SI9600120A - New and improved fermentative procedure for the production of clavulanic acid and its salts - Google Patents

Abstract

A process for preparation of clavulanic acid comprising fermentation of a clavulanic acid producing species of Streptomyces in a fermentation broth containing assimilable sources of carbon and nitrogen, wherein the concentration of phosphorus in the fermentation broth is less than 0.15 % w/v.

Description

The present invention is within the scope of the pharmaceutical industry and relates to a new and improved process for the preparation of clavulanic acid and its salts by fermentation of the microorganism Streptomyces sp. P 6621 FERM P 2804.

A technical problem

There is a continuing need for a new and improved microbiological process for the preparation of clavulanic acid and further processing into its alkali salts, such as potassium clavulanate, to give the desired compound in high yield in aqueous culture broths obtained after fermentation with a clavulanic acid-producing microorganism.

Stanie techniques

Clavulanic acid is a generic name for (2R, 5R, Z) -3- (2-hydroxyethylidene) -7-oxo4-oxa-1-azabicyclo / 3.2.0 / heptane-2-carboxylic acid of the following formula:

HO

HH ₀ / CH ₂ OH j /

F = C \

H '' COOH

Its alkali salts and esters act as inhibitors of β-lactamases produced by certain gram positive and gram negative microorganisms. Clavulanic acid and its alkali salts have a synergistic effect in combination with β-lactam antibiotics of the penicillin and cephalosporin species in addition to the inhibitory activity of 8lactamases. Therefore, clavulanic acid and its salts are used in galenic preparations to prevent deactivation of β-lactam antibiotics. Commercial preparations contain the more stable potassium salt of clavulanic acid (the acid itself is quite unstable) in combination with amoxicillin trihydrate, its salts or in combination with other antibiotics.

Clavulanic acid is prepared by fermentation of a clavulanic acid-producing microorganism, such as various microorganisms belonging to different Streptomyces strains, such as S.clavuligerus NRRL 3585, S.jumoninensis NRRL 5741, S.katsurahamanus IFO 13716 and Streptomyces sp. P 6621 FERM P 2804.

The use of S.clavuligerus NRRL 3585 in the process of the preparation of clavulanic acid by fermentation is described in patent GB 1 508 977, where the medium containing between 0.1% and 10% of an organic nitrogen source, such as yeast hydrolyzate, is most suitable for fermentation, meat and fish extract, seed proteins, corn syrup and various hydrolysates (in the case of a defined medium, urea, valine, asparagine, glutamic acid, proline and phenylalanine) are used, between 0.1% and 5% of organic carbon (starch hydrolyzate, dextrin , sucrose, lactose, glycerol and its esters, vegetable oils and animal fats). A particularly suitable and inexpensive medium was the combination of soybean flour, dried filtrate from beer production and dextrin. Inorganic salts can also be added to chemically defined media: sodium, potassium, magnesium, ferric and zinc chlorides, and sodium, magnesium and ferrous sulphates, and phosphoric acid sodium and potassium salts. Manganese, nickel and cobalt salts and vitamins can also be added to the culture medium, and antifoaming agents are required to control foaming. The result of fermentation is fermentation broth rich in clavulanic acid and its salts.

After fermentation, the resulting aqueous culture broth can be cleaned and concentrated by conventional methods comprising e.g. filtration and chromatographic purification as illustrated in patent GB 1 508 977, which describes a fermentation process for the preparation of clavulanic acid with the Streptomyces clavuligerus microorganism and its isolation from the filtrate culture. Among other things, it also shows that the salts of clavulanic acid can be obtained by adsorption of the clavulanate anion from the filtered soup onto an anion exchange resin, eluting the clavulanate anion with an electrolyte, and the resulting solution being salted and then the solvent removed. This process yields the acceptable yields of the desired substance, but the process is based on the demanding purification by chromatographic methods, as is well known, and the use of resin columns requires considerable investment, which limits the large-scale use.

