The method of obtaining tylosin

 

(57) Abstract:

Usage: biotechnology, receiving antibiotic tylosin, finds application in the field of agriculture. The essence of the invention: production method of tylosin culture-producer grown in optimal conditions, separate the mycelium, the filtrate of the culture fluid absoluut, absorb from the desalted filtrate tylosin in the acidic cation exchange resin, elute tylosin phosphate salt and dried directly from the eluate. 4 C.p. f-crystals, 1 Il.

The invention relates to the field of biotechnology and related to tylosin is a macrolide antibiotic of broad-spectrum antibiotics used in agriculture.

For disease prevention and treatment of farm animals and poultry tylosin can be used orally ( in the form of feed preparations containing tylosin raw low degree of purification) or parenteral (in the form of drugs tylosin base and its water-soluble salts containing not less than 80% tylosin).

Depending on the nature of the use of the drug received tylosin differ and the ways of getting from a relatively simple way of extracting Ustashi method for the production of tylosin is to crop producer on a suitable nutrient medium under optimal conditions and subsequent drying of the culture fluid, resulting in a receive feed product containing 2-5% of antibiotic raw remnants of the culture medium and mycelium producer [1].

The method allows to obtain the drug tylosin, called "Traditinal", with a low content of the active agent and having a high hygroscopic property. In the drying process of the drug are a fire hazard conditions, because drying the drug is prone to fire.

In most cases, in practice, there are methods for obtaining purified tylosin, and for a long time for these purposes applied extraction method.

Described, for example, a method of obtaining tylosin, providing for the crop producer, the separation of the mycelium, the extraction of the target product from the filtrate of the culture liquid butyl acetate, reextraction antibiotic from the organic phase acidified water, re-extraction of the aqueous system with butyl acetate in alkaline medium, the processing of the extract activated carbon and precipitation of the desired product in the form of a base [2].

This way though, and allows you to get tylosin high purity (90%), but is characterized by a multi-stage, significant losses of the desired product (50%) and obrazovanie.zamuzhem additional stage sorption of the antibiotic.

Thus, in particular, a patented method of obtaining tylosin, which consists in the fact that culture-producer is grown in a nutrient medium, separating the mycelium, the cultural filtrate liquid is extracted with an organic solvent (chloroform, acetone, benzene), organic extract of tylosin adsorb on these sorbents like activated charcoal, gel, aluminum hydroxide, or on such ion-exchange resins, as XE 64, IRC 50 (weakly acidic cation exchange resin), Dowex 50 (strongly acidic cation exchange resin), or resins based on carboxymethylcellulose. The target product in the form of Telesina-base elute lower alcohols or lower alcohols containing up to 50% lower ketone, and get tylosin 70-80% purity [3].

Despite the reduction in volumes of used organic solvents, challenges remain for their regeneration and other disadvantages of the extraction methods.

Further improvement of the methods of obtaining tylosin consisted in the rejection phase extraction and sorption of the antibiotic from the aqueous phase.

Known, for example, a method of obtaining tylosin, comprising the steps of growing a crop producer in suitable facilities the product on nonionic polymeric resin type ER-180, amberlit, duolith and elution of water-alcohol or water-acetone solution. The solution tylosin base then discolor on macroporous resin and precipitated by salting out with sodium chloride.

To obtain water-soluble tartrate or phosphate tylosin to a solution of tylosin base add equimolar amount of the appropriate acid and the solution is dried [4].

The relative disadvantage of this method is the multistage and the need to use organic solvents as eluents, which again raises the issue of regeneration of solvents.

The purpose of the invention is to simplify technology for tylosin and getting the fodder drug - phosphate tylosin high quality.

This goal was achieved due to the fact that the eluate from the ion exchange resin without any additional processing dried, obtaining the target product.

The essence of the proposed method is as follows.

Culture-producer grown in optimal conditions, separate the mycelium, the filtrate of the culture fluid absoluut, absorb from the desalted filtrate tylosin on acidic cation exchanger, elwer is used any culture, biosynthesise tylosin, mainly active strains Str.fradiae.

