihydrostreptomycin bases to netoo1n. respectively forms the streptomycin and dihydro-streptomycin salts of notomycin; it is convenient for this reaction to use an organic solvent such as ethyl acetate. Other amine sols which can be used include those which are used therapeutically in all form of salts of penicillin, for example procaîns,
 EMI21.7
 of N-bansyl-bata-phdndthylamine, of hydrabamins, of phenomine. of N, N '- <Hbons: yl6thylenediaroine, dehydroab16tamine

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 and dicyclohexylamine.

   The acidification of an aqueous solution of the sodium derivative of notomycin is removed to precipitate the purified notomycin in the form of free acid.



   A preferred method of isolating notomycin from fermentation broth is to extract the entire broth at pH 6.0 with half of its volume of methyl ieobutyl ketone, to be re-extracted at pH 10.0 in liquid. water (a quarter of the volume of the methyl isobutyl ketone phace), and to be re-extracted at pH 6.0 in ethyl acetate.



    / the or in methyl isobui-yl ketone (one third of the volume of the last aqueous phase), The final concentrated solution of notomyoine in the organic solvent is then concentrated by vacuum distillation to tenth or twentieth of its original volume. The notomycin is coated on precipitated and if it does not precipitate spontaneously, adding mixed lower alkanes, for example by adding 10 volumes of Ukellysolve B.



   The notomyoine is also easily recovered from the broths filtered by adsorption on activated charcoal (Daroo KB), then by eluting the latter, but this way of operating is not superior to the extraction of the total broth. with a solvent, since in many broths most of the notomyoine is in the mycelium.



   Microbiological studies them- Notomycin antibacterial activity in vitro
The minimum inhibitory concentration (MIC) of notomycino in varied microorganisms was determined by the dilution method or dune agar, using horse arm agar for streptococci and.

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 EMI23.1
 the tiresNoooquea h'olft1qu,

  ut of jeagar with SJUOOO * T with yeast extract and 1 lu peptone for the feaoillts 140tiquois nutritive agar for other baot'r1. "from Ilagar de Sabouraud to 2 Glucose for the ohamp1SAoni and broth of K1rhn.r for tuber- culosis bacilli The results are collated below in table format $ Same as those obtained for uovo.bioony, which was used as oopara18o.

 <Desc / Clms Page number 24>

 
 EMI24.1
 



  -et 1% lAiiufriori ien tJr ani som oUt (tac., 1 'jDrfianlBm da l 8fll tpNMim ## MVMM G1 "am-nl.hsatl.t.



  Racherichia ooli NIHJ eup. & 0 ldilin M1495 aup. To 50 90 Kl'ébâlplia tlttuütal7.t, l Type A 5 13 *? #? a "? tt'1 I! I¯t xl t3 Ï, 1 ± 1. tix7. t 6,2 'riexnorî lup.à; 0 6, a, 1.' 5CifJOlfM A stip at 50 5 (j "i'fl \ op. L-1î) tbup.é 0 12, 'àgr2lialli felt in aup at 50 <t) p. & P0 ####### ji'2UU 4bly Pceudor.10no.o 116.L'u! ': InODI aup.à 50 <upt & 50 Vi'6rlo "v IlwtchTIi1'kovi1 1 U 0.025 0.1 Orwn-polJl tU' leDx-2D9p 0.003 0.049 f3. Aurleus iDA 209P ; 3: u1, UT-il) 0.003 0.049 â 'clult3ua FDA 209P (UB-H) it56 6t23 St aureus 52-34 <T0, 14, CM, P0-R) 0.003 0.1 t'7' hUr'l '\ U3 Siaith utrain 0.006 0.049 ST aureua 193 (pot Ta-11) 0.003 0.049 ST nurcu3 193 ÇPO 1' C, t Ùt OM-R) 0.003 0.1 ST aurou3 193 PO, TO, Ï34-R) 0.003 0.049 ST 3ur6uo Terajima 0.003 0.012.



