SU561521A3 - The method of obtaining antibiotic - Google Patents

Description

with the properties of other gray cultures are given in Table. 1 (for all cultures, Sm spore surface, RA morphology, gray color).

M o r f o l o g.

Uefiii spores formed on the aerial pinion are branched monodnally and sympodially to form straight chains, loops, hooks, stretched and irregular shaped gray spirals (3-4 turns), also having a paniculate sweep. Chains are both short and long, but for the advantage} but they contain more than 10 spores. A sufficient density is observed on most media. The spores are beats; they are shaped like ovals and cylinders, the surface of the spores is smooth.

Physiologists. Soluble p and g me n g

On aiap - agar with JKCTpaKTaMM, yeast and malt are left behind; brown pigment.

The culture is chromogenic (forms melanin) on agar with peptone and iron and tyrosine, as well as on broth with tryptome and yeast extract.

Cultured (incubation period 4 days at 28 ° C) and 4 iziolo1 characteristics of cultures of Streptomyces Sp. X-5i08 giveivedeny in table. 2 and tab. 3 accordingly.

The organic nitrates are not reduced in the broth; starch is actively hydrolyzed, and gelatin lipids are very weakly liquefied after 14 days; xopoiJJO digested with L-arabinose, d-fructose, d-mannitol, 1 - rampose, d-raffinose and sugar; poor d-Kx4iJio3a and L-inositol and cellulose are not completely absorbed.

The culture of Streptomyces Sp.xopoino develops at 24 and 37 ° C, but does not develop at 42 or 50 ° C.

The nature of the assimilation of carbon sources and azog Streptomyces Sp. X-5108 is shown in the incubation period at 28 ° C.

The assimilation of 1-arabinose) 1, L-pa.viHO3bi, d-xylose, d-minita, L-in () ziIa, glucose, fructose, sucrose, acetate, citrate, malaga, (zsalata, salicylate, succinate, 1art;) ata, phenol (0.1L), raffinose, starch, d-ribose, d-galactose, d-mannose, nej.iio6H03bi, lactose, maltose, dextrin, dulisht, E) itri1a, sorbitol and glycerin is 1, 3 3, 3, 3, 3, 3, 3.2 2, 1.0, 2.0.0, 3.0-1.3.3.3, 3.3.3.0.0, 0, 1.0-1, and the absorption of ammonium sulfate, sodium nitrate, sodium nitrite acetamide, asparagine, tyrosine n D, L -tryptophan -1,1,1,1,3,3 and 2, respectively (negative control O-1).

Various methods of fermentation are used to cultivate the producer of Streptomyces Sp, X-5108.

When the enzymes in flasks, a portion of the spores, taken from a slice, on agar-agar, are inoculated in 100 ml of nutrient medium in a 500 ml Erlenmeyer flask and incubated C on a Rotating shaker for 7 days. Broth samples are taken on the third, fifth and seventh day.

When cooking broth portions of broth, the inoculum product is prepared in Erlenmeyer flasks with a capacity of 6 liters. n HHjxKcoBbix bottles x with a capacity of 16 liters or tanks equipped with devices for aeration, sampling, etc. For aeration through the fermentation medium in the tanks, sterile air is generated. When using tanks x-1 for additional mixing, mechanical blade mixers are used. To prevent peporoobrazovanie, a vilicultural agent, such as pork lard and soybean oil, is introduced. Streptomyces Sp. X-5108 is cultivated in various liquid media, especially in media that contain sources of assimilation, such as starch, glucose, melas, and a source of assim; Milled nitrogen, such as proteins, protein hydrolysates, Lolipets} schy, amino acids, liquid obtained by processing corn cobs, ammonium salts, sulfates, phosphates, potassium, sodium, calcium, magnesium chlorides, and P | 5) 1.5s cobalt, copper, iron, molybdenum, boron.

