MXPA94007171A - Process for the recovery of natamic - Google Patents

Abstract

The present invention relates to a process for the recovery of highly pure natamycin from fermentation broth by extraction with methanol. In one embodiment the process comprises the steps of: (1) adding methanol to a feed stream of natamycin comprising at least 2 g / l of solid natamycin suspended to form an extraction medium, and maintaining the extraction medium at a temperature from 0-25 ° C, preferably not higher than 15 ° C; (2) adjust the pH of the extraction medium from 0.1 to 4.5 whmaintaining the temperature at 0-25 ° C for 0.5 to 30 hours, but preferably no more than 0.5 hours when the temperature it is 15-25ºC, (3) remove the solids from the extraction medium to form an extraction liquid, (4) raise the pH of the extraction liquor to 6.0-9.0, and (5) recover the natamycin precipitates.

Description

"PROCESS FOR THE RECOVERY OF NATAMYCIN" Inventors: PHILLIP TERRY OLSON, North American, domiciled at 3220 Waldo Boulevard, Manitowoc, Wisconsin 54220, E.U.A .; JAMES R. MILLIS, North American, domiciled at 512 Audubon Road, Kohler, Wisconsin 54220, E.U.A. and MICHAEL HENRY REIMER, North American, domiciled at 1308 South 24th Street, Sheboygan, Wisconsin 53081, E.U.A.

Causahabiepte: BIO-TECHIMICAL RESOURCES L.P., limited partnership of the State of Lüi scapsin, E.U.A., domiciled at 1035 South Sevepth Street. apitouioc, Lüiscapsin, E.U.A. »> - 2 - SUMMARY OF THE INVENTION The present invention relates to a process for the recovery of highly pure natamycin from fermentation broth by extraction with methanol. In one embodiment the process comprises the steps of: (1) adding methanol to a feed stream of natamycin comprising at least 2 g / L of solid natamycin suspended to form an extraction medium, and maintaining the extraction medium at a temperature 0-25 ° C, preferably not greater than 15 ° C; (2) adjust the pH of the extraction medium from 1.0 to 4.5 while maintaining the temperature at 0-25 ° C for 0.5 to 30 hours, but preferably no more than 0.5 hours when the temperature is 15-25 ° C; (3) remove the solids from the extraction medium to form an extraction liquor; (4) raise the pH of the extraction liquor to 6.0-9.0; and (5) recovering the precipitated natamycin.

FIELD OF THE INVENTION This invention relates to a cheap fast process for recovering natamycin from fermentation broth.

BACKGROUND OF THE INVENTION Natamycin (also known as pimaricin teneoetin) is a well-known antibiotic (Flower "Analytical Profiles of Drug Substances", Vol. 10, 1981; Me Index, 8th ed., "Pimaricin", page 834). Although its valuable antibiotic properties were recognized, there was little research or marketing of natamycin due to its extremely high manufacturing costs. Due to its solubility in several liquids, the proc of recovery of natamycin has not been economical. Exi the need for an economic recovery natamic process. Natamycin has been prepared by fermentation as described in British Patent No. 846.9 using streptomyces qilyosporeus. In this process, natamycin is recovered by extraction with methanol followed by tedious steps of adsorption and elution. Bridger, North American Patent No. 3,378,441, describes the recovery of Natamycin by salting the fermentation broth, extracting with methanol, removing the solids, and then evaporating the liquid. Struyk, US Patent No. 3,892,850, describes the recovery of natamycin by extraction with afied butanol followed by distillation and precipitation. Struyk also describes calcium chloride dissolved in methanol to improve the solubility of natamycin. Each of these processes requires an expensive recovery step, such as adsorption and elution, distillation, or evaporation. British Patent No. 844,289 shows the precipitation of the natamycin from acetic aby the addition of water. Millis, PCT publication WO 92/10580, describes a process for the recovery of natamycin from a fermentation broth containing at least 2 g / L of natamycin comprising the addition of methanol and adjusting the pH of the extraction medium to 1.0 to 4.5. to dissolve the precipitated natamycin; remove the solids; and increase the pH to 6.0-9.0 to precipitate natamycin. However, the natamycin recovered by this process contains natamycin methyl ester, an undesirable by-product.