  Ir. ' 91bu; l "0.006 0.049 rrcrococcua elavus 0.006 0.049 .'C I11 logea lot 1001 010.5 0.1 i1e SLl) '. 4' 219 0.1?. SpherrEs 122 0.78 0.1 0 mycoides seen ()" ot? 8 0.39 r cereùs ÀTQG 10702 3. 5 0.76 TT 'unthr'1ci t 115 0, i6 0.1 r? Ctol)> jcï "nu8 cosel .ti'00 769 ########### IUP $ À 50 0.1 L . aGic3orihiina B-406-1 eup. to 50 0.1 ro'i) 'tbco.ccu9 fflgc.alla mt "Óptococua' ttlCZSl 8 = 55205 3 $ 13 0.39 S. ²YOfpne ± 1 Typo z 0.78 imrinvoccut3 pnouiiion ne 0 Il it 156 0 78 t '' G t: r 111II1 W'y'1 Lycobactcgluia pe 50 12.5 Fungus AstHrr: i 11u2 nigex; yan U2Zhec% 'rril up. aup.à 50 Nup.à 50 1'enicilliulIl M aupoà 50 eup.t 50 candida "al.V.1 cr tia aupià 50 tUptà 50 Sacçharoiayc 9 a ceravik3ingL tacçharoYcQ 7.'MC'97 8up.A 50 aup. à 50 frlchophytpq F! enta. (r t'l1: L-te! supea 50 t; up.à 50 Abbreviations t Iî # resistant PO t p'n101111n.



  OR . ntI'optomyciDe EU. r) 'th%' omyc1ne ST tstreptothrloine OU * 04rboo1n.



  NO a noôbioo1ne 2C tûtrnoycl1ne

 <Desc / Clms Page number 25>

 
 EMI25.1
 The minimum concentration * iDh1- bïtrîceo of notomyoiM was also determined by the method of
 EMI25.2
 double dilution in tube, working with nu- broth
 EMI25.3
 tritif, except for streptococci and pn.umooo, u8. flat) 1: 1cl'l. \ ", which we try to have a boil10D ... of 1Dtua: Loza. of brain and heart.

   The results are grouped below in a table, together with the ratio 4'aot1v1t 'of the noto1n' and of the novobiooinet t9..pR ....... omA.a.rhc.,. PM. at the
 EMI25.4
 Org1etlu,! BSa !! p9YCIn, .lJpQ;: i Klebsiella pneumoniaq 3.15 og / al 6.29 og / sal 2 "'" "" "#'" "" ihs "y9p 0.0019 0.049 52 sïêh :, oCOOQU eraj1m & 0.00075 0.012 StMhyloooQoua aweus r Stehx220QUUI itn- a 0.0015 0.024 16 ffayqinfl luq POl 1001 0.006 0.049 0 ^ x ,, aat 0.0007 '0.024 53 "K-1IU 0.00075 0.024 Baollluc oereui ATCO ... 10702 0.78 0.78 pso11us enthroi8 115 o, 9 0.199 1/2 '"ype 3 0.049 0,' 9 8 R10900uO oUliat! J <1 l 0.098 0.098 1
 EMI25.5
 Lu notomyuina brr9tar development! bacterial dot diveratw 0: L1U1I1-pou1UvOQ, except for 13 ,,, b. ,,: 11! ot lactic buo111oD, which are doniblen to novobiooine.

   Lu l1otolll; yclnlt out part1ou116rowont activated against leo atuPtlYlocuclude this uotivity being 10 in

 <Desc / Clms Page number 26>

 
 EMI26.1
 30 times greater than that of novobiocins However, it shows reduced activity towards * atapb71o-, cocci which have been made resistant to novobiocin in the laboratory.
 EMI26.2
 



  XntlMen2e- de ula JU-11 ggaaegu: 9n tnibttr9 Mipimal! Using (Smith strain) and 6taphth aureus (strain 193) as the test organism * in nutrient broth, dilutions were made.
 EMI26.3
 tubes with various dimensions of inoulua, The results $ of which gathered below in the form of 'table and it can be seen that the influence of the important inocula is similar to that obtained with novobiocin.
 EMI26.4
 



  Influca, de, the dIncMi on inooulum on lft .. ,, O, | ij [
 EMI26.5
 Onanism lm ce of Ilicoeulum (cell / rn) test AatioiotiQ XO ', 10 10 LO 4 10g 10 (019 a Uog./MX.)
 EMI26.6
 
<tb> S. <SEP> aureus <SEP> Notomyoine <SEP> 0.012 <SEP> 0.012 <SEP> 0.003 <SEP> 0.003 <SEP> 0.003 <SEP> 0.003
<tb>
<tb> Smith
<tb>
 
 EMI26.7
 elderberry Novobiooine 0.39 $ 0> g otl95 0.098 0.098 0.098 amith 8.

