MXPA96004372A - Procedure for the recovery of natamic - Google Patents

Abstract

The present invention relates to a new process for the recovery of high purity natamycin from a fermentation medium containing natamycin, comprises the steps of adjusting the pH of the medium above about 10 and the addition of an amount of a miscible solvent in water, such as isopropanol, sufficient to dissolve natamycin in the medium, followed by the removal of the insoluble solids from the medium with the adjusted pH, followed by the decrease in the pH of the medium to a level to precipitate natamycin and the removal of natamycin from the med

Description

PROCEDURE PñRfl Lñ RECUPERflCION-DE NRTflrlICINñ BACKGROUND OF THE INVENTION The natapuena, also known as pinai icitia, was initially isolated in 1955 from the fermentation medium of a culture of 0+ re toinyce natalensis obtained from an earth + naught near the city of Pietermari t, burg in Na- »al, Rfpca del 'r. (ñ.P. í: t r uyt-- et al., fln * -? b? o »? e (Inriual, 11) < 78 (J957-1958). The pirnaricma, designated natarnicma by oi WHO, e produces b in from C? r entomycos natalonsis or of the St r eptornyces yilvosporeu. The first patents described recovery procedures that require multiple stages of f) ur? ? c, ('i »n and Lh involving unit operations rel ively ca '- .. the British patent GT) 84fi "933 describes > , recovery by ndsorcíon / the nati-uni ciña ucion a part of the fermentation medium using miscible polar solvents in? < Jua talos corno inet nol, tanol and acetone, the patent of lo rst lo, H) United States No. 3,892,850 discloses the recovery of natamic ina to + from the fermentation medium by extraction of an organic solvent having a misci ility in imitated water, followed by recovery in the solvent, the United States patent no. 3,378,441 claims the R) recovery of natarnicma by the addition of salt «< it is outside the processing medium, followed by the dissolution of the solvent and the precipitation by evaporation. A patent, US Pat. No. 2,136,498, describes the concentration V.K lo or t extraction in butanoi of the filtered fermentation medium to obtain a crude anti fungal complex, from which the natarnu ina can be isolated. UO 92/10500 discloses the sun ubi 11 ation of natamycin ton ethanol < ? ? l-1 lid jo, followed by the removal of the solids from the middle. Under these conditions, the natamicma is susceptible to a specific degradation, with the possibility of a reduced recovery performance and a reduced product purity. Thus, the above technique does not describe an efficient method for the recovery of high quality na-micin.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to a novel, simple and efficient process for recovering quality naphthamycin from a fermentation medium using a solvent substantially miscible in water at an alkaline pH. Specifically, the natamycin is recovered in the fermenting medium through a process comprising the steps of: a) adjusting the pH of the medium to a greater than approximately 10 and adding an amount of an aqueous solvent. a sufficient quantity in enough water to dissolve the natarnic in the middle; b) The removal of the insoluble solids from the medium with tl pl adjusted; c) the pH difference of said medium makes a sufficient level to precipitate the natamic in the medium; and d) the ex trac ion of the natarnicma of the medium .. In its preferred embodiment, the f > H of the medium is adjusted to between 10 and 11 in step aJ and up to < between 5.5 and 7.5 in stage o). In Steps b) and d), any of the various methods may be used or, for example, centrifugation, depth infertility and cross-flow fi ltration, the preferred procedure being cross-flow filtration. It is preferred that, prior to the recovery process, the fermentation medium be concentrated, for example, to a solids concentration of 10 to 50%, on a weight / volume basis. For an increased stability of natarnicin during the recovery procedure, an antioxidant such as BHR, BHT, ascorbic acid or sodium eptorhate can be added.

