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    • 专利摘要:
    In an electrostatographic copying system where a toner image on a photoreceptor is transferred to a copy sheet with transfer charges, which charges are removed from the copy sheet by passing the copy sheet under a detacking corona generator, the body of the copy sheet is supported to insure its stripping from the photoreceptor at a point under the detack corona generating electrode where the transfer charge thereon is only partially neutralized, while a restricted minor area of the lead edge of the copy sheet is stripped further downstream, where it has been substantially fully neutralized, at a fixed position controlled by a mechanical stripper finger.
    公开号:SU751332A3
    申请号:SU772485600
    申请日:1977-05-23
    公开日:1980-07-23
    发明作者:Хайнц Михель Карл；Юджин Хичченс Кэльвин
    申请人:Эли Лилли Энд Компани (Фирма)；
    IPC主号:
    专利说明:

    one
    This invention relates to microbiology and concerns the production of antibiotics.
    The proposed antibiotic complex and the method for its preparation are not described in the patent and scientific-technical literature.
    The purpose of the invention is to obtain an antibiotic complex A-35512.
    This goal is achieved by growing the strain of Streptomyces candidus NRRL 8156 on a nutrient medium containing sources of carbon, nitrogen and mineral salts under aerobic conditions, followed by isolating the complex and / or dividing it into individual components.
    The strain of Streptomyces candidus NRRL 8156, which is used to produce the antibiotic complex, was extracted from soil samples collected on the Enolvetok atoll and is stored in the crop collection of the United States Northern Regional Research Center under the number NRRL 8156.
    The strain has the following cultural and morphological characteristics.
    Sporophores elongated wavy shape. Spores form
    a chain of 10-50 spores, have a cylindrical shape, the sizes can vary from 0.7 to 1.05 / xj-1.4 to, 3.5 | u. Solid and dense structures are formed on the growth medium.
    Yeast Yeast Agar. Intensive growth. Substrate mycelium-changing grayish yellow color, abundant aerial mycelium and formation
    10 dispute, white. The pigment is insoluble.
    Oat agar. Moderate growth, substrate mycelium — varying pale yellow-green, pale aerial mycelium, and sporulation.
    15 Pigment insoluble. The formation of continuous structures is observed.
    Agars Emerson. Growth is good. Substrate mycelium — there is no variable light olive aerial mycelium; there is a soluble pigment, light brown, and no sporulation.
    Agar Bennett. Growth is intense. Substrate mycelium is moderately yellow,
    25 abundant aerial mycelium and sporulation, white, insoluble pigment.
    Agar čapek. Growth is good. Substrate mycelium pale yellow-green
    thirty . well developed aerial mycelium
    and spore formation, white, pigment. the insoluble formed mycelium is surrounded by villi
    Lgar of tomato paste and oat flour. Intensive growth, substrate glycelium is greyish yellow, abundant aerial mycelium and sporulation, white, non-soluble pigment.
    Nutrient agar. Growth is moderate. Substrate mycelium - variable pale yellow-green, well-developed aerial mycelium and sporulation, white, soluble pigment light brown,
    Tyrosine agar. Growth is good, substrate mycelium is white, well developed aerial mycelium and sporulation, white, soluble brown pigment, formation of solid structures is observed.
    Physiological properties.
    Milk coagulates within 14 days. Nitrates restores. Melanin does not form, dilates the gelatin. It grows well and forms spores at 26-30 ° C. Absorbs glucose, inositol, D-mannitol, fructose, xylose. Does not absorb arabinose, sucrose, rhamnose, raffinose.
    The medium for growing the antibiotic producer may be different. As a carbon source, using mouth sucrose, glucose, dextrin, fructose, mannitol, maltose, lactose. Meat peptone, soy bean flour, flour made from pig blood, amino acids such as glutamine are used as a nitrogen source. Inorganic salts such as chlorides, carbonates, sulfates, nitrates of zinc, sodium, magnesium, calcium, and ammonium can be introduced into the nutrient medium. The medium can include trace elements necessary for the growth and development of microorganisms,
    In the fermentation process, when a large amount of foam is formed, you can add a small amount (for example, 0.2 ml / l) of an agent that suppresses the formation of foam, such as polypropylene glycol,
    The cultivation of the producer is carried out under aerobic conditions. The vegetative grafting material is prepared by grafting in a small amount of cpepf to grow a spore culture. The vegetative grafting material is then transferred to a tank of large size.