It is preferable to use bred at the research Institute of genetics and selection of industrial microorganisms highly productive strains Str. fradiae Vniigenetika-29A and Str. fradiae Vniigenetika-29S, deposited in culture Collections research Institute of genetics and selection of industrial organisms under registration numbers PMBC-S-851 and PMBC-S-1112, respectively. These strains currently provide for the sustainable biosynthesis of tylosin on the level 7000-12 500 u/ml in the laboratory and at the level of 5000-7500 IU/ml in an industrial environment when using nutrient media of relatively simple composition.

The producer is grown on a suitable nutrient medium suitable for the culture conditions.

After completion of the fermentation stage is separated by known techniques mycelium and culture filtrate liquid absoluut.

Desalting can be carried out, for example, by electrodialysis.

Preferably obessolivanie the cultural filtrate liquid ion-exchange method, for which the filtrate is first passed through a column of anion exchange resin in th environment.

Sorption of the antibiotic is carried out on srednekaloriynom the cation type was made the KRF-2 (phosphorylated polystyrene blended with 2% divinylbenzene) in the sodium-hydrogen form weak acid carboxylic cation exchanger type KB-2E in the sodium-hydrogen form or in a similar resins.

For the preparation of a mixed sodium-hydrogen form cation-exchange resin in the hydrogen form is treated with two volumes of buffer solution, which represents a 1 n sodium hydroxide solution, neutralized with phosphoric acid to pH 3.5.

Alualu tylosin from the cation exchanger can be of various phosphate salts, phosphate buffer, phosphate salts of potassium and sodium varying degrees of substitution, but the most suitable solution disubstituted phosphate. In all these cases in the eluate goes phosphate tylosin.

The pH of the eluting solution is not critical and may be in the range of 8.5 to 10.5.

Dry the eluate are different methods, but the most suitable method is spray drying.

Be subjected to drying can the entire eluate or a separate faction.

The output from the ion exchange column of tylosin phosphate and inorganic FOSFA the ins and inorganic phosphate cation exchanger KB-2E 1 N. a solution of disubstituted phosphate.

As clearly seen, first in the eluate from the column is only phosphate tylosin and only then appear inorganic phosphates. According to the schedule it is easy to calculate that by passing the eluent in the same volume as the resin, the ratio of the concentrations in the eluate of tylosin phosphate and inorganic phosphate is approximately 4:1. This ratio is very favorable for extraction and further purification of phosphate tylosin, but in this case desorbed only half of tylosin.

By passing 1.5 volume of eluent ratio of tylosin phosphate and inorganic phosphate in the eluate is reduced to 1:1 and the amount of elution solvent desorbed already 80% tylosin.

In case of further increasing the volume of eluent used tylosin desorbed completely, but the ratio in the eluate of tylosin phosphate and inorganic phosphate is reduced to 1:3,5.

The first fraction of the eluate enriched Telesina, can be subjected to drying, and the second fraction of the eluate enriched in inorganic phosphates, can be used as eluent in the following cycles of production. It is particularly expedient if the received tylosin phosphate ro equally can be subjected to drying the entire eluate, especially if you get the product in the future be used as a feed additive.

The dry product obtained by drying the entire eluate contains 35-40% phosphate salt tylosin, 50-60% of the inorganic phosphate salts, 1.5 to 2.5% of water and 6.0-10.0% of organic compounds.

Product obtained by drying enriched Telesina fraction contains 45-55% phosphate salt tylosin, 45-50% of the inorganic phosphate salts, 1.5 to 2.5% of water and 3-5% of organic compounds.

Get in any case the product is not hygroscopic, is not flammable and soluble in water.

An additional advantage obtained by the proposed method of drug tylosin is the presence of significant quantities of inorganic phosphate is a necessary component of feed for livestock and poultry.

Thus, when the simplified method of obtaining water-soluble drug tylosin by excluding independent stage of converting the base tylosin soluble salt get tylosin in feed medication of high quality.