The activity of antibiotic X-5108 is determined by m vitro by the diameter of the zone of inhibition of the gram-positive bacteria of Bacillus N. yu Bacillus simplex OH) metol. 1i | ffuz and in agar agar with tea and plastic,

It is conditionally accepted that the activity of the antibiotic (e.VMJi) of the solution corresponds to the diameter of the intibi zone;) Ovan - 20 mm. The test culture of NttiKpoopraHn3Ma is grown in nutritional broth, for example in broth with trypticase and soy, for 24-42 hours at 28 ° C on a rotating cachypcha (10 ml of medium in a 500 ml Erlenmeyer flask). 4 ml in size } {Noah culture (0.25-0.5 / 0 concentration) is poured on frozen pi A1e; 1Hp 1H arap-aiapa layer in Petri tea, put them in holi.pn.chk at least 2 hours before sampling and filling The test solution, the antibiotic, is then incubated for 18 hours and the diameter of the inhibition zone is measured with an accuracy of 0.5 mm. The antibiotic activity How are calculated from standard curves.

For production of antibiotics, it is advisable to use complex nitrogen-containing substances, for example, vegetable (soybeans or cotton flour, oats for flour, solid tomato paste, soluble substances formed during the fermentation of the grain) or animal (fish meal, honey and milk decoction, products gadrolysis of amino acids of origin and microbial cells (torul yeast).

Carbon sources are, for example, glucose glycerin, dextrin and corn starch. In addition to inorganic salts, which are usually included in the composition of natural media, other substances are introduced, such as potassium phosphate, calcium carbonate, wean sulphate, and micro elements.

It is advisable for the production of antibiotic X-5108 to ferment the enzymes into the medium containing

1% defatted flour from cotton seeds (Proflo), 0.5% liquid concentrate obtained by leaching maize, 1% corn starch, 0.1% calcium hydrogen phosphate K2HPO4.0.1% calcium carbonate CaCO3.

In tab. Figure 4 shows the effect of the nutrient composition on the yield of the antibiotic Streptomyces Sp. X-5108 during the experiment in flasks (incubation period of 5 days).

All media contain 1% dextrin, 0.1% KJ NRO4.0.1% CaCO3 and 1% nitrogen source.

1 ml of Streptomyces Sp. Spore suspension is added to each 100 ml of inoculum. X-5108.

The results obtained when growing Stromyces Sp. in shake flasks for 4 days on media containing 0.1% K2HPO4.0.1% CaCO3 and the components listed in Table. 5 (1% each) are listed in Table. 5. To inoculate 100 ml of each medium, use 1 ml of a suspension of spores of Streptomyces Sp. X-5108.

The effect of the composition of the nutrient medium on the yield of the antibiotic Streptomyces Sp. X-5108 in shake flasks and in aerated tanks is shown in Table. 6

During fermentation in various apparatus, the results are summarized in Table 1. 7.

Each medium contains 0.5% CaCO3 and 0.1% K2NU4. Bacillus simplex is used to determine the activity of the antibiotic.

Producer Streptomyces Sp. X-5108 also grows on some chemical synthetic media containing ammonium salts, nitrates or amino acids (glutamate, arginine, glycine) serving as sources of nitrogen, glucose, dextrin, starch, citrate, acetate, etc., which serve as sources of carbon, with the addition of salts, such as potassium phosphate, calcium carbonate and magnesium sulfate, and trace elements, including divalent ions of iron, copper, manganese, cobalt and zinc.

Ferptomyces Sp. X-5108 on various synthetic media are shown in Table. eight.

The yield of anti-iotic X-5108 increases with good aeration of the medium. Antibiotic X-5108 can be obtained in any temperature that causes a satisfactory growth of the microorganism. So, for example, when Streptomyces Sp. X-5108 in flasks at 24, 28, 30 and 32 ° C (analysis of the antibiotic yield after 3,4,5 and 6 days), the output of the antibiotic reaches a maximum after 5-6, 4, 3-4 and 3-4 days, respectively .

During fermentation, the medium remains almost neutral or becomes slightly acidic. The final pH value depends on the presence of buffer substances and on the initial pH value of the medium (the medium must be neutral before sterilization).

To isolate and purify antibiotic X-5108, various methods are used, for example, solvent extraction (intermittent, continuous countercurrent extraction) carried out in extraction columns, and chromatography based on the penetration of a crude antibiotic gel from the culture medium. 5From the culture medium antibiotic X-5108

Mice is separated by filtration or centrifugation followed by extraction of a filtrate or fugate with a water-immiscible ester, for example ethyl acetate, 0 amyl acetate, butyl acetate, preferably butyl acetate, at pH 3-7.5.