    Strain producing antibiotic complex, grown at 20-40 ° C, preferably at 30-34s
    When the culture is immersed under aerobic conditions, sterile air is blown through the medium. For effective growth of the culture, air is preferably supplied to the growth reservoir with about 0.1 volume of air per 1 volume of medium for growing the culture in 1 minute. For efficient cultivation of the culture, air is supplied to the reservoir at about 0.25 rpm.
    The production process of the complex, which takes place during the fermentation process, is accompanied by the selection of test samples of the medium or extracts of particles of the mycelium to test the antibiotic activity.
    against organisms known to be sensitive to antibiotics. One of the Organisms that is used in antibiotic tests is BaciPPus subtiEls ATCC 6633. A bioassay is performed by placing a paper disk,
    on which the sample is placed, on flat agar containing culture medium with a limited amount of nutrients.
    0 The maximum extraction of antibiotic complex A-35512 is achieved by pre-filtering to remove the mycelium mass. Filtered medium for
    5 The culture is purified by known methods, preferably using an adsorption process of the filtered broth on a polyamide column and elution.
    Q columns with water and a mixture of water and alcohol. The eluted fractions that exhibit antibiotic activity are combined, resulting in the antibiotic complex A-35512.
    Further purification of the individual components of the complex includes additional procedures for adsorption and extraction. For this purpose, materials such as alumina, silica gel, and ion exchange resins are used.
    The components of the antibiotic complex are formed during the fermentation in the form of dihydrochlorides. As a result of polyamide separation
    5, component B is formed, and the remaining part contains the mixture in the form of dihydrochlorides. Each individual component can be turned into its free base (not containing
    Q of chlorine ions) by known methods, such as chromatography over a weakly basic ion exchange resin.
    Component B The aglycone is prepared by hydrolyzing the component B in a weak acid. Component B is most easily available as dehydrochloride. It is also possible to use component B or the salt of component B formed by the addition of an acid. Hydrolysis in acid is carried out in the usual way, preferably using hydrochloric acid, while the component B aglycone is obtained in the form of a salt of hydrochloric acid, in the preferred embodiment, the hydrolysis is carried out
    in water at reflux temperature for 1-2 hours.
    The antibiotic kog / tlex A-35512 components of complex A, B, C, E, H, component B aglycone suppress the growth of some pathogenic microorganisms, in particular gram-positive bacteria. The minimum inhibitory concentration at which the components of the complex inhibit the growth of bacteria is determined in a known manner.
    The number and proportions of the various components contained in the complex may vary depending on the fermentation conditions. Component B is major in the A-35512 antibiotic complex.
    The components are formed in complex A-35512, they are extracted separately from the mixture in the form of hydrochloric acid salts. Each component can be separately transformed into its free base that does not contain chlorine ions, for example, by using chromatography on a weakly alkaline ion exchange resin.
    The components of the complex are soluble in water, partially dissolved in an alcohol, such as methanol and ethanol, and not soluble in other organic solvents, such as benzene, chloroform, acetone.
    Component A of the antibiotic complex.
    Component A, a white amorphous alkaline compound, has the following elemental composition,%: carbon 54.29, hydrogen 5.19, nitrogen 5.58, oxygen 33.76, chlorine 1.69.
    Component A dihydrochloride, a white amorphous hygroscopic compound, has the following elemental composition,%: carbon 51.03, hydrogen 5.10, nitrogen 4.75, oxygen 34.20, chlorine 4.80.
    Infrared absorption spectrum has a maximum at the following frequencies (cm-), KBr: 3405 (intense), 3300 (bending), 2950 (weak), 1750 (weak 1670 (intense), 1625 (bending), 1602 (intense), 1520 ( intense), 1470 (weak), 1440 (weak), 1405 (weak), 1345 (bend), 1312 (moderate), 1225 (moderate), 11801130 (weak), 1080 (intense) and 1020 (bend).