BRIEF DESCRIPTION OF THE INVENTION Highly pure natamycin can be recovered from a fermentation broth containing natamycin by Extraction with methanol under controlled pH and temperature conditions. The process comprises the steps of: (1) adding methanol to the feed stream of natamycin, comprising at least 2 g / L of suspended solid natamycin to form an extraction medium, and maintaining the extraction medium at a temperature of 0 -25 ° C, preferably not higher than 15 ° C; (2) adjust the pH of the extraction medium from 1.0 to 4.5 while maintaining the temperature at 0-25 ° C for 0.5 to 30 hours, but preferably no more than 0.5 hours when the temperature is 15-25 ° C; (3) remove the solids from the extraction medium to form an extraction liquor; (4) raise the pH of the extraction liquor to 6.0-9.0; and (5) recovering the precipitated natamycin.

In another embodiment the process comprises the steps of: (1) adding methanol with a content of about 10-50 g / L of salts to increase the solubility to a feed stream of natamycin comprising the minus 2 g / L of solid natamycin suspended to form an extraction medium; (2) remove the solids from the extraction medium to form an extraction liquor; (3) adding water to the extraction liquor to precipitate natamycin; and (4) recovering the precipitated natamycin.

In any embodiment, highly pure natamycin (1) can be obtained by washing the precipitated natamycin recovered in the last step with water, (2) treating the extraction liquor with activated charcoal before the precipitation of natamycin, or (3) by a combination of those steps. The preferred salt to increase the solubility is calcium chloride.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the recovery process under controlled pH and temperature conditions. Figure 2 is a schematic representation of a recovery process using a solubilizing salt.

DETAILED DESCRIPTION OF THE INVENTION Extraction with methanol Natamycin Fermentation Broth Feeding Stream The production of natamycin by fermentation is described in U.S. Patent No. 5,231,014 to Eisenschink, British Patent No. 846,933, and U.S. Patent Applications Nos. 07 / 740,545 and 07 / 740,536, both filed. on August 5, 1991. The fermentation broth produced in these fermentations comprises suspended solid natamycin, biomass and water. The process described by Eisenschink, for example, typically produces a broth containing about 7-12 g / L of natamycin. The broth is used as the feed stream for the recovery process, either directly, or after its concentration by removing something or almost all of the water. The feed stream should contain at least 2 g of natamycin per liter, although higher concentrations are preferred. Since the broth comprises water, the suspended solid natamycin, and the biomass solids, some, or substantially all of the water can be removed by any conventional, convenient solid / liquid separation technique, such as centrifugation, filtration, or decanting ( see, Example 1, which exemplifies the filtration). If desired, the solids can be further dried by conventional means such as with hot air, by drying or by spray, or by pressing the water out by mechanical means. If the solids are dried by heating, a temperature of approximately 20-80 ° C should be used. Temperatures above 95 ° C should be avoided. The solubility of natamycin in the extraction medium (feed stream and methanol) decreases as the water increases. Since the water content of the extraction medium depends on the amount of water in the feed stream, it is desirable to remove as much water as possible from the feed stream before Step 1. If the feed stream contains approximately 70% in water volume, approximately three times more methanol is required than when starting from a substantially dry feed stream. If the feed stream contains approximately 40% by volume of water, approximately twice as much methanol is required. The solids content of the feed stream can be as low as about 20% by weight or less, if little or no water is removed, or, if the water is removed, as high as about 98% by weight. To achieve the concentration of natamycin required to a high recovery, it may be necessary to concentrate the broth before the addition of methanol. Depending on the availability of equipment, particularly for the recovery of methanol, it may be desirable to remove substantially all the water from the broth before the methanol addition. This minimizes the amount of methanol needed for recovery. If the economy is imposed, most of the water should be removed from the broth. This allows the process to be carried out at a higher pH without excessive formation of by-products. In a preferred embodiment, the removal of water produces a feed stream containing at least 50% by weight solids. Addition of Methanol Referring to Figure 1, in Step 1, the methanol is added to the feed stream. Under the right conditions, natamycin has excellent solubility in this inexpensive solvent, and simple methanol recovery techniques are available. For optimum recovery of natamycin, sufficient methanol is added to the feed stream to produce an extraction medium containing approximately 20-150 g of natamycin per liter. Since natamycin tends to precipitate from the acidified extraction medium at high concentrations (Step 2), a concentration of 20-120 g of natamycin per liter of extraction medium is preferred.