For countercurrent distribution extraction, a mixture of ethyl acetate, isopropanol, and 0.1 is used. Nao aqueous solution of NU.

5 D.GI of the final purification of the antibiotic X-5108 uses a chromatography method based on penetration into the gel of the previously purified extractant of the preparation, on a cross-linked dextran gel of l / w or on a polymerizable 0 dextran gel.

For chromatography, take Sephadex LH-20 and elute with alcohol.

After separation of the antibiotic by means of centrifugation or centrifugation of the culture medium, its 5 is analyzed by thin layer or paper chromatography with the method of spotting by fluorescence and bioautography. For chromatography, it is better to use silica-coated glass plates.

0 For thin layer chromatography using a mixture of chloroform, methanol and ammonium hydroxide.

After purification, antibiotic X-5X8 is an amorphous yellow substance.

5 Found,%: C 63.63; H7.81; N3.48; O25,08

(by difference).

The antibiotic Kh-5108 is soluble in alcohols,

for example, metshgale, ethanol, 1 and 2-propanol,

 tert-butanol; water-immiscible esters, for example ethyl acetate, amyl acetate,

butyl acetate, and in chloroform.

The antibiotic is not soluble in water.

IR spectrum: 3400 (shnrok), 1660 (strong), 1580 (wide), 1580, 1380.1250, 1100, 1040 and 995 (distinct.

UV spectrum (0.1 N hydrochloric acid), Xmax (E j)) 334 (403), 233 (610) and 206 (SQOV

UV spectrum (isopropanol buffer - K2HPO4 PH7), Hmax (EfJ ;,), mmk: 327 (423), 231 (660).

UV spectrum (0.1 N potassium axi), Hmax (E) mmk: 327 (416), 231 (647).

NMR spectrum (deuterochloroform, internal standard tetramethylsilane): distinct signals at 0.92, 1.66-2.03, 3.16, and 3.43 5 and in the area of olefin signals.

The microbicidal activity of antibiotic X-5108 was determined by the plate and plate method, with diffusion in agar-agar medium.

7

, ntabiotik X-5I08 has low toxicity when administered orally and parenterally, has significant activity against streptococci in systematic (oral) and local (subcutaneous) administration, activity against pneumococci and intestinal cancer. During experiments on micel, it was established that the antibiotic is practically non-toxic when administered orally and subcutaneously (LDso no more than 2000 and 1320 mg / kg, respectively). Antibiotic aiCTHBCH with oral (LDso 52 l {G / c-g) and sub-condom (CD5o 44 mg / kg) administered against Streptococcus pyognes, active against Diptococcus pneumoniije (CD5o 807 and 283 mg / kg with oral and subcutaneous administration, respectively) and against gram negative bacteria.

The antibiotic X-5108 npsi introduced into Maiiuien feeds stimulates the growth of nosi.

Usually aHTiiOHOTHK X-5108 add} SCO to nutritionally balanced feeds for poultry balanced in nutrition (per 100 parts by weight of feed 0.0001-0.01 parts by weight of antibiotic). / A further increase in antibiogic concentration does not lead to improved results.

Example. Suspension snor Streptomvces Sp. X-5108 from a test tube with agar agar medium ipokuluyut the contents of the tank of 1shreks with a capacity of 16 liters, equipped with a device for aeration, which is loaded with 15 liters of medium, containing (in%): 1 soy flour, 1 yellow sugar, 0.25 solids in the processing of corn cobs, 0.1 K2HPO4, 0.1 CASE2 and 0.05 lard fat (a. reader.).

The period inc bashi 4; 1 l at 28 ° C under aeration conditions. The grown fibrous material is transferred to a 378 l fermenter made of stainless steel which contains 192 l of medium from% defatted cotton seed meal, l / f cornstarch, 0.25% of solid matter obtained from processing corn cobs , 0.1% CaCO3 0.1% PRO., And 0.5% pork fat.