    UV absorption shows absorption maximum and 282 nm in neutralized methanol, and 292 nm in alkaline methanol.
    The component A dihydrochloride has the following specific rotations (1, H20), M5f-400 ° (1, Hj, 0). .
    The molecular weight of Component A Dihydrochloride is about 2106. Soluble in water, partially soluble in alcohols, such as methanol and ethanol, and insoluble in other organic solvents, such as benzene, chloroform, acetone, etc. Stable for 72 hours in aqueous solutions having a pH in the range of 3-10.
    According to amino acid analysis, there are five amino acid residues, one of which is glycine.
    Component B of the antibiotic complex A-35512.
    Component B, white amorphous basic compound. The empirical formula has the form С „.„ Н „,.,„, Мв., 0, С Е and has the following percentage composition,%: carbon 53.98, hydrogen 4.75, nitrogen
    .5.25, oxygen 34.29, chlorine 1.59.
    The UV absorption spectrum shows an absorption maximum of 282 nm in acid and neutral methanol, and 292 N in alkaline methanol.
    Component V. has the following characteristic rotations: M -123 ° (1, P, O), D -44b (1, H ,, 0).
    The molecular weight determined by titration is about 2,143.
    The component B dihydrochloride, a white crystalline compound, is hygroscopic and does not have a specific melting point. It has the following elementary composition: carbon 52,57,
    hydrogen 4.80, nitrogen 5.66, oxygen 32.86, chlorine 4.51.
    The UV spectrum in acidic and neutral methanol shows an absorption maximum of 282 nm, az of mainly methanol 292 nm.
    Component B dihydrochloride has the following characteristic rotations: M-128 (1, H20), W | -475 ° (l,). The molecular weight determined by the titration method is about 2027.
    Amino acid analysis by acid hydrolysis indicates the presence of five amino acid residues, one of which is glycine.
    An analysis of the hydrolysis products in acid shows that Dihydrochloride of component B contains the following sugars: glucose, fructose, mannose, rhamnose, and 3-amino-2,3,6-trideoxy-3-C-methyl-xylohexopyranose. When hydrolysis is carried out in a weak acid, glucose, fructose, mannose and rhamnose groups are extracted and an aglycone-derived compound is obtained.
    Component B dihydrochloride has one hydroxyl group capable of participating in etherization.
    Dissolved in water, partially soluble in alcohols, such as methanol and ethanol, and insoluble in other less polar organic solvents. Stabilize for 72 hours in aqueous solutions with a pH in the range of 3-10.
    Component C of the antibiotic complex.
     Component C, a white amorphous basic compound, has the following composition,%: carbon 53.93, hydrogen 5.15, nitrogen 5.80, oxygen 32.35, chlorine 1.0
    Component C has a molecular weight of approximately 18b2,
    KoivinoHeHTa С dihydrochloride, a white amorphous compound, has the following elemental composition,%: carbon 51.76, hydrogen 5.07, nitrogen 5.61, oxygen 30.29, chlorine 4.88.
    The empirical formula for component C dihydrochloride is in the region of Cg3.35Hgj. | O, Ng037.qCE3
    The infrared absorption spectrum of component C dihydrochloride in KB g has the most significant absorption maxima at the following frequencies, 3370 (intense), 3280 (bending), 3040 (bending), 2980 (bending), 2920 (weak), 1740 (weak), 1658 ( intense), 1620 (weak), 158-9 (moderate), 1503 (intense), 1460 (weak), 1428 (moderate), 1385 (weak), 1330 (weak), 1295 (moderate), 1210 (intense), 1162 (moderate) 1120 (weak), 1060 (intense), 1005 (moderate).
    The UV absorption spectrum of component C dihydrochloe shows an absorption maximum of 282 nm in acidic and neutral methanol and 292 nm in alkaline methanol, calculated using a molecular weight of 200
    The component C dihydrochloride has the following characteristic rotations: -16 lM 1.05, H.O.), -614 ° (1.05,).
    The molecular weight of component C dihydrochloride, determined by titration, is 198.2,
    Amino acid analysis of the dihydrochloride of component C indicates that it contains five aglyc acid residues, one of which is glycine.