   Aurella Uotomycin 0.024 0.003 O, OOJ.; O, 001.5 O, u ,, "0.0015 etrain it aurtug Novobigoine 0OV8 0.195 0.098 0.098 01098 0.098
 EMI26.8
 
<tb> strain <SEP> 193
<tb>
 
 EMI26.9
 Fluenpe mil i or on Minimal Inhibitory Oonoentgation
 EMI26.10
 J: i Jl'tl'l
The MIC of notoayoin is determined by the broth dilution method, the pH of the media being
 EMI26.11
 adjusted to various values.

   Nutrient broth and brain and heart infusion broth are used as water media and a dilution is inoculated into each tube.

 <Desc / Clms Page number 27>

 
 EMI27.1
 10 ** 4 'um cultur. of 16 collided with Btpun. aureue Smith (105 c.ll./#l). as can be seen C1-480 'OR' the activity of notomwine increases in acidic pH and decreases in alkaline pH. The comparative results obtained with novobioocin indicate that notomycin is influenced by the pH of the media to a greater extent than novobiooine.



     Influenced medium pH on CIM
 EMI27.2
 Not9oin â2voblo2ipl pp du .. IM and tcg./ar1.



  Medium N 'i3Ca HN ln
 EMI27.3
 
<tb> 6.3 <SEP> 0.0001 <SEP> 0.00005 <SEP> 0.024 <SEP> 0.024
<tb>
<tb> 7.0 <SEP> 0.003 <SEP> 0.006 <SEP> 0.098 <SEP> 0.098
<tb>
<tb> 7.6 <SEP> 0.049 <SEP> 0.098 <SEP> 0.195 <SEP> 0.39
<tb>
<tb> 8.2 <SEP> 0.39 <SEP> 0.78 <SEP> 0.78 <SEP> 0.78
<tb>
 
 EMI27.4
 605 .r5 '3113 1.56 .6 - BN - nutrient broth BCC = brain and heart infusion broth
 EMI27.5
 1 & .tlll2nge., D! L, mllllâ, jsur Conaentpation Inhibitrioe tf1tql !.



     This influence was investigated using four species of media 1 nutrient broth, brain and heart infusion broth (Difoo), tryptioase soy broth and 1% yeast extract broth.

   The OIM is not influenced by the media used for the determination, as long as the media fold is controlled.
 EMI27.6
 nt uennu 8! Ium our OoX \ q, otrntion Inh1bt1'1o. lUnil11) 10 Concetitrations are added cross-content of serum

 <Desc / Clms Page number 28>

 human to nutrient broth containing a phosphate buffer at the final concentration of 1/10 M, to maintain the pH of the media at 7.2.

   We use as a boredom organism
 EMI28.1
 of dt2l) hyloopc22.o auèt the dil1l.o! U.ion of Ilincoulum being 105 that / ml @ Corne indicates it Io table oi-deesoue, the 011-1 is clear Marnent influenced by the serum Influence, serum on the CIM
 EMI28.2
 CIL en mok; olmlb xonoentr! Tion of the sacrum pot9myo1n bi22ln2
 EMI28.3
 
<tb> without <SEP> serum <SEP> 0.003 <SEP> 0.098
<tb>
<tb> 2.5% <SEP> 0.024 <SEP> 0.195
<tb>
<tb> 5 <SEP>% <SEP> 0.098 <SEP> 0.195
<tb>
<tb> 10 <SEP>% <SEP> 0.098 <SEP> 0.39
<tb>
<tb> 20 <SEP>% <SEP> 0.098 <SEP> 0.18
<tb>
<tb> 50 <SEP>% <SEP> 0.195 <SEP> 3.13
<tb>
 
 EMI28.4
 ot1té d! notomyaine against stqoQUS ftléj. \ U:

  Tfü Q \ 1emfJn Various cultures of Si1! .. ur'l (1 *> strains), isolated from clinical sources, against notomycin and six antibiotics of comiuerce 1, namely bontylp6nieillinst ten1-dxoutreptomyoîne, are examined in vitro. , tetracycline, ilerythromyoinel, kanamycino and novobiooine.