Not known to forage drugs tylosin, enriched with phosphates, as well as ways of proizvodstva.

Compared with the method of the prototype, which allows to obtain a basis of tylosin a high degree of purification, the proposed method aims at the preparation of phosphate tylosin moderate frequency, having an independent consumer value.

The degree of purity of the obtained phosphate tylosin (i.e., the content of active principle in the preparation of tylosin) is significantly higher than that in known feeding drugs tylosin and significantly simplify the method further purification, if there is a desire to obtain a crystalline phosphate tylosin a higher degree of purification.

In fact, it was found that previously known as an intermediate product in the eluate from the ion exchange column can be used after drying as a final target product with novel properties. Contrary to popular belief that to obtain high-quality dry drug tylosin you need to remove excess inorganic salts, intentionally stored in a drying solution of excess phosphates and the obtained target product with new properties. Developed technical solution is not obvious, and the proposed method sooty tylosin proposed method has the following advantages:

is more simple and high-tech, because it provides separate (independent) phase transfer of tylosin base in salt form;

eliminates the possibility of fire, burning and sticking of the product during the drying process, as it dries the product is not flammable because of the high content of inorganic phosphates;

reduces the likelihood of inactivation of tylosin in the process of drying and storage, as inorganic phosphates have on antibiotic stabilizing effect;

results in a well-granular and crystalline non-caking product, due to the high content of inorganic phosphates.

In addition to the direct use obtained by the proposed method tylosin as a feed additive target product can serve as a source of crystalline tylosin, intended for parenteral use.

Example 1. The culture of strain Str.fradiae Vniigenetika-25A sown in the fermenter with a volume of 100 l of a medium containing (wt.%) flour fish (2,0), molasses (2,0), potato starch (1,5), one-deputizing the ammonium phosphate (0,02), sodium chloride (0,2), magnesium sulfate (0,2), chalk (0,3), soybean oil (1,0), water water the speed of the mixer 220 rpm The air supply is 1 volume of air to 1 volume of medium per minute. Foam breaking is carried out by continuous addition of soybean oil. Total consumption of soybean oil in the process of defoaming amounted to 2% of the volume of the medium. The duration of fermentation was 7 days.

There was obtained 70 l of culture liquid with a concentration of tylosin 7 g/l Culture fluid fed into the reactor with a capacity of 100 liters and bring back 350 ml of a solution of phosphoric acid. The mixture was incubated for 1 h at a temperature of 50omake it an 8.4 l of a 20% suspension, one-deputizing calcium phosphate and heated at the same temperature and under stirring for another 1 h, the Suspension is filtered through a belting fabric under vacuum. Get 8,6 kg sludge with a moisture content of 55% 73,6 l of leachate concentration of tylosin 6.2 g/L.

The filtrate is alkalinized to pH 9.5 and at a speed of 30 l/h is passed through a closed system with electrodeionization consisting of 5 parallel-connected standard cells, when the voltage of 10-20 V and current of 2 A. After 5.5 hours the content of cations in the circulating solution decreased from 0.15 to 0.01 mol/L.

75 liters of desalted solution containing tylosin 5.7 g/l and a pH of 6.5 with a speed of 15 l/h is passed through ion-exchange Colo is orodno form.

Alualu tylosin carried out with a column passing through it at the rate of 2 l/h 6 l 1 N. of one-deputizing solution of sodium phosphate with a pH of 9.5. The appearance in the eluate tylosin determined spectrophotometrically at a wavelength of 283 nm. The selection of the active fractions begin when the value of the extinction of 2.0. During elution the value of the extinction increases dramatically, reaching a maximum value of 2200, and then decreases. The first fraction of the eluate has a volume of 7.5 l and contains 42.6 g/l of tylosin phosphate and 39.4 g/l of inorganic phosphates.

The resulting fraction is enriched Telesina of the eluate is dried by spray method and receive 634 g of the drug tylosin phosphate with a moisture content of 3% and a content of tylosin 50,3%. Output tylosin is 65% of the content in the culture fluid.