    Component C dihydrochloride is soluble in water, dimethyl sulfoxide and aqueous solution of dimethylformamide, poorly soluble in alcohols, such as methanol, ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene. Dihydrochloride of component C is stable in aqueous solutions, having a pH from 3 to 10 for 146 hours
    Component E.
    Component E, a white amorphous basic compound, has the following elemental composition,%: carbon 54.84, hydrogen 4.73, nitrogen 5.26, oxygen 32.67, chlorine 1.72.
    Hydrochloride. Component E, a white amorphous compound, has the following elemental composition,%: carbon 52.6 hydrogen 4.59, nitrogen 5.55, oxygen 33.51, chlorine 3.62.
    The infrared absorption spectrum of hydrochloride of component C in KB g is the most significant absorption maximums of im-1.
    3360
    em on the following frequencies, see
    (intense), 3220 (bend), 2900 (weak), 1725 (weak), 1650 (intensive), 1580 (moderate), 1498 (intensive), 1450 (weak), 1419 (slaby), 1295 (moderate), 1205 (moderate), 1172 (moderate), 1110 (slaby), 1060 (intense) and 1000 (weak).
    The UV absorption spectrum of hydrochloride component E detects absorption maxima in neutral methanol at 270 nm and 359 nm, in acidic methanol at 286 nm and 310 nm, mainly methanol at 270 nm, 300 nm and 354 nm.
    Hydrochloride of component E has the following characteristic rotation: Y –108.3 (1,).
    The molecular weight of the hydrochloride component E, determined by titration, is approximately 201
    Component E hydrochloride is soluble in water, partially soluble in alcohol such as methanol, ethanol, and insoluble in benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, etc. Component N.
    Component H, a white amorphous basic compound, has the following composition,%: carbon 53.76, hydrogen 5.32, nitrogen 5.53, oxygen 33.48, chlorine 1.59.
    Component H has an empirical formula in the woo-wise field. OzvchoS E. The molecular weight is approximately equal to 1908.
    Hydrochloride of component H, white amorphous hygroscopic compound. Hydrochloride of component H has the following composition,%: carbon 53.10, hydrogen 5.37, nitrogen 5.35, oxygen 30.12, chlorine 3.78.
    The infrared absorption spectrum of hydrochloride of component H in KBr has the most significant absorption maxima at the following frequencies, cm 3410 (intense), 3240 (bending), 2940 (weak), 1670 (intense), 1630 (bending), 1505 (intense), 1520 (intense), 1470 (weak), 1442 (weak), 1400 (weak), 1345 (bend 1310 (moderate), 1225 (moderate), 1180 (weak), 1135 (weak), 1080 (intensive) and 1020 (bend ).
    The UV spectrum absorbed by hydrochloride of component H exhibits an absorption maximum of 282 nm in acidic and neutral methanol, and an absorption maximum of 292 nm in basic methanol.
    Hydrochloride of component H has the following characteristic rotation: AND "-123, 5 (1, HgO).
    The molecular weight of coponent H hydrochloride, determined by titration, is 1660.
    Amino acid analysis of the hydrochloride component H indicates that it contains five amino acid residues, one of which is glycine.
    Hydrochloride of component H is soluble in water, partially soluble in methanol, ethanol, and insoluble in organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane
    Hydrochloride of component H is stable for 72 hours in aqueous solutions, has from 3 to 10.
    Components A, B, C, E, H, as well as the less significant components F and G can be easily separated using paper chromatography.
    Component B Aglycone. Component B is an aglycone hydrochloride, a white amorphous compound having the following composition,%: carbon 54.29, hydrogen 4.34, nitrogen 7.40, chlorine 5.02, oxygen 28.95.
    The infrared absorption spectrum of the hydrochloride component B of the aglycone has the most significant absorption at the following frequencies, cm: 3440 (qzg), 3340 (bend), 3515 (intensive.), 2950 (bend), 2910 (intense), 2840 (intensive), 2640 (bend), 1735 (weak), 1655 (intense), 1590 (moderate), 1500 (intense), 1460 (intense), 1378 (moderate), 1365 (bend), 1298 (moderate), 1215 (moderate), 1155 (moderate), 1120 (bend), 1105 (weak), 1060 (weak), 1040 (weak), 1008 (moderate), 925 (weak 875 (weak), 765 (bend) and 718 (weak).