The great novelty and improvement that enables the use on a large scale is the method of isolation of clavulanic acid and / or PCT application WO 95/23870 described in our Slovenian patent P-9400107 or equivalent PCT application WO 95/23870. its pharmaceutically acceptable alkali salts, such as potassium clavulanate, based on the removal of mycelium and other suspended particles from the fermentation broth by microfiltration and then by ultrafiltration. The purified broth is then concentrated by reverse osmosis and can be directly extracted with a water-immiscible organic solvent, yielding the desired high purity compound after the isolation process is completed. This method avoids the time-consuming conventional methods of isolation and chromatographic purification of the product.

It is known from the literature that in order for fermentation to be as successful as possible, the conditions in the fermenter need to be optimized. Article by Yung T.B. et al., Ferm.Technol.1972, Today 163 describes batch fermentation as a dynamic process in which the environment is constantly changing so that microbial cells are constantly subject to change. Changes in the environment of the cell are expressed as significant changes in the cell's physiological state, so that the cells live constantly under certain stress factors that prevent their optimal growth and the production of the desired metabolites in them.

For example, patent GB 1 543 563 describes a modified fermentative process for the preparation of clavulanic acid using the strain S.clavuligerus NRRL 3585, maintaining the pH of the medium in the range of 6.3 to 6.7, thereby increasing the yield of the desired compound.

In addition to physical parameters such as pH, temperature, oxygen content, the amount of assimilating nitrogen and carbon sources and the amount of various mineral sources can be controlled and maintained in the fermenter. Such guided fermentation is called nutritional fermentation, which is well known in the literature. The use of such fermentation is described in the book by Crueger W. et. al., BIOTECHNOLOGY 1984, p. 197-237, which describes, among other things, the usefulness of nutritional fermentation in the production of antibiotics.

An example of controlling and maintaining the amount of an assimilating source of nitrogen and carbon in a fermentation broth is described in an article by LEE J.S. et. al., Cor.Jour.Microbiol. 1978, Vol. 1, p. 21-29, which describes that the production of penicillin can be improved by the controlled addition of an assimilating source of nitrogen and carbon by constantly following the needs of the microorganisms in the fermenter.

In an article by Lilley G. et. al., J.Chem.Tech.Biotehnol. 1981, Vol. 31, p. 127-134. it is described that the production of certain antibiotics by bacteria of the Streptomyces species can be regulated by changing the concentration of the assimilating source of nitrogen, carbon and phosphorus source accordingly. Thus, for example, the production of thienamycin in the fermenter begins only when the amount of phosphorus drops to zero.

The use of feeding and controlling the amount of an assimilating carbon source in the process of producing clavulanic acid is first described in EP-B-0 182 522, which describes a method for the preparation of clavulanic acid by batch fermentation of the microorganism S.clavuligerus, finding a significant improvement in the process if the source carbon, like glycerol, is added to the medium during fermentation, either continuously or at intervals, and it is very important to maintain the carbon level at a sufficiently low concentration below 0.5% (w / v), but in no case should the carbon level grow over 2%. The examples illustrate that a significant improvement in the increased yield of clavulanic acid was observed when a carbon source, such as glycerol, was not present in the starting nutrient medium and was added continuously or intermittently during fermentation. They state that, after 160 hours, the concentration of clavulanic acid in the fermentation broth was around 1400 / µg / ml, a marked improvement over the previous procedures.

From Butterworth D., Drugs Pharm.Sci.1984, Vol 22 (Biotechnol. Ind. Antibiot.) P. 225-35, showing an overview of published fermentation processes for the production of clavulanic acid with known species of Streptomyces microorganisms, it is apparent that the processes for the production of clavulanic acid by S. clavuligerus microorganisms published in the patent literature are based solely on pilot devices and that the processes for producing clavulanic acid with other microorganisms of the genus Streptomyces are limited to a small or laboratory scale. Also, the fermentation process for the production of clavulanic acid described in EP-B-0 182 522 is shown only on a pilot plant scale, so that the published literature so far does not describe the fermentation of clavulanic acid by Streptomyces microorganisms on an industrial scale (large-scale operation).

Description of a solution to a technical case problem

The invention is based on the task of substantially increasing the yield or concentration of clavulanic acid in an aqueous culture broth prepared by fermentation of the microorganism Streptomyces sp. PP 6621 FERM P 2804.