The pH of the medium before inoculation is 6.8. The fermenter is aerated and the contents are unloaded for 5 days, filtered using diatomite earth. 1S, the filtrate is acidified to pH 3.5, extracted with half the volume of butyl acegate, the clarified extract is centrified in vacuum at a temperature below 40 ° C to form a brown syrup, after rubbing which with. etrol.). m injected () t solid iiopoirioK a} 1 X-5108 tiotic with activity against Bacillus E. 120. UMg. fl p and m e p 2. The antibiotic X-5108 is purified by periodic-x-scraps with a solvent. After the in vitro tests, the batches of the culture medium after fermentation (11340 l), KOTOJbie show activity / v 130 units / ml against Bacillus simplex, filtered, the filtrate is extracted with butyl acetate at pH 7.5, the extract is washed with water, and concentrated using the instant method.

rhenium at a temperature below, add i-lonat dizti; Sodium to pH 9.0, the yellow precipitate is formed is filtered off, washed with butylapest, treated with water-butylapetag and diluted with pH to 4. The organic phase is separated, treated as above, but added to the disodium sodium malonate until pHv8.5 is added. The resulting precipitate is acidified, extracted with fresh butyl acetate, the extract is taken with an extract from 1 to G-60 and

The sodium salt of the antibiotic X-5108 is precipitated by adding diethyl sodium malonate to pH 8.0. The resulting antibiotic yellow sodium salt is washed and dried. When tested in vitro, co-activity gi 430 u / mt against Bacillus

simplex.

Technological diagram of the distribution of the angiotic i-X-5108.

Step 1. 11340 l of broth in divide (collection after 5-6 days) is filtered.

Stage 2, KPO4 is added to 9804 L of filtered broth (pH 7.5) at 1 eubhodism and extracted with 2268 L of butyl acetate.

Herd 3. 2192 l of extract are washed with 2 x 94 l of water and concentrated by flash method.

Stage 4. Diethyl sodium was added to 302 L of concentrate to pH 9 (1-1.5 L, 7N of malonate solution), the precipitate was filtered and washed with 3.7 L of butyldedetate.

Herd 5. The resulting precipitate was dissolved in 94 l of WATER, added with Nz ROC to rP 4 and extracted with two portions of butyl acetate (75 + 37.8 l).

Step 6. 106 l of the extract are washed with 2 x 7.5 l of water and the end} is ground by flash evaporation.

Step 7. Diethyl sodium malonate is added to a pH of 8.5 to 15 liters of concentrate, the precipitate is filtered off and the solution is fed with 3.7 liters of butyl acetate.

A hundred; 1, and 8. The isolated precipitate is dissolved in 75 l of water, pf 1 is adjusted to 5 with 15% water from PO and extracted with two portions of butyl acetate (56.7 28, 3 l).

Herds 9. I wash the 78 liters of the extract with 2 x 7.5 liters of water and concentrate by flash evaporation.

Stage 10. Concentrate 15L of concentrate from 50 g of lightly G-60 for 1 hour at room temperature, filter and concentrate by flash evaporation.

Step 11. Add 7.5 liters of concentrate.

diethyl sodium malonate to pH 3.0, the precipitate is filtered off, washed with 3.7 l of butyl acetate, then 3.7 l of petroleum ether (b.p. 30-60 °), dried at 56 ° С / Ь, 5 mm for 36 hours and get the sodium salt of the antibiotic X-5108 in the form of a yellow A. morphic substance.

PRI me R 3. Purification of antibiotic X-5108 by countercurrent distribution extraction with the number of tubes 2 (Yu, volume of the upper and lower phases 40 ml (each ;; a).

5 g of antibiotic X-5108, extracted by extraction from raw broth after fermentation, are dissolved in 40 N01 of the upper phase and the eshyl acetate – isogrotanol 0.1 MNa2HPO4 system (12: 9: 20 by volume) is used. The number of transfers 200, the peak for pipes after 200 transfers 159, the coefficient distributed 3.88.

Fractions 150-170 are collected, 1-butanol is added to the antibiotic extract in the organic phase, the aqueous phase is discarded, the organic is washed with four portions of water, water is distilled off as azeotrset, petroleum ether is added and 3 g of antibiotic are added, the activity of which is 2 times the activity of the source material.