    Component B hydrochloride aglycone has a molecular weight of about 1282.
    Hydrochloride of the component B of the aglycone has the following characteristic rotation SocHo, CH3), Mls-l 8 ° (5, CH3).
    The UV absorption spectrum of the hydrochloride component B of the aglycone shows an absorption maximum of 282 nm (102.62) in acidic, neutral methanol, and in 302 nm (182.09) 1 in alkaline methanol.
    Hydrochloride of component B, an aglycone, is soluble in water and methanol, HQ is not soluble in benzene, chloroform, acetone, distil ether, ethyl acetate, toluene, hexane, acetonitrile, and dioxane.
    The components of antibiotic complex A-35512 are relatively non-toxic. For example, LDjo for component A hydrochloride is 8000 mg / kg. The LOjo index for the hydrochloride of component B in the tishys when administered intraperitoneally is 1365 mg / kg, and when administered intravenously it is equal to 10OO mg / kg. Dihydrochloride component C, as well as
    Component E hydrochloride has a strong toxicity when administered intraperitoneally to mice, so the LD exceeds 300 mg / kg.
    The antibiotic complex and its components are effective when used to increase the release of propionates and thereby increase the efficiency of feed use when using ruminant animals in doses from 0.15 mg / kg to 10.0 mg / kg per day.
    0 The drug is mixed with animal feed, but it can be used, for example, in the form of tablets, pellets, capsules. Each individual unit dose should contain an amount of the antibiotic complex and its components close to the daily dose for animals.
    The method is illustrated by the following examples.
    Example 1. Freeze Dried
    0 tablet of the strain Streptomyces candidus NRRL 8156 is dissolved in 1-2 ml of sterilized water and a solution of DD1I is used for grafting a culture on a slanted agar containing extract of yeast5 and yeast Bacto C1SP No. 2.
    The grafted agar is grown for 7 days at. The culture matured on the agar is coated with water (2 ml) and removed using a sterile syringe to loosen the spores. A 0.1 MP portion of the aqueous spore suspension is used to graft another oblique ISP No. 2 agar, grown for 7 days at 30 ° C. Matured
    5 culture pour 5 ml of water and remove With a sterile pipette to loosen the spores. A portion of the resulting spore suspension (2.5 ml) is used as a graft
    0 graft material 50 ml of vegetative medium having the following composition: soy broth (Trypticase) 30 g, water (deionized) 1 l. The grafted culture medium is incubated at 30 ° C for
    5; 48 hours in a 250 milliliter Erlenmeyer flask on a vibrator with a rotation speed of 250 rpm.
    The resulting 0.5 ml vegetative medium is used for grafting.
    0 50 ml of medium for growing the culture, with the following composition: tapioca dextrin 25.0 (g / l), glucose 10.0, MNDOM 2.5; KCE 1,5; MgS04 1.1; FeCl2-4H2 0 0.03, tnCl2 0.03, KNgP04
    5 0.1; L - glutamic acid 1.0, DL - citrulline 0.1; CaCO-j 5.0; deionized water 1l.
    The grafted medium is grown for about 8–10 days in a 250 ml Erlenmeyer flask on a vibrator.
     To provide a large volume of inoculum, 20 ml is prepared. grafted vegetative medium and use it for grafting 400 ml
    five
    medium for growing the second stage of vegetative growth, having the same composition as the vegetative medium. Growing lead in a 2-liter flask for 24 h at 32 ° C on a vibrator. 800 ml of the obtained vegetative material is used for grafting 100 liters of sterile medium. The medium thus grafted is used in a fermentation process in a 165-liter tank for 8-10 days at З2с. In the fermentation process, sterile air is fed at a rate of 0.25 l / v / min with constant stirring at a speed of 200 rpm.
    Example 2. A grown vegetative medium, according to example 1, can be stored for further use in the vapor phase of liquid nitrogen. To do this, place 2 ml of medium of the following composition,%, in a small tube with a closing lid,%:
    Glycerin 20 Lactose 10 Water deionized 70
    To this medium, add 2 ml of the vegetative medium prepared according to example 1 and grow for 48 hours. The resulting suspension is cooled and kept in the gas phase in a tank with liquid nitrogen.