This target was achieved in such a way that the microorganism Streptomyces sp. P 6621 FERM P 2804 described in JP Kokkai Application No. 79-72 085 (Early-Disclosure No. 80-162993) and produces clavulanic acid grown at a temperature between 20 ° and 30 ° C, preferably at a temperature between 23 ° and 25 ° C and at a pH of the medium between 6.5 and 7.5, preferably in the controlled maintenance of the pH of the medium between 6.8 and 6.9 with an aqueous solution of an alkali agent, under aerobic conditions and in a nutrient medium containing a carbon source, a source of nitrogen and mineral salts.

The term cultivation means the free aerobic growth of an organism that produces clavulanic acid in the presence of a carbon source, a nitrogen source, and assimilable mineral salts. Such aerobic growth takes place in a nutrient medium in which the nutrients are dissolved or suspended. Growing can be done on an aerobic surface or submerged. The nutrient medium can be composed of complex nutrients or chemically defined ingredients.

Nutrient media used for cultivation of Streptomyces sp. P 6621 FERM P 2804 are complex sources of organic nitrogen in the amount of 2% to 15%, such as yeast, fish and meat extracts, vegetable proteins such as soybean flour, protein hydrolysates, such as peptones. Optionally, chemically defined nitrogen sources such as various amino acids may also be used.

Any assimilating carbon source, preferably one with a chemically defined structure, can be used as a carbon source in the amount of 1.5% to 7.5% for the nutrient medium. Starch or starch hydrolysates such as dextrin, sucrose, lactose, maltose and other sugars or glycerin and glycerol esters such as glycerol trioleate (Estol) may be used. Various vegetable oils such as cottonseed oil can be used as a carbon source. The addition of anti-foaming agents, such as Synperonic (commercial name of I.C.I., GB), is essential for controlling the foaming in the fermenter medium.

Mineral salts are added to the abovementioned media, in an amount of about 0.05%, such as MgCl2, FeCl2, ZnCl2, CuCl2, MnSO4, FeSO4, Na2SO4 and phosphoric acid sodium or potassium salts, such as NaH2PO4 traces may include other elements such as cobalt et al.

Streptomyces sp. P6621 FERM P 2804 can be grown in the media described above in glass conical flasks where aeration is performed by shaking on rotary shakers or in conventional stainless steel aerobic fermenters equipped for mixing and supplying sterile air. The working volume of the fermenter ranges from 25% to 85% of the total capacity, and the fermentation can be done batch or continuous.

The highest yields of clavulanic acid are obtained up to a maximum of 6 days.

According to an object of the invention, where we describe a batch fermentation of Streptomyces sp. P 6621 FERM P2804 First, the most appropriate clone of the micro-organism is isolated by a selection process. Next, we prepare a laboratory inoculum, which is a high-yield culture on slopes or rubble.

This is followed by fermentation of the micro-organism, with the first inoculation of the laboratory inoculum into a propagator (pre-pre-fermentor) with a pre-prepared sterilized medium for best results. The growth medium at this stage is vegetative, vegetative and is intended only for the multiplication of the micro-organism and not for the production of clavulanic acid. When the culture reaches the desired growth rate (18 to 25% discoloration in 0.5 to 1.5 min) (percentage discoloration is a measure of the vitality of the culture), it is germinated in a pre-fermenter with a pre-sterilized culture medium. This stage is also vegetative and is intended only for the multiplication of the micro-organism. It is only after the culture reaches the desired growth rate that it is inoculated into a sterilized fermenter medium, where the microbiological process of obtaining clavulanic acid is then carried out.

A nitrogen source, a carbon source and mineral salts such as the above are used for the composition of the culture medium, and the growth and microbial production of clavulanic acid is illustrated in the example.

It is known from EP-B-0 182 552 that the carbon content of the inoculum medium is between 0% and 2% (w / v) (weight / volume), preferably from 0% to

0.5% (w / v). This importance is also illustrated in Examples 1 to 3, where they get the best results if they are not present at all in the initial medium of the carbon source. In accordance with Example 1 described above, after fermentation was complete, high yields or concentrations of clavulanic acid of about 1400 pg / ml were achieved after 160 hours.