Acid addition. In Step 2, the pH of the extraction medium is adjusted to approximately 1.0-4.5 by the addition of an acid. This makes the natamycin highly soluble in the extraction medium. Hydrochloric acid is an excellent acid, although any conventional compatible acid material can be used. Although a technique is not preferred, Steps 1 and 2 could be combined by adding acid directly to methanol. The formation of natamycin methyl ester, an unwanted byproduct, depends on the pH, the contact time between natamycin and methanol, and the temperature (see Table 1). It is important that this step be carried out at a temperature of about 0-25 ° C, since the formation of natamycin methyl ester and other undesirable byproducts is minimized. The higher the temperature or the longer contact time, the greater the production of by-products at a given pH. To avoid excessive formation of natamycin methyl ester at 15-25 ° C, short contact times should be used, ie 0.5 hours or less. At approximately 15 ° C, the extraction time should not exceed approximately 1.5 hr. An extraction temperature of about 0-15 ° C is preferred. In general, at a pH close to 2 and at approximately 0 ° C, the extraction can continue for approximately 12 hr without excessive formation of by-products. To approximately ° C, extraction should be discontinued after approximately 0.5 hr. As the pH increases to 4.5, longer times are acceptable. At a pH close to 4 and at a temperature of about 4 ° C, the time may be about 30 hours.

TABLE 1 Formation of Natamycin Methyl Ester in Methanol For commercially acceptable production, it is desirable to keep the methyl ester content below about 5% by weight of the total product (natamycin, methyl ester and any impurities), and preferably below about 3% by weight. It is possible to conduct the extraction of the methanol at the necessary low temperature by cooling the stream and / or the methanol so that the extraction medium is in a low temperature range at the time of the addition of the acid.

The appropriate time / temperature / pH ratio for the extraction can be easily determined by testing, noting the times and temperatures that will give the desired purity of the product at a particular pH, and having in mind the optimum economy for the particular equipment that is used. The pH depends to some degree on the water content of the extraction medium and the desired yield. To obtain a precipitation yield of approximately 90% or more, the concentration of natamycin in the extraction medium should be approximately 40 g / L. In the absence of water this requires a pH of about 4.5. At about 30% water / 70% methanol (by volume) this requires a pH of about 2.5 (Table 2).