The resulting product is a light yellowish brown hygroscopic powder, completely soluble in water.

After separation of the enriched Telesina fractions of the eluate process elution continue and finish the process by reducing the extinction to the value of 3.0. This fraction of the eluate has a volume of 6.7 l and contains 12 g/l of tylosin phosphate and 83 g/l of inorganic phosphates.

Example 2. The culture of strain Str.fraliae Vniigenetika-25S vysehradsky (1,0), sugar beet molasses (2,5), yeast BVK (0,25), one-deputizing the ammonium phosphate (0,2), soybean oil (1,0), chalk (0,2) and tap water (up to 100.0). In the fermentation process for defoaming add a 2.0% soybean oil. The fermentation process are as described in example 1.

After completion of the fermentation process receive 61 l of culture fluid containing 7.5 g/l of tylosin. The culture fluid is subjected to coagulation and filtration as described in example 1. The resulting precipitate obtain 64 g of a filtrate of the culture fluid containing 6.8 g/l of tylosin. The filtrate absoluut by passing it at a speed of 30 l/h through a system of 10 ion exchange columns loaded anion exchange resin AV-17 in the hydroxide form and the cation exchanger Ku 2 x 20 in hydrogen form. Column with anion exchange resin and cation exchange resin arranged in series. The displace liquid from the column with distilled water.

The fraction containing tylosin, determined spectrophotometrically at a wavelength of 283 nm. Collect the solution having values of extinction from 2.0 (at the beginning of the process) to 3.0 (after reaching the maximum value of the extinction and decline). The content of inorganic salts is controlled by the conductivity of the solution. The desalted gnosti 0.2 MS. 71 l of desalted solution is passed at a rate of 20 l/h through the ion exchange column with a diameter of 8 cm and a height of 100 cm, filled with weak acid cation exchange resin KB-2E in a mixed sodium-hydrogen form. The solution was ousted from the column with water, and the whole tylosin sorbed on the cation exchanger, alualu tylosin performed using 6 l of 1 M solution of disubstituted phosphate, which is passed from the bottom with a speed of 3 l/h Contents tylosin in the eluate control spectrophotometrically at a wavelength of 283 nm. Collect the eluate, which is characterized by extentia from 2.0 (at the beginning of the process) to 3.0 (after a maximum of extinction and decline). There were collected 14,2 l of the eluate containing 27.5 g/l of tylosin and 48.6 g/l of inorganic phosphates. The pH of the eluate was 6.8. The eluate is dried to dryness on the spray dryer.

Received 1026 g of light yellowish brown hygroscopic powder with a moisture content of 4%, containing 34.7% of tylosin phosphate and 60.5% of inorganic phosphates. Output tylosin is 78,0% of the content in the culture fluid.

Thus, clearly shows the advantages of the proposed method of obtaining tylosin prior known methods.

Sources of information

1. Ha is And Minidrama the USSR. Vol.1 1991 - 52 C.

2. Auth. mon. NRB N 20.697, CL C 12 D 9/16, 1978.

3. U.S. patent N 3178341, CL 424-120, 1965.

4. U.S. patent N 4568740, class C 07 H 17/08, 1986. the prototype.

1. The method of obtaining tylosin, providing for the crop producer under optimum conditions, the separation of the mycelium, the sorption of the target product from the filtrate, alualu and drying, characterized in that before sorption conduct a preliminary demineralization of the filtrate, and sorption was carried out in the acidic cation exchange resin.

2. The method according to p. 1, characterized in that the demineralization of the filtrate is carried out by successive contact with the anion exchange resin type EDE-10p or AV-17 in the hydroxyl form and type cation exchange resin Ku 2 20 in the hydrogen form.

3. The method according to p. 1, characterized in that as acidic cation exchange resin using weak acid carboxylic cation exchanger type KB-2E in the sodium-hydrogen form.

4. The method according to p. 1, characterized in that alualu spend solution disubstituted phosphate with a pH of 8.0 to 9.5.

5. The method according to p. 4, characterized in that the drying is subjected directly to the eluate.

 

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