Unlike the microbiological process for producing clavulanic acid by batch fermentation of S.clavuligeris, as described in patent EP-B-0 182 522, where they have been markedly improved in such a way that they are introduced into the initial medium where the carbon source was not present fermentation was added continuously or intermittently to the carbon source in such a way that the carbon concentration was maintained below 0.5% (w / v) or at best below, but in no case the concentration of the carbon source should at any time exceed 2%, we now surprisingly find that clavulanic acid is produced in significantly higher yields or in higher concentrations in aqueous culture broths by fermentation of the microorganism Streptomyces sp. P 6621 FERM P2804, where an assimilation carbon and nitrogen source and a phosphorus source are present in the initial culture medium and additional amounts of phosphorus source and assimilating nitrogen source are added during fermentation.

According to the invention, it has further been found that the absence of a carbon source in the source medium, as stated in EP-B-0 182 552, is not significant and that an initial medium can be used where the carbon content (eg glycerol, glycerol trioleate and maize) starch) is more than 5% (w / v). In order to achieve the highest yields of clavulanic acid, it is important to have a sufficiently high phosphorus concentration in the initial medium, but in any case higher than 0.02% (w / v) and less than 0.15% (w / v). In the course of fermentation, until a maximum of forty hours of fermentation is maintained, a sufficient phosphorus concentration is maintained between 0.15% and 0.005% (w / v) by adding a phosphorus source, then allowing the phosphorus concentration to decrease to zero (w / v). This regulates the growth of biomass and prevents the early production of clavulanic acid. (When the production of secondary metabolites begins in the fermentation process, which certainly is clavulanic acid, the production of live biomass slows down.)

In accordance with the invention, it was further found that the addition of ammonium hydroxide to the fermentation broth from the beginning to the end of fermentation as the sole assimilating source of nitrogen and at the same time the pH regulator did not have a positive effect on the fermentation process. Too much ammonia is a poison to the cells and too low a pH to lower production of clavulanic acid, so first soybean meal is added to the fermentation broth as an assimilating nitrogen source, then ammonium hydroxide is added.

If we used ammonium sulfate instead of ammonium hydroxide as the assimilating source of nitrogen, and sodium hydroxide as the pH regulator, we found that biomass starts to fall much later than when the only assimilating nitrogen source and pH regulator ammonium hydroxide, as can be seen in graph 1. Keeping the biomass as high as possible is extremely important when using continuous fermentation to produce clavulanic acid.

During fermentation for 5 to 6 days, the phosphorus source concentration is maintained between 0.005% and 0.15% (w / v) during the biomass growth phase. During the whole fermentation, the concentration of the assimilating source of nitrogen is maintained between 0.5% and 15% (w / v), preferably about 1%, and the concentration of the assimilating carbon source ranges between 7.5% and 1.5% (w / v). Thus, in a fermentation broth, a clavulanic acid concentration of about 3500 mg / l (3500 pg / ml) is obtained, which is significantly higher according to the methods known in the literature.

We consider such a marked increase in the yield of clavulanic acid in a fermentation broth following a microbiological process with Streptomyces sp. P 6621 FERM P2804 is achieved by both favorable selection of the strain and the best selection of the source medium and maintaining the pH by a suitable combination of alkaline agents and maintaining the concentration of phosphorus and nitrogen sources during fermentation in defined areas.

To the best of our knowledge, it has not yet been described in the literature that the addition of a phosphorus source and an assimilating nitrogen source can regulate and guide the production of clavulanic acid by Streptomyces microorganisms.

Achieving such high yields of clavulanic acid in fermentation broth was truly surprising and unexpected.

The fermentation broth is then treated according to the procedure described in our Slovenian patent P 9400107 or the equivalent PCT patent application WO 95/23870, after which high purity potassium clavulanate is obtained as the desired desired product.

The invention illustrates, but is in no way limited by, the following examples which illustrate the fermentative process of obtaining clavulanic acid.