TABLE 2 Solubility of Natamycin in Methanol and Mixtures of Methanol / Water Removal of Suspended Solids. In Step 3, the remaining suspended solids are removed by any convenient solid / liquid separation technique, such as filtration or centrifugation leaving an extraction liquor. After being removed from the extraction medium, the solids can continue to contain a significant amount of natamycin. Accordingly, in a preferred embodiment, the solids are washed with methanol with a mixture of methanol and water to recover at least a portion of the contaminated natamycin in the solids. The washed solution is added to the extraction liquor. Preparation and Recovery of Natamycin. In Step 4, any compatible basic material can be used to raise the pH of the extraction liquor to about 6.0-9.0. This causes the precipitation of highly pure natamycin, which is removed and dried. The basic and cheap compatible materials, typically useful, are the hydroxides of sodium and potassium. If desired, water may also be added in Step 4 to aid in the precipitation of natamycin. This may be preferable where the feed stream has a high concentration of natamycin and a small amount of water. The remaining liquid byproduct (waste liquid) containing valuable natamycin, fermentation residues, methanol, inorganic salts and water is sent to methanol recovery. The methanol can be recovered by conventional distillation techniques for the recovery of solvents. The process produces natamycin of at least 80% by weight of purity and often of at least about 90% by weight of purity. In a preferred embodiment, the natamycin precipitate which is recovered in Step 5 is washed with water and then dried resulting in natamycin of at least about 90 wt.% Purity. The process can be carried out effectively by discrete or batch extraction. However, for large-scale commercial extraction, a continuous process may be preferred. In one technique, the natamycin fermentation broth, preferably concentrated at a low water content, is continuously fed to a mixer and thoroughly mixed with methanol at a low temperature. The pH it is controlled by the addition of acid and natamycin dissolves rapidly. The resulting extraction medium is then fed to a filter to separate the cell mass and other solids from the extraction liquor. The extraction liquor is fed to a crystallizer that operates at a higher pH and precipitates natamycin. The natamycin precipitated and the residual liquid are continuously removed from the crystallizer. The extraction time, water content, temperature and pH can be adjusted for the economic production of natamycin.

Adding a Salt to Increase Solubility The rapid and high recovery of natamycin is also possible without the addition of acid to lower the pH, followed by the addition of a base to raise the pH and precipitate natamycin. In this process a mixture of methanol and a salt to increase solubility are mixed with the feed stream of natamycin fermentation broth (with or without concentration). The resulting extraction medium is filtered to remove the cell mass and other solids and produces an extraction liquor. Water is added to the extraction liquor to precipitate natamycin.

Any salt that increases the solubility of natamycin in methanol under the extraction conditions and does not react with or otherwise interfere with the extraction, recovery or purity of the natamycin product can be used. Calcium chloride (CaCl2) is the salt to increase the preferred solubility. This is more effective in amounts of approximately 10-50 g / L per liter of methanol. For optimal recovery of natamycin, the water is typically removed from the feed stream as described above. The feed stream should contain sufficient natamycin so that, after the extraction liquid (methanol / saline) is added to the feed stream, the resulting extraction medium contains at least 2 g / L of natamycin. When the concentration of natamycin is less than 2 g / L, it is difficult to precipitate natamycin by the addition of water. To precipitate a high percentage of natamycin, the water content of the extraction liquor should be increased to at least 50% by volume. Table 3 indicates the solubility of natamycin in a methanol / water / calcium chloride solution as a function of the water content.

TABLE 3 Solubility of Natamycin in Methanol / CaCl2 * Extractor at Various Water Concentrations g / L CaCl, This process can be carried out at pH 2-9, preferably at pH 6-8. If the pH is low, shorter extraction times should be used because the acid conditions promote the formation of natamycin methyl ester. (See Table 4) If the pH is higher than 6, the formation of the ester is slower. Any reasonable temperature (ie, 20-30 ° C) can be used.

TABLE 4 Formation of Ester in Methanol with 40 g / L CaCl.

Figure 2 schematically shows the process. In Step (a), a methanol / salt solution is added to the feed stream. In step (b), the remaining suspended solids are removed from the extraction medium by any convenient solid / liquid separation technique, such as filtration or centrifugation, to get an extraction liquor. The solids may still contain a significant amount of natamycin and may be washed with methanol or a mixture of methanol and water to recover at least a portion of the natamycin contained in the solids. The methanol wash solution is added to the extraction liquor. In Step (c), water is added to the extraction liquid to precipitate natamycin. In step (d), the precipitated natamycin is recovered. The recovered precipitate contains at least 80% by weight of natamycin and usually at least 85% by weight of natamycin. A highly pure product with up to at least 90% by weight of natamycin can be obtained by washing the precipitate recovered in Step (d) with water or by treating the extraction liquor with activated charcoal before Step (c) or by a combination of those steps.

INDUSTRIAL APPLICABILITY The process is a fast, cheap process for the recovery of natamycin from fermentation broth, especially from broths containing more than 2 g / L of natamycin.