    Before use for fermentation, the medium is melted. For this, it is placed in a vial which is subjected to a water bath with a temperature of 43 C. A portion of the melted solution (1 ml from the vial is used to graft a vegetative medium, 50 ml in volume, having the same composition as the medium from Example 1. The grafted vegetative medium is used for fermentation in a flask that is shaken periodically, or used as a graft material, which is then used in the fermentation process.
    Example 3. I conduct fermentation according to example 1 using a medium of the following composition, g / l:
    75 40
    tapioca
    ya mnoy
    15.0 0.5 2.0 Else
    Example 4. 950 l of culture fluid with fermentation products obtained in example 1 is filtered using earth diatoma at pH 6.8-7.2. The resulting clear filtrate is passed through a column containing 10 ml of a polymeric adsorbent per 100 ml of filtrate at a rate of 150 ml / min. Received
    fractions are examined for biological activity against Sarcina lutea. Biologically inactive fractions are discarded. The column is then washed and the inactive wash water is discarded. - Then the column is eluted with a 50% aqueous solution of methanol (600 l) at a rate of 200 ml / min. The eluate containing the antibiotic complex is concentrated under vacuum to a volume of 15 liters, which contains about 200 g of the antibiotic complex per liter.
    Example 5. The antibiotic complex (about 3000 g dissolved in 15 liters of methanol), obtained according to example 4, is subjected to chromatography on a polyamide column. The column is then eluted with deionized water at a rate of about 80-120 ml / min.
    Fractions are examined using cellulosic thin-layer chromatography or paper chromatography, and the mixture used is a mixture of n-butaNOL: pyridine: acetic acid: water (15: 10: 3: 12) and carried out additional biosamorbtion. Sarcina Lutea.
    The first 100 l of the eluate is discarded, then the flow rate is changed to 1 2 ml / min. 12 l fractions are then taken. Twenty 12-liter volumes are selected in this manner. In this case, the percentage composition of the elution solvent is changed.
    Based on the results of bio-recording, the resulting volumes are combined and evaporated to dryness under vacuum. The result is the dihydrochloride of component b and the following enriched mixture of components (see table).
    Example 6 Partially purified dihydrochloride of component B (400 g) obtained in accordance with Example 5 was dissolved in 1.2 l of a 50% aqueous solution of methanol and chromatographed on an alumina-containing column, which was prepared as follows. The acid-forming alumina oxide (10 kg) is stirred in a 50% aqueous solution of methanol. After the mixture has settled down, the solution formed from above is drained and removed. Aluminum oxide is again mixed with a 50% aqueous solution of methanol, and then placed in a column. The alumina-containing column is then washed with a 50% aqueous solution of methanol until a clear eluate is obtained. The column is eluted with 50% methanol at a rate of 810 ml / min. At the same time take a fraction of 240-300 ml. The fractions follow according to Example 5. Based on the data obtained, the fractions are combined. The yield of the purified dihydrochloride of component B is distributed as follows depending on the fraction number: fractions 17-21 - 9.6 g, 32-20 - 72 g, 30-37 - 117 g. Each of these fractions is crystallized from a concentrated 50 % aqueous solution of methanol at 4 ° C. The hydrochloride of component B thus purified contains about 4.6% chlorine. The dihydrochloride solution of component B in a 60% aqueous solution of dimethylformamide has a pH of about 6.5. Example 7. 1g of the purified dihydrochloride of component B of example 6 is dissolved in 40 ml of water and passed through an ion exchange column 2.5 to 18 cm in size at a rate of 0.5 ml / min. Then the column is eluted with deionized water, 5 ml of the initial eluate is discarded. The next 50 ml of eluate is evaporated to dryness under vacuum and 0.76 g of component B, which does not have chlorine ions as a white powder, is obtained. The chlorine content is 1.59%. The solution of this component in a 66% solution of dimethylformamide has a pH of 9.13. Example 8, 1g of the partially purified dihydrochloride of component A prepared in Example 5 (fractions 1-10), is dissolved in 5 ml of a 50% aqueous solution of methanol. Then, chromatography is carried out on a column of size 3-16 cm, containing acid-forming alumina. The column was