EXAMPLE 1

A) FARMING OF STREPTOMYCES SP. P 6621 FERM P 2804

Selection and maintenance of strain

Selection procedures ensure that the most productive clones of the Streptomyces sp.P 6621 micro-organism are isolated. Only the most productive cultures are stored and destined for use in subsequent selection cycles.

The colony of Streptomyces sp. P 6621 is aseptically transferred into sterile chase with 2 ml of sterile water and homogenized. The mycelium fragments were then transferred to a slanted agar medium and incubated on a thermostat at 25 ° C until maturity, which lasted for 10 to 14 days.

After 8 to 10 days, the bacterium outgrows the agar surface with its mycelium in gray-green. From the surface of the slanted agar, the spores are scraped and the vegetative phase aseptically inoculated and incubated on a shaker for 24 hours at 250 rpm. and 25 ° ± 1 ° C.

At the same time as the vegetative phase is inoculated, a homogeneous agar spore suspension is stored in skim milk suspension, which serves as a protective culture medium for up to two months.

After the vegetative phase is completed, a portion of the culture is aseptically transferred to the fermentation medium and incubated under the same conditions on a rotary shaker for 96 hours. After the fermentation phase is completed, the content of clavulanic acid is analyzed. High yielding cultures represent fermenter (laboratory inoculum) grafts.

All described procedure is conducted under aseptic conditions.

The strain is stored on slanted agars and ruches at 4 ° C for up to 4 weeks, skim milk spores for up to 2 months at 4 ° C, freeze dried for 4 years at 4 ° C.

n

The composition of the culture medium for selection of the strain for naceplianie in the fermenter

Medium for slashes, rubble and petri plates:

Composition amount of dextrin 10 g

KH ₂ PO ₄ 1 g

MgSO ₄ . 7 H ₂ O 1 g

NaCi 1 g (NH ₄ ) ₂ SO ₄ 1 g

CaCO3 4 g trace elements * 1 ml agar 20 g water (demineralized) up to 1000 ml

All the above ingredients except agar are mixed in 1000 ml of demineralized water. The agar is dissolved in the culture medium so prepared only at the end. Prior to sterilization, adjust the pH to 7.00 to 7.40 with 30% aqueous NaOH. To prepare 1000 ml of medium, use a 2000 ml erythema flask, which is capped with a cotton wool and paper and elastic. The thus prepared erythema tube was sterilized in an autoclave for 20 min. and at 121 ° C.

The breeding ground for petri dishes and rubella is the same as for slashes.

Before pouring 15 to 20 ml of media into a 25 x 250 tube, boil the medium. The tubes were capped with a cotton wool and sterilized in an autoclave for 20 min. at 121 ° C. After sterilization, the medium should be mixed well to distribute the calcium carbonate evenly.

Petri dishes and rootstocks are prepared by sterilizing them and then aseptically pouring into them 30 ml of sterilized medium with the above composition.

Composition of mineral solution (trace elements ^)

Ingridients quantities CaCI ₂ . 2H ₂ O MgCI ₂ . 6 H ₂ O NaCl FeCIg. 6 H ₂ O ZnCI ₂ CuCI ₂ .2 H ₂ O MnSO ₄ . H ₂ O 10.0 g 10.0 g 10.0 g 3.0 g 0.5 g 0.5 g 0.5 g

water - demineralised up to 1000 ml

Vegetative selection medium

Ingridients quantities corn starch soy flour KH ₂ PO ₄ estol (Priolube 1435) tap water 10.0 g 20.0 g 0.6 g 5.0 g up to 1000 ml

The ingredients were poured into the water while stirring and the pH of the medium thus prepared was adjusted to 7.00 with a 30% aqueous NaOH solution and then divided into aliquots with 50 ml in 300 ml conical flasks each. The latter are capped with a cotton wool, covered with paper and elasticized, and thus prepared in an autoclave for 20 minutes. at 121 ° C.