EXAMPLE 1 This example illustrates the recovery of natamycin using controlled pH and temperature conditions. The fermentation broth containing natamycin, biomass, water and minor amounts of nutrients and impurities, prepared as described in Eisenschink, U.S. Patent No. 5,231,014, was concentrated to about 45% by weight solids by filtration in a Buchner funnel. Celite® 545 was added to increase the filtration rate. The filter cake contained about 7.5% by weight of natamycin. Approximately 74 g of filter cake was mixed with about 96 mL of methanol to form a suspension of extraction medium and the temperature of the suspension was reduced to about 4 ° C with an ice-bath jacket. Hydrochloric acid was added to the suspension to reduce the pH to about 2.4 and the suspension was maintained for about 3 hours at this temperature and pH. The suspension was filtered on a Buchner funnel and approximately 105 mL of clear liquid (extraction liquor) with a content of approximately 40 g / L of natamycin was obtained. The pH of the extraction liquor was raised to approximately 7 by the addition of 5N sodium hydroxide and maintained at this pH with moderate agitation for approximately 1 hr. During this time a thick white precipitate formed.

The precipitate was isolated by filtration and dried at about 40 ° C under vacuum. Approximately 4.1 g of dry product was obtained. The product was approximately 92% by weight of natamycin (on anhydrous basis) and 1.7% of methyl ester.

EXAMPLE 2 This example illustrates the recovery of natamycin using controlled pH and temperature conditions. The process of Example 1 was repeated except that the temperature of the extraction medium was about 22 ° C instead of about 4 ° C. About 3.9 g of dried product were obtained after drying. The product was approximately 83% by weight of natamycin (on anhydrous basis) and 10% by weight of methyl ester.

EXAMPLE 3 This example illustrates the recovery of natamycin using a salt to increase solubility. The fermentation broth was concentrated to 45% by weight solids by filtration on a Buchner funnel. Celite® 545 was added to increase the filtration rate. The filter cake was further dried to 98% by weight solids at 60 ° C in a fluidized bed dryer. The natamycin content of the dried cake was 18% by weight. 40 g of dry cake were added to a solution of 3 g of calcium chloride in 150 mL of methanol, and the suspension was mixed for 30 minutes. The suspension was filtered on a Buchner funnel and the clear filtrate was collected (extraction liquor).

The water content of the filtrate was increased to 30% by volume by slowly adding water while stirring. A thick matte white precipitate formed. The precipitate was separated by filtration, washed with water, and dried at 30 ° C under vacuum. The precipitate was 88% by weight of natamycin (on anhydrous basis).

EXAMPLE 4 This example illustrates the recovery of natamycin using a salt to increase solubility. The fermentation broth (1 L) was heated at 70 ° C for 15 min, cooled to room temperature, and allowed to settle. 500 mL of the top layer was decanted and discarded. The remaining solids were concentrated by filtration on a Buchner funnel. 20 g of Celite® 545 were added to increase the filtration rate. The filter cake was washed twice with 50 mL of methanol to remove most of the water and leave about 10% by weight of water. The filter cake was 9% by weight of natamycin. They were added 40 g of filter cake to a solution of calcium chloride 2 g in 50 mL of methanol and mixed for 0.5 hr. The suspension was filtered on a Buchner funnel and the clear filtrate was collected (extraction liquor). 3 g of activated charcoal were added to the filtrate and the mixture was stirred for 1 hr. The charcoal was removed by filtration and the clear filtrate was collected. The water content of the filtrate was increased to 50% by volume by slowly adding water with stirring. A thick white precipitate formed which was filtered, washed with water, and dried at 30 ° C under vacuum. The precipitate was 95% by weight of natamycin (on anhydrous basis).