   The MIC values are obtained as follows by serial ten-fold dilution of the agar:

 <Desc / Clms Page number 29>

 
 EMI29.1
 Penalty of Isolated Clinical Cultures (etiaphyloqqquea! Ja jaajtom'yoine, a <: nmc 6ntibioin'ueB co
 EMI29.2
 , A.nt'U.otlaux 019 ta-àxlm, 1'Qmrçi1J. gag .ami :: g, 1:

  1 read at 100 0 0 3 0 0 0 0
 EMI29.3
 
<tb> 100 <SEP> 0 <SEP> 1 <SEP> 16 <SEP> 2 <SEP> 1 <SEP> 0 <SEP> 0
<tb>
<tb>
<tb> 10 <SEP> 0 <SEP> 7 <SEP> 103 <SEP> 0 <SEP> 5 <SEP> 0 <SEP> 1 '
<tb>
<tb>
<tb>
<tb>
<tb> 1 <SEP> 0 <SEP> 89 <SEP> 2 <SEP> 8 <SEP> 117 <SEP> 123 <SEP> 0
<tb>
<tb>
<tb>
<tb> 0.1 <SEP> 1 "<SEP> 21 <SEP> 0 <SEP> 114 <SEP> 3 <SEP> 3 <SEP> 122
<tb>
<tb>
<tb>
<tb>
<tb> 0.01 <SEP> 52 <SEP> 7 <SEP> 2 <SEP> 2 <SEP> 0 <SEP> 0 <SEP> 1
<tb>
<tb>
<tb>
<tb> 0.001 <SEP> 48 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 2 <SEP> 0 <SEP> 2
<tb>
<tb>
<tb>
<tb>
<tb> 0.0001 <SEP> 21 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0 <SEP> 0
<tb>
 
 EMI29.4
 0.00001.

   1 "4 0 0 .0 0 0 0 same strain
 EMI29.5
 PO Benzylpenioillin DU Dihydrootreptomyoint 10 Tetracycline Eu Erythromyoin KM - kansmycin
 EMI29.6
 NB Novobiooie These results indicate that KOto # yoin is extremely active against * staphylococci and that it does not have antlgon1.t activity. wine vis-à-vis other antibiotics, only one strain in 126
 EMI29.7
 strains, resistant to novobioony, and which is less sensitive to notomyo1ne. but the IMO value of notomycin for this strain is 011 mog / mI. i-q - viv2-.ÊUL. lu notosycin Toxicity
Notomycin is a weak antibiotic
 EMI29.8
 toxicity.

   It was found that the LD50 by the intramuscular route is 500 ag / kg for mice, either druue or has been studied in vivo nativity of the antomyoine.

 <Desc / Clms Page number 30>

 in the stable, against infection, experimental by Bouche de Smith. We infected
 EMI30.1
 smiled intra-peritoneally, with 100 liver the LD 50 of the pathogen and the antibiotic was administered intramuscularly or perorally, immediately after the attack
 EMI30.2
 bacterial.

   A single intra-molar OD 50 value (50 / c of the curative dose) of 0.3 mg / kg # and a single peroral OD of 415 Mg / kat was obtained In = comparative trial, we found than intramuscular and peroral values
 EMI30.3
 of OD5, are respectively 6.0 mg / kg and 30.0 mg / kSI The results are shown below. Chemothtapeutic effect of notoM.yne 'X'tlp., UtiJ1! a intramungulaire
 EMI30.4
 
<tb> Dose <SEP> in
<tb>
 
 EMI30.5
 57 - s JOVOItY $ 'nt {9yoQç! Nt
 EMI30.6
 
<tb> 50 <SEP> 5/5 '<SEP> 6/6
<tb>
<tb>
<tb>
<tb>
<tb> 25 <SEP> 5/5 <SEP> 6/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1, <SEP> 5 <SEP> 5/5 <SEP> 4/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 6.25 <SEP> he / he <SEP> 4/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> ', the <SEP> 6/6 <SEP> 1/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 1,

  56 <SEP> 6/6 <SEP> 0/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0.78 <SEP> 12/12 <SEP> 0/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0.39 <SEP> 4/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0.19 <SEP> 1/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0.10 <SEP> 1/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> 0.05 <SEP> 0/6
<tb>
<tb>
<tb>
<tb>
<tb>
<tb>
<tb> Witness <SEP> 0 / iL, <SEP> 0/12
<tb>
 
 EMI30.7
 * Number of ourla iàyiuit lost / Number of "ourla infest

 <Desc / Clms Page number 31>

 
 EMI31.1
 Effect çhimiot, héàn2¯utiLug, of the 'Uot2rnyoîL r ligus nel2Eal2
 EMI31.2
 