Fermentation medium for selection

Ingredients cornstarch soybean meal

KH ₂ PO ₄ Estol (Priolube 1435) Glycerol Morpholinopropane Sulfonic Acid Trace Elements * Water (Plumbing)

Quantities

9.6 g 38.5 g

1.2 g

23.0 g

5.0 g

12.0 g 10.0 ml to 1000 ml

The ingredients were added to the water while stirring and divided into aliquots of 25 ml into 300 ml conical flasks, covered with a cotton wool, covered with paper and elasticated, and autoclaved at 20 min. at 121 ° C.

Preparation of laboratory inoculum

The source cultures for the preparation of the laboratory inoculum (clamp) are cultures on slopes or rubble. The selected slash or ruffle is prepared by aseptically pouring 10 ml of sterile water, scraping the spores, which are homogenized in sterile potter. The spore solution is a laboratory inoculum.

VEGETATIVE PHASE IN THE PROPAGATOR (pre-referees

A breeding ground for propagators

Vol. propagator = 500 I

Volumes = 350 I

Prepare a nutrient medium with the following composition:

Ingredients of corn starch 7.0 kg of soybean meal 7.0 kg

NaH ₂ PO ₄ 0.185 kg estol (dec. 1435) * 0.7 kg synperonic 0.150 kg water (drinking) up to 350 I * The culture medium contains the same amount of soybean oil instead of estol.

Inoculate the laboratory inoculum into a culture medium sterilized in a propagator and cooled with sterile air at 28 ° C. The vegetative phase is run for 50 to 70 hours at a temperature of 28 ° ± 1 ° C with stirring, with a pressure of 0.3 bar and with the introduction of sterile air.

The growth of the culture is monitored by pH, PMV%, methylene dye discoloration and microscopic examination of the samples. When the culture reaches the desired growth parameters, it is sucked into a pre-fermenter.

Pattern of movement of growth parameters in the propagator

MON (h) PH PMV% discoloration min. 0 7.20 - - 4 7.25 10 > 5 10 7.35 8 > 5 16 7.30 10 > 5 22 7.20 16 4 28 7.02 17 2.5 34 6.85 18 0.5 39 6.66 20 0.3 45 6.60 21 0.5 51 6.52 22 1.0 56 6.39 22 1.0 61 6.45 20 1.3

Legend:

PON = culture growth length pH = pH of the sample

PMV% = volume% of culture in sample discoloration = time of discoloration of methylene dye

VEGETATIVE PHASE IN THE PREDFERMENTOR

Medium for pre-fermentorie

Predfermenter Vol. = 7500 I Vol. Medium = 4500 I

Prepare a nutrient medium with the following composition:

Quantity ingredients

corn starch 90 kg soybean meal 90 kg NaH ₂ PO ₄ 2.4 kg estol (decl. 1435) * 9 kg synperonic 0.5 kg water to 4500

* The medium variant contains an equal amount of soybean oil instead of estol

The vegetative phase of the propagator was applied to the medium sterilized in the pre-fermentor and cooled with sterile air introduction and stirred at 28 ° C using sterile air pressure. The air is sterilized through 0.2pm pore size filters.

The vegetative phase is run for 10 to 20 hours at a temperature of 28 ° ± 1 ° C with the introduction of sterile air and stirring at a pressure of 0.3 bar.

Growth was monitored by pH, PMV%, methylene dye discoloration and microscopic examination of samples.

When the culture reaches the desired growth parameters, it is pumped into a previously prepared fermenter.

Pattern of movement of growth parameters in pre-fermentorio

MON (h) pH PMV% discoloration (min.) 0 7.20 - - 6 7.10 15 > 5 12 6.87 20 1.5 16 6.65 22 0.3

Legend:

PON = culture growth length pH = pH of the sample

PMV% = volume% of culture in sample discoloration = time of discoloration of methylene dye

B) Batch Fermentation of STREPTOMYCES SP. P 6621 FERM P 2804 IN FERMENTOR

Breeding grounds for fermentors

Fermenter Vol. = 90,000 I Vol

An initial medium of the following composition is prepared in the fermenter:

Quantity Ingredients

corn starch 570 kg soybean meal 2300 kg NaCI 6 kg estol (Priolube 1435) * 2380 kg glycerol 1640 kg NaH ₂ PO ₄ 5 kg MgCI ₂ 6 H ₂ O 7 kg FeCl3. 6 H ₂ O 1.6 kg ZnCI ₂ 0.5 kg MnSO ₄ . H ₂ O 0.1 kg synperonic 25 kg water up to 60 m ^:

Legend: - Estol is a generic name for glycerol trioleate; (Priolube 1435 commercial name of Unichem G.m.b.h., Germany)

- Synperonic (trade name I.C.I., GB) is a propylene glycol-based antifoam * The culture medium contains an equal amount of soybean oil instead of estol.