EXAMPLE 4 To purify antibiotic X-5108 by chromatography on Sephadex LH-20, a solution of sodium methylate (wet 1-x paper) to pH 8-9 was added to a 300 mg sample of an antibiotic previously cleaned with a counter current distribution. added to column with Sephadex LH-20 (290 x 41 mm), equilibrated with alcohol FOR. The column is developed with THO alcohol and the antibiotic is removed from the column as a solution with a volume of 950-1200 ml (active fractions). Subsequent fractions contain a number of colored areas with little biological activity. The active fractions are drained, evaporated to a small volume, ether and petroleum ether are added to the residue, and the antibiotic is precipitated as a yellow sodium salt, giving only one yellow spot during thin-layer chromatography (F-254 skipping), detected in UV light with Rf 0 , 29 (silica gel / diatomaceous earth, bioautography).

I p e m i r 5. 0.464 g of the sodium salt of antibiotic X-5108, obtained by chromatography on Sephadex LH-20, is dissolved in 4.6 ml of ice-cold water, 10 ml of ethyl acetate is added, transferred to a separatory funnel and added 1 ml of NaH2PO4 solution (50 g of NaH2PO4H2O in 100 ml of water) The precipitate formed initially dissolves upon shaking. The organic phase is washed with four portions of ice water (1 volume by volume), the azeotrope is distilled from it, the concentrate (2 ml) is diluted 2. With a volume of ethyl acetate, diethyl ether is added dropwise until cloudy and 30 ml are added.

MO

petroleum ether. The precipitate of antibiotic X-5108 is filtered off and taken at room temperature with t11at.

PRI me R 6. The basic ration is prepared using the following ingredients (wt.%):

Crushed Yellow

corn grain56,075

M with and bone

flour (50% protein) 4,000

Fish meal

(60% of proteins) 4.00 (

Soyeva flour

(50% discount) 28,000

Dehydrated

alpha alpha flour1,000

Animal Fat4,000

Methionine 0,200

Mineral phosphate 0,250

Calcium carbonate., 1,200

Iodized salt 0,250

Vitamins, 000

Mineral components0,025

To this diet, antibiotic X-5108 was added to the norm of 50 g of antibiotic per 1 kg of diet.

llpoBOiViT experiment by non-forcible feeding of poultry (one-day-old chickens) with an ration supplemented with antibiotic X-5108 for 15 days. Then they were weighed taking into account the amount of KopNia eaten, and the feeding efficiency was determined by comparison with control groups.

The average additional weight gain for each; experimental group was divided by the average weight gain of the control group and the quotient multiplied by 100.

The weight gain for 2 weeks (average value for 40 kg) for the control and test (50 g antibiotic per 1 kg of diet) group is 154 ± 8 and 185 12 g (considering the rms error) for the average value), or 100 and 120% respectively. The feeding efficiency is 1.55 and 1.39, the feeding efficiency improvement is 12%.

With the introduction of the various coats of the antibiotic X-5108 into the diet, the results are shown in Table. 9.

From the data table. 9 shows that chickens fed a diet supplemented with 50 mg of the antibiotic X-5108 per 1 kg of the ration grow faster than in the control group.

Table 1

Good growth on all the listed liquid media, the runway with tryptone (dark pigment, negative gel reaction to indole after 3 days); nutrient broth GDA, Chapek's broth with sucrose, Krainsky broth with glucose and broth with glycerin and calcium citrate.

Good growth for 8 days at 24, 28, 32 and 37, but not at 42 ° C.

Broth with organic nitrate 8

13

Gelatin (15%) at 18 ° C

12

Agar-agar with peptone and iron (Yushgper)

Litmus milk

13

Egg firapDsfset

YU

No nitrate reduction is observed, no gas is formed.

Also

No liquefaction of gelatin

Weak liquefaction

Chromogenic - melanin is formed

The color of the litmus does not change; no cloudiness or clarification

Light brownish color; no clouding or lightening

A little bit of honest dark pigment

Nitrogen source

Decoction of m sa flour

VU-100 (Commercial Solven Corporation)

Proflo (Treders Oil Mill

Company)

Oats on Flour (Kusker Oats

Company)

Soyloz (defatted coeabLx bean flour) Belkova peanut flour

Protein powder from coconut

nuts

Solid tomato

pasta

Flour with flax butter

Corn flour

Dried extract from corn grain with soluble substances (X Worker)

Saludri (Shinley Distiller Fish Meal (Gorton) Fish Waste (Wilknson)

A mixture of milk sugar with albumin

Torul Dried Yeast Autolysate National Yeast

Soybean Gndrolizat, formed under the influence of knowledge

Whey Protein Hydrolysis for Acid Lysis (Turon Mills)

Homogenized pressed

Protopepton N 366 (Wilssch Laboratories)

Hard corn cobs

 The incubation period is f days.