<tb> Dose <SEP> on <SEP>
<tb>
 
 EMI31.3
 M & 4th Notom, yoin â2veblegln
 EMI31.4
 
<tb> 100 <SEP> 5/5
<tb>
<tb>
<tb>
<tb> 50 <SEP> 11/11 <SEP> 6/6
<tb>
<tb>
<tb>
<tb>
<tb> 25 <SEP> 6/6 <SEP> 6/6
<tb>
<tb>
<tb>
<tb>
<tb> 12.5 <SEP> 6/6 <SEP> 5/6
<tb>
<tb>
<tb>
<tb>
<tb> 6,

  25 <SEP> 10/12 <SEP> 1/6
<tb>
<tb>
<tb>
<tb>
<tb> 3.12 <SEP> 1/6 <SEP> 1/6
<tb>
<tb>
<tb>
<tb>
<tb> 1.56 <SEP> 0/6 <SEP> 0/6
<tb>
<tb>
<tb>
<tb> 0.78 <SEP> 0/6 <SEP> 0/6
<tb>
<tb>
<tb>
<tb>
<tb> Witness <SEP> 0/12 <SEP> 0/12
<tb>
 
 EMI31.5
 Notoayoine is a useful agent for the treatment of mastitis in cases of diarrhea in cattle.
 EMI31.6
 or calves; one leaves for example with this effect of ausponation. in a vegetable oil, to fall in * -Ion teat $ to treat mastitis, and containing from 1 to 1000 ag / ml, and preferably about 50 mg of the antibiotic, or exen
 EMI31.7
 .utt1oamm.nt of capeulec to Garnish a total dose of 0, to 2.08 for peroral administration, for example for diarrhea of calves.



   When desired for specific applications
 EMI31.8
 Oils and once they have been made pharaMeutically compatible, one can eat with the compounds of the present invention other drugs such as antihistani. nique $$ sulfonamides (eg sultadiatins aulfabentamide, eult.c'tù4., sultanilamide, .ult.p141n't aultathlacolt aulfapyratin, cult- suanidinst sultathalîdinob la tultaouxidint # null-

 <Desc / Clms Page number 32>

 
 EMI32.1
 tinoxasol, sultaM11on, phthalyleulfacetamidei U '.', 4-dimethylbenzoyl-Dullan11amide.

   the bQnI11-lullanrla-
 EMI32.2
 mido and the agents
 EMI32.3
 lipotropics (in particular the methionic agent ehlolne, inositol and beta-aitootdrol and their mixtures *),
 EMI32.4
 central nervous system stimulants (e.g.
 EMI32.5
 caffeine, amphetamines, local unenthenics, analogs (for example aspirin, salicylamid and sodium Sontisate, p-aoetylaminophenol, phenacetinst codeine), don s6dat1ts (pure example of barbiturates,
 EMI32.6
 bromides), benzyl penicillin sala (for example
 EMI32.7
 the potassium derivative of penicillin G, prooaTne penicillino 0, l-ÓphJnW: l1no penicillin 0, diboncylamine
 EMI32.8
 penicillin G, and others are described in the
 EMI32.9
 United States of Brick NO J.627.491 of;

   this*
 EMI32.10
 mix are particularly interesting to allow
 EMI32.11
 to vary the rates Quinquina obtained), ph'nox; ym'thI1- penicillin, In phanethieillins methicillin and oxacil- A * cloxoillin, natoillin, c4pho.lotb1ne and other penicillins tlYlltih.jt.1quo and their Mêla, others like untibiut.iqueo lhll1 oKcunplo lu streptomycin * dihydrOD1ireptOL, yc1ne, bacitracin, polyuixint, tyrothricin, tl 1 {; rhythNl, uyc 1ua chlorotütruoyullne, o.tl '; ncyc11nu, lu tutr'oyclinu, ol: u1 (1ol41c1no, cblormnph6nicol.