eluted with a 50% aqueous solution of methanol at a flow rate of 0.5 ml / min. Fractions having a volume of 5 ml were collected and analyzed according to Example 5. Based on these tests, fractions 7-15 were mixed, concentrated under reduced pressure to a small volume , lyophilized, and 0.3 g of component A dihydrochloride with a chlorine content of 4.71% is obtained. Example 9, 200 ml of the component A hydrochloride prepared in Example 8 are dissolved in 10 ml of water. This solution is passed through an ion exchange column 1.5-10 cm in size at a rate of 0.5 ml / min, then the column is eluted with deionized water, 10 ml of the initial eluate is discarded, the next 20 ml of eluate is concentrated to a small volume under reduced pressure, lyophilized and 115 mg, of component A, not containing chlorine ions are obtained. Example 10. Partially purified dihydrochloride of component C (15 g), prepared as described in example 5 (fractions 25-31), was dissolved in 40 ml of deionized water and passed through a polyamide column of 4 115 cm in size. Before use, the column is washed with water for 15 hours, then the column is eluted with deionized water at a rate of 3 ml / min. 250 ml of the initial eluate is discarded, then fractions of 24 ml are collected. The fractions obtained are examined by thin layer chromatography biosolamination. Cellulose plates are used for thin layer chromatography and the solvent is secondary butanol: pyridine: acetic acid: water in a ratio of 10: 10: 3: 8 and bacteria of the strain BaciP us subt i Ei s, based on the obtained results, fractions 1-33 are mixed, concentrated under vacuum to a volume of 150 ml and lyophilized. Two additional batches of partially purified component C dihydrochloride are examined by chromatography under the same conditions. In this case, each time the fractions 1-33 are mixed, concentrated under vacuum to a volume of 150 ml and lyophilized. The three lyophilized samples obtained on the three columns are mixed and 12.3 g of the partially purified dihydrochloride of component C is obtained. It is then purified by chromatography on another polyamide column, but a 15 ml volume is selected, with a flow rate of 1 ml / min. The fractions obtained are re-examined as described above. Fractions 36-58 are mixed, concentrated under vacuum to a volume of 150 ml, and lyophilized, to obtain 5.3 g of the dihydrochloride of component C, which is further purified. The final purification of the desired product is carried out by chromatography on a 5 41 cm column containing acid-forming alumina. The prepared column is washed with a 50% aqueous solution of methanol. After washing, a solution containing 5.3 g of component C in 30 ml of a 50% aqueous methanol solution is passed through the column. The kilon was then eluted with a 50% aqueous solution of methanol at a rate of 1 ml / min, and 12 ml fractions were collected, which were then examined as described above. Fractions 22-74 mixing-. are concentrated under vacuum to a volume of 250 ml, lyophilized, and 3.86 g of component C dihydrochloride are obtained.
    Example 11. 200 mg of the dihydrochloride of component C, obtained in example 10, are treated using chromatography on a weakly basic ion exchange resin, according to example 9. 156 mg of component C, not containing chlorine ions (chlorine content 1.9%), is obtained.
    Example 12. 8.1 g of the partially purified component E obtained in Example 5 (fractions 32-44) are dissolved in 40 ml of deionized water. The resulting solution is passed through a 5-HO cm polyamide column prepared according to Example 10. The column is then washed for 16 hours with water. After the solution containing component E is passed through the column, the column is eluted with deionized water at a rate of 20 ml / min and selected fractions having a volume of 20 ml. During selection 118 of the fraction, the elution solution is changed to a 50% aqueous solution of methanol, and the obtained fractions are tested as in Example 10.
    Fractions 148-195 containing component E are mixed, concentrated under reduced pressure to a volume of 150 ml, lyophilized and 2, 7 g of hydrochloride of component E are obtained.
    615 mg of a partially purified preparation is dissolved in a 50% aqueous solution of methanol (5 ml) and containing acid-forming alumina through a 1.5-50 cm column and washed with 50% aqueous solution of methanol to obtain a clear eluate. Then the column was eluted with a 50% aqueous solution of methanol at a rate of 1 ml / min and a fraction of 10 ml was selected.