 Concentration of nitrogen source 2%.

Table 4

Activity antifaiOTHKa. u / ml

50 45

23

32

thirty

41

50

57 37

33

74 51 25 16

50 38 J6

33

12

27

18 26

Component nutrient medium

Table 5

The activity of the antibiotic, u / ml

OiBap from m with flour and dextrin

The residue after the fermentation of grain (VU-100) and dextrin, Proflo and dextrin

Fish waste

Dried extract from grainfish with soluble (X. Walker) and dextrin

Proflo and dextrin Proflo and dextrin Proflo and dextrin Proflo and dextrin

Proflo and glucose Proflo and starch Proflo and glycerin Proflo and brown sugar Proflo and maltose Proflo and lactose Proflo and mannitol

 Incubation period of 6 days

BezSaSOz.

 Without CaCO3 or KjHTO.

 The concentration of Proflo is 0.25%.

 The concentration of Proflo is 2.0%.

40 67

52 40

138

nineteen

34

12 47

21

21

35

14

22

26

25

one

Starch profile

12

1 BZjicyuieiffloro ra, a soluble substance after extraction of maize kernels, 1 dextrin, 0.1 K2HP04 and 0.1 CAS02

13

1 L-glutamata sodium 1 dextrin, 0.1 hardness of the substance of corn cobs, 0.15 Kj HPO4 and 0.05 MgSO4

eleven

1 solids of tomato paste, 1 dried soluble substance after extraction of maize kernels, cumin starch, 0.5 glucose, 0.5 CAC02 and 0.1

K2HP04

four

0.5 Profio, 0.5 dried soluble substance, extraction of corn zerin, 0.5 tomato paste solid matter, 0.5 iiacibi from meat extract (Progupepton N 366), starch I, 0.1 CASO3 and 0.1 KgNPO

1. Profo, 0.5 liquid, obtained when processing corn cobs,, 0 corn starch 0.1 CAC0 and 0.1 K2HP04

Fermenter volume of 454 liters (loading 295 liters of medium), made of stainless steel. Fermenter volume of 19,000 liters (load 14,500 liters of medium), made of carbon steel. Fermenter volume 3400 l (load 2220 l of medium), made of carbon steel.

Table 7

67

100

200

91

260

23

1.0 sodium glutamate, 1 dextrin, 0.1 K2H10.0.0 MgS04, 1.0 mixture of amino acids and 1 dextrin,

0.1 К2НРО4,0,1 salts VSP№ G, ВВ1, Chapek-Doks broth

1.0 arginine, 0.1 dextrin, 01 K-2 NU4, 0.1 CAS2 and 0.05 MgSO4

1.0 D, 1-aspartic acid, 1, dextrin, 0.1 К2НРО4,0,1 СаСОз 0.05% MgSO4

0.1% sodium glutamate, 0.6 (NN4) 2NU4 1 glucose, 1 dextrin, 0.1 K2NU4.0.1 CaCO3, 0.1 KC1 and 0.05 MgSO4

0.33 (NN4) 2 SO4, 1.5 glucose, 0.1 sodium citrate, 0.05 sodium acetate, 0.5 sodium chloride, 0.025 MgS04-7H20, 0.01 K2HU4, 0.01 KH2PO4.0.3 CAS02 , 0.001 MnS04-4H2O, 0.004 ZnSO4 VHjO, 1.6-10 K, Cr, 03% sucrose, 0, nitrate, sodium, 0.1% K, HP04, 0.05% u, uui / I ferrous sulfate.

24 Table 8

7,6

11.5 4.4

0.8

6,6

20 MgSO ,. 0.05% potassium chloride,

48

Control

Antibiotic

1.54

149 (100)