It is noted that in relation to this date, the best method known by the applicant to implement said invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (18)

1. A process for the recovery of natamycin, characterized in that it comprises, in order the following steps: (1) adding methanol to a feed stream of natamycin comprising solid natamycin to form an extraction medium containing 20-150 g of natamycin per liter , and keep the extraction medium at a temperature of 0-25 ° C; (2) adjust the pH of the extraction medium from 1.0 to 4.5 while maintaining the temperature at 0-25 ° C for 0.5-30 hours; (3) remove the solids from the extraction medium to form an extraction liquor; (4) increase the pH of the extraction liquor to 6.0-9.0; and (5) recovering the precipitated natamycin.

2. The process according to claim 1, characterized in that the feed stream contains at least 50% by weight solids.

3. The process according to claim 1, characterized in that the temperature of the extraction medium is maintained at 0-15 ° C.

4. The process according to claim 1, characterized in that steps 1 and 2 are combined by the addition of acid to methanol before step (1).

5. The process according to claim 1, characterized in that it additionally comprises, after step (3) and before step (4): (6) washing the solids with methanol and adding methanol to the extraction liquor.

6. The process according to claim 1, characterized in that it additionally comprises, before step (4), the step of: (7) treating the extraction liquor with activated carbon.

7. The process according to claim 1, characterized in that it additionally comprises, before step (1), the step of: (8) removing the water from the feed stream of natamycin so that the stream of Natamycin feed comprises less than 10% by weight of water.

8. The process in accordance with the claim 7, characterized in that the extraction medium contains 20-150 g of natamycin per liter.

9. The process in accordance with the claim 8, characterized in that the temperature of the extraction medium is maintained at 0-15 ° C.

10. The process in accordance with the claim 9, characterized in that the temperature of the extraction medium is maintained at 0-15 ° C for 0.5-1.5 hours.

11. The process in accordance with the claim 10, characterized in that it additionally comprises, before step (4), the step of: (7) treating the extraction liquor with activated carbon.

12. A process for the recovery of natamycin, characterized in that it comprises, in order the following steps: (1) adding methanol with a content of approximately 10-50 g / L of a salt for increasing the solubility to a feed stream of natamycin comprising at least 2 g / L suspended solid natamycin to form an extraction medium; (2) remove the solids from the extraction medium to form an extraction liquor; (3) adding water to the extraction liquor to precipitate natamycin; and (4) recovering the precipitated natamycin.

13. The process in accordance with the claim 12, characterized in that the salt to increase the solubility is calcium chloride.

14. The process in accordance with the claim 13, characterized in that the feed stream contains at least 50% by weight solids.

15. The process according to claim 13, characterized in that it additionally comprises, before step (3), the step of: (7) treating the extraction liquor with activated carbon.

16. The process according to claim 13, characterized in that it additionally comprises, before step 1, the step of: (6) removing the water from the feed stream of natamycin so that the feed stream of natamycin comprises at least 10% in weight of water.

17. The process in accordance with the claim 16, characterized in that the extraction medium contains 20-150 g of natamycin per liter.

18. The process in accordance with the claim 17, characterized in that it additionally comprises, before step (3), the step of: (7) treating the extraction liquor with activated charcoal. In testimony of which I sign the present in this City of Mexico, D.F., on September 19 of? SSk. BIO-TECHNICAL, RESDURCES L.P. SUMMARY OF THE I NVENCÓN The present invention relates to a process for the recovery of highly pure natamycin from fermentation broth by extraction with methanol. In one embodiment the process comprises the steps of: (1) adding methanol to a feed stream of natamycin comprising at least 2 g / L suspended solid natamycin to form an extraction medium, and maintaining the extraction medium at a temperature 0-25 ° C, preferably not greater than 15 ° C; (2) adjust the pH of the extraction medium from l.o to 4.5 while maintaining the temperature at 0-25 ° C for 0.5 to 30 hours, but preferably no more than 0.5 hours when the temperature is 15-25 ° C; (3) remove the solids from the extraction medium to form an extraction liquor; (4) raise the pH of the extraction liquor to 6.0-9.0; and (5) recovering the precipitated natamycin.