    The fractions were examined as described in VESPIA, fractions 5-8 were mixed, concentrated under reduced pressure to a volume of 10 ml, then 50 ml of deionized water was added and the resulting solution was basified. Get 480 mg of the hydrochloride component E.
    Example 13. Hydrochloride of component E (200 mg, obtained in accordance with example 12, is subjected to chromatography over a weakly basic ion exchange resin, according to example 9. Obtain 170 mg of component E, not containing chlorine ions (chlorine ion content 1.72%).

    Example 14. Partially purified hydrochloride of component H (30 g), obtained in accordance with example 5 (fractions 1-10}, is dissolved in iv with the minimum amount of methanol: water solution (7: 3). The resulting solution is adsorbed on a 3 eO cm column containing acidic alumina, the column is filled with methanol and eluted to those
    until the eluate becomes transparent. Next, the column is eluted with methanol at a speed of 4} ll / mnn and 24 ml fractions are collected. With the selection of 59 fractions, the eluting solvent is changed to a solution of methanolhyd (1: 1). Next, the fractions are subjected to thin layer chromatography using chloroform solvent: methanol: ammonium hydroxide (2: 3: 1) and examined for BaciPJus subtiEis using bio-recording under alkaline conditions. Fractions 51-118 are mixed and evaporated under vacuum to obtain 6.4 g of purified hydrochloride of component N.
    Example 15. 200 mg of the hydrochloride of component H obtained in example 14 are chromatographed on a weakly basic ion exchange resin of example 9. 143 mg of component H are obtained which does not contain chlorine ions (chlorine concentration 1.59%).
    Example 16 5 The dihydrochloride of component B, prepared in Example 6, is dissolved in 200 ml of water. The resulting solution is acidified with 14 ml of 4N HCE solution and refluxed for 2 hours. Then the solution is cooled, evaporated under vacuum to 3/4 of the original volume. The resulting solution is then added dropwise to bN hydrochloric acid until a precipitate forms. The resulting precipitate is separated and dried. 3.56 g of crude A-35512 hydrochloride agiglone B are obtained. The filtrate is concentrated and analyzed. It contains glucose, fructose, mannose and rhamnose.
    The crude aglycone is further purified by chromatography on alumina, washed with an acid, using water: methanol (1: 9) as the solvent. The eluted fractions containing aglycone are mixed and evaporated under vacuum to obtain 398 mg of a partially purified preparation. 100 mg of a partially purified preparation is subjected to chromatography on polyamide and eluted with water. After analysis, the eluted fractions are mixed and lyophilized. Get 64 mg of purified aglycone hydrochloride total yield of 5.08% of the original component B.
    权利要求:
    Claims (1)
    [1]
    Example 17. 9.0 mg of the hydrochloride component B of the aglycone prepared according to Example 16 is dissolved in 30 ml of a mixture of methanol: water (1: 1). The resulting solution is neutralized using an ion exchange resin. The resulting solution is stirred for 15 minutes at room temperature. The resin is then removed by filtration. The x: tent filtrate under vacuum at a temperature of at least up to the volume constituting. half of the original, then lyophilized, to obtain 68 mg of the component B of the aglycone in the form of a free base. The proposed method allows obtaining an antibiotic complex, the use of which will improve the efficiency of food digestion in ruminants and domestic birds. 18 Claims of the invention A method for producing an antibiotic complex, characterized in that the Streptomyces candidus strain NRRL 8156 is grown on a nutrient medium containing carbon, nitrogen and mineral salts, in aerobic conditions, followed by separation of the complex and / or its separation into individual components.
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    同族专利:
    公开号 | 公开日
    DE2723426A1|1977-12-08|
    NL7705611A|1977-11-28|
    GB1579893A|1980-11-26|
    US4058306A|1977-11-15|
    FR2353086A1|1977-12-23|
    JPS6252299B2|1987-11-04|
    DE2723426C2|1985-10-24|
    CA1104195A|1981-06-30|
    FR2353086B1|1983-06-17|
    JPS52145226A|1977-12-03|
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    法律状态:
    优先权:
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