4700 I Vegetative stages of culture of Streptomyces sp. PP 6621 FERM P 2804 is sterile injected into a 60,000 I initial medium in a 90,000 liter stainless steel fermenter equipped with a mixer and inlets for the introduction of sterile air through 0.2 μηη pore filters. The medium and all fermenter inlets are sterilized. The medium was then cooled to 24 ° C with sterile air and inoculated with the vegetative phase from the pre-fermenter, starting fermentation, which was conducted at 24 ° to 25 ° C with stirring and pressure of 0.3 bar and controlling the pH of the medium in the range between 6.8 to 6.9 with a 25% aqueous ammonia solution. The fermentation was conducted at 24 ° C while stirring and supplying sterile air for 96 hours to give a clavulanic acid concentration in the fermentation broth of 3580 mg / l.

During fermentation, the culture of Streptomyces sp. P 6621 FERM P 2804 A sterile phosphorus source and an assimilating nitrogen source (500 kg of soybean meal per 5000 I of water), a 25% aqueous ammonia solution, are sterile added to the initial culture medium, while monitoring important parameters for fermentation.

Pattern of movement of concentration of phosphorus source and assimilating nitrogen source in fermentorium

MON common resource P common resource N 0 0.035 1,73975 8 0,030625 1,692286 16 0,0095 1,331 th most common 24 0,005188 0,9785 32 0,004638 0,5527 40 0,003638 0,69945 48 0,000863 0,9128 56 0 0,8475 64 0 0.709 72 0 0,653625 80 0 0,571 th most common 88 0 0,47675 96 0 0,53825 104 0 0,7555 112 0 0,77025 120 0 0,673375 128 0 0,78725 136 0 0,734625 144 0 0,8985

Legend:

MON = hours after vaccination

P = concentration of soluble total phosphorus in sample v (w / v) N = concentration of total nitrogen source in sample v (w / v)

Note: the concentration of soluble total phosphorus in the fermentation broth falls within 51 hours below the limit of detection

In the initial hours of culture growth, the pH rises to almost 7.5. During this time, the phosphorus source is consumed, then clavulanic acid begins to form, which lowers the pH value of 19. Without maintaining the stated pH value, the medium would be reduced to a level where the active substance would not be generated.

EXAMPLE 2

EXTENDING THE BIOMASS GROWTH PHASE BY USING AMMONIUM SULPHATE AS AN ASSIMILATING SOURCE OF NITROGEN AND SODIUM HYDROXIDE AS A PH REGULATOR

Two media were prepared in two fermenters (500 I). The fermentation media were prepared in the same manner as in EXAMPLE 1 B, except that proportionally smaller amounts of ingredients were used. In the first fermenter fermentation was carried out as described in EXAMPLE 1 B, and the fermentation process in the second fermenter differed from the fermentation described in EXAMPLE 1 B only in that as a source of assimilating nitrogen from PON = 40 (PON = 40 means 40 hours after inoculation into the fermentor) from to PON = 60 was added an 11% solution of ammonium sulfate at a flow rate of 9 ml / min, and the pH level was adjusted with sodium hydroxide. After PON = 60, the assimilation source of nitrogen was no longer added. In both cases, we measured the viscosity of the fermentation broth, which is proportional to the amount of biomass.