   In m.-iiiumycliio, 1: "wJyoù1oc1no, la oyolo'- e61'lno. Lu nl.om $ 1u (j. La i; ... IJC1ul, 'fIJin4.1; (I. & IIU oort.J1ho case these melUf'- ± 1; elJ abluquolit uno t;:. 1IJ1JJO pluo,) t <JnJuu Ù 'ol'gau1aw "to or else make proof of an ofl'1ow =! tJ s, yu <.Irt; é. -tiqUat. or have less toxicity z effioacitu ÓIl. & h), vitamins (for example u, A, ali 13it B, B t deM ruproawntttnta of this family, acid i'oUque ot roprécojitants of oatt @

 <Desc / Clms Page number 33>

 
 EMI33.1
 vitamin family cl, D2, D5 and bzz hormones (e.g. oortisone, hydrocortisone, 9 aûaw'xuara
 EMI33.2
 cortisone, 9 alpha-fluorohydrooortisone, predaisoaa and prednisolone), aobolic agents (eg, i ,, x7d, hydraxy ..alpha s.uara l alpha "methyl-4-aAdpostene-5-on6;

   17 ulpha-ethyl-19 nor-alloi3terons), and agnta antifungiquoa (eg myooatatin).



  The antibiotic of the present invention constitutes
 EMI33.3
 an ozont useful for detecting contamination by Qrum-n6at1veg bacteria, ahump, nane, yeast *, Ito, during the industrial production of the ensymot btreptokinase and streptodornase by culture of 3txeptooaquea and the production of amylase by fermentation of 'wsubt1l! or from. cereua. Thus, adding from 1 to 1000 mcg / ml and preferably about 10 mog / ml of the antibiotic of the invention to an aliquot of inoculated medium, followed by incubation, can be used. develop unwanted impurities and detect them with the naked eye.
 EMI33.4
 



  B..J.JL! LJ1JI A. A method of producing an antibiotic substance,
 EMI33.5
 referred to as notontyoine, characterized by the following points, taken alone or in combinations 1) A strain of trtPomY'1 1thi1 .: 11a is cultivated in an aqueous solution of carbohydrates denying a nitrogenous nutrient, in submerged aerobic environment, - until significant activity against bacteria
 EMI33.6
 arumpaaiivaa is communicated to said solution, then the notomyoine is recovered from the solution.



  2) The organism had the iltreptoluyope rblr1eOB '.



  A.TtO.C. 14.812.



  3) One aépure 1, nnt1b1ot! .11UO notomyaine from the broth

 <Desc / Clms Page number 34>

 of fermentation by extracting at an acidic pH of this insecticide using a solvent immiscible with water, in which said antibiotic is soluble $
4) The solvent containing the
 EMI34.1
 oto = 1Cin ..



  B. As a new industrial product, an antibiotic substance effective in inhibiting the development of Gram-positive bacteria, characterized by the following points, taken alone or in combinations:
5) Bile is chosen from the group consisting of
 EMI34.2
 an acidic substance, notomyoine, which is readily soluble in acetone, dioxane and alkaline water moderately soluble in ethanol, butanol, ethyl acetate, butyl acetate and methyl isobutyl oetone, less soluble in benzene, methanol and chloroform, and insoluble in carbon tetrachloride,

   petroleum ether and acidic water, which gives positive Fehling and Molisch reactions, discolors bromine and gives
 EMI34.3
 a reaction with ninhydrin, a reaction of Tollens and a reaction with the anthrone negative, and which in the form
 EMI34.4
 purified melts at 222-224 * 0, has an alpha 20de -134 * (0. 1.0, acetone), present in ethanol absorption spectra in the ultraviolet having maxima at 275 millimierons' ( m am - 595) and z 335 milliolierons (3 1 P. 498) 9 dan.



  H01 N / 10 of the maxima at 275 millimicrons (1 im. 287) and at 349 mil, im3, aranr (111 t or w 277) and having dirir fet3i N / 10 a maximum at te30 millimiorons (1 i: # 766) , a UA equi-! are worth of neutralization of 548 and whose mean elementary analysis is as follows 0 "99.1 96i H, 3% # N - 5.90% and O (by difference): 29.65%; and which when she

 <Desc / Clms Page number 35>

 
 EMI35.1
 is Paettllée with potancium bromide Present $ dan. the infra-red region of the spectrum an absorption characteristic
 EMI35.2
 rintique and leu solo of this acidic substance.



  6) The sodium salt of this compound.
 EMI35.3
 7) The ouloitm sol of this compound.



   8) A salt of compound and a non-toxic organic base.



   9) An amine salt of this component.



   10) The streptomycin salt of this compound *
 EMI35.4
 11) The soil of 41hydx'oatrePtomyo1n. of this oompond.