MON (h) ferm.1, viscosity (m Pa * s) term.2, viscosity (m Pa * s) 0 / / 8 / / 26 474 551 44 728 714 62 948 998 80 995 1076 98 936 1226 116 824 863 128 628 873

Claims (14)

A method for the preparation of clavulanic acid and its pharmaceutically acceptable salts, characterized in that the microorganism Streptomyces sp. P 6621 FERM P 2804 is grown at a temperature between 20 ° and 30 ° C, preferably at 23 ° to 25 ° C and at a pH of the medium between 6.5 and 7.5, preferably in controlled maintenance of the pH of the medium between 6.8 and 6.9, under aerobic conditions, and a nutrient medium containing from the start of fermentation a source of phosphorus, assimilating sources of nitrogen and carbon and mineral salts; during the fermentation until the formation of clavulanic acid begins as a growth regulator, a phosphorus source is added to the culture medium and care is taken to maintain the appropriate concentration of the nitrogen source throughout. Method according to claim 1, characterized in that the pH of the medium is maintained between 6.5 and 7.5. Process according to claim 1, characterized in that the concentration of the phosphorus source is maintained between 0.00% and 0.15% (w / v) during fermentation. Process according to Claims 1 and 3, characterized in that the concentration of the phosphorus source is maintained between 0.005% and 0.05% (w / v) up to forty hours of fermentation. Process according to Claims 1, 3 and 4, characterized in that during the fermentation the concentration of the assimilating nitrogen source is maintained between 0.5% to 15% (w / v). Process according to claim 1, 3, 4 and 5, characterized in that the initial concentration of the assimilating carbon source is higher than 5% (w / v). Process according to Claims 1, 3 and 4, characterized in that sodium or potassium phosphate, or sodium or potassium dihydrogen phosphate, or disodium or dicalium hydrogen phosphate is added as a source of phosphorus. Process according to claims 1 and 5, characterized in that soy flour or derivatives derived from soy flour are added as a source of nitrogen; or add other types of vegetable flour, such as cottonseed meal. Process according to claims 1, 5 and 7, characterized in that soy flour or derivatives derived from soy flour are added as a source of nitrogen during the vegetative part of the fermentation; or add other types of vegetable flour, such as cottonseed meal. Process according to claims 1 and 5, characterized in that ammonium hydroxide is added as a source of nitrogen. Process according to claims 1 and 5, characterized in that an ammonium salt, such as ammonium sulfate, is added as a source of nitrogen. The process according to claims 1 to 10, characterized in that the fermentation method is batch fermentation or pre-fermentation or continuous fermentation. Process according to claim 1, characterized in that the fermentation method is continuous fermentation and ammonium sulfate is added as an assimilating source of nitrogen during fermentation. 14. A process for the preparation of clavulanic acid, characterized in that the process comprises the steps of: a) selection of the strain and selection of the culture medium b) inoculating the medium with the microorganism Streptomyces sp. P 6621 FERM P 2804 c) fermentation of the medium with a microorganism in the presence of a phosphorus source, assimilating carbon and nitrogen sources, and mineral salts, and adding a phosphorus source and assimilating nitrogen sources to the medium during fermentation d) a fermentation broth containing a high concentration of clavulanic acid is obtained. ABSTRACT A new and improved process for the preparation of clavulanic acid and its pharmaceutically acceptable salts is described, whereby a substantially increased yield of the desired compound is obtained in a fermentation broth, according to which the Streptomyces sp. P 6621 FERM P 2804 is grown at a temperature of 20 ° to 30 ° C, preferably at 23 ° to 25 ° C and at a pH of the medium between 6.5 and 7.5, preferably in a controlled maintenance of the pH of the medium between 6.8 and 6.9 with an aqueous solution of an alkaline agent , under aerobic conditions and a nutrient medium containing assimilating sources of nitrogen and carbon, a source of phosphorus and mineral salts from the start of fermentation, and during the fermentation to the onset of clavulanic acid, a phosphorus source is added to the medium as a growth regulator and care is taken to maintain the appropriate source concentration at all times. nitrogen. The initial concentration of the assimilating carbon source is higher than 5% (w / v), and during fermentation, the concentration of the phosphorus source is maintained between 0.00% and 0.15% (w / v) during fermentation. The yields of clavulanic acid in fermentation broth are extremely high and amount to around 3500 mg / l.