DETAILED DESCRIPTION OF THE INVENTION The production of natarnicina por fermentation is well known. The British patent GB 046,933, mentioned above, is representative and describes the production of natamycin through fermentation using S t reptoinyees gi 1 vosporeus. The fermentation process f > or if it is not eritic for the present invention. \ "half the faith that you can not -jne 1 t natarnicina can concentrate-, 1 - is desired, &t through" uatquior pr-oced nniento proper such & , -o? The most common method is cross flow filtration, preferring the concentration up to a range in solids of the same type. 10 to 50% on a weight / volume basis During the concentration, the medium can be heated to a range of 50 to 70oC for the improvement or evaporation or the velocity of 1 t ration. at a pH greater than about 10, preferably between approximately 10 and approximately 1 1, using a suitable basifying agent such as, for example, Na 2 CO 3, K 2 CO 3, KOH, NaOH or a combination thereof. of water in the concentration stage, it may be necessary to dilute the concentrate with water or an iscible solvent in water to adjust agitation during pH adjustment. Traditionally, a thinner solvent is added in the water to the concentrate with the adjusted pH in an amount sufficient to solubilize the cream. suitable water-soluble solvents include, for example, ethanol, propanol t isopropanol, acetone, tet rahi ro urane and combinations of the foregoing. Of these, isopropanol is preferred .. Normally, one or two volumes of isopropanol are required per- / olutiten of concentrate. Although the popularity of the "low" is low in both water (Spanish and Spanish) ("So 1 ubi 1 i" and pntibiotits in Twenty-Six Solvents ", Dournal or '< OssocLAtion of Official Rnalytical Oherni ts, vol. O, No. 2, 19b7), in a solvent mixture at high pH, this is soluble 5 from 40 to 120 g / 1 or more.Rungue the literature shows that natarnicin it is unstable at alkaline pH (eg, "Rialytical Pr-ofiles of Drug Substances"), in reality it is reasonably stable under the conditions of the present invention. If desired, it can be further improved 1 ment The stability to the addition of an + ix and dant is such as ascorbic acid, eptorbic acid, BUR and BHT at levels up to about 0.2 weight percent of the natamycin activity. the conditions of recovery procedures at ba or pH such as those describe in the document (JO 92/10580, the ice cream is susceptible to a rapid loss of activity and to the formation of natarnicin analogues which do not separate in a simple way - natamycin. the Natarnicin solution by any of several methods such as deep filtration, cross luxury filtration or cent p leakage. ion. If desired, the solids can be washed, for example, with aqueous isopropanol from 40 to 100%, to reduce the activity of the product. residual and improve the performance of the recovery. Cross-flow filtration is a preferred method for the removal of environmental media. The film, in Huj "cr-uzado, as defined by M'Uallor and M Cox -MI" The I anuuage of Bio + echnology "(R eri'-an Themi al V? Ety , Washington, DC, 198R) is "an operation regime for a filtering device in which the main liquid flow is parallel to the filter, such that < = > The liquid passes through the pendular filter to the main flow. This regimen minimizes the formation of the filter paste and also the consequent reduction in the filtration rate. This allows rapid filtration without the need for filtration or loculant aids. "In the procedure here, cross-flow filtration provides fast filtration rates and high final solids concentrations without the addition of filtration aids. , the ceramic filtering elements that are used allow high cross flow velocities, which provides an effective cleaning of the filtering surfaces. These can be used at high temperatures, allowing the flow to be boosted. Its stability to the severe conditions of pH and temperature facilitates frequent sweeping and these can be re-opped during operation to minimize plugging. The pH of the solution rich in product can then be adjusted with a suitable acid such blunt hydrochloric acid to carry out the precipitation of the natarnicin. the choice of acid is not critical; for economy and duplicity, hydrochloric acid is effected. the precipitated natarnic aps + ales are then isolated by a suitable method such as, for example, depth filtration, cross-flow filtration or centrifugation, but additional steps of known purification can be followed, for example, washing with a mixture of * yua-isopropanol, followed by - a drying. The treatment of natamycin by centrifugation or cross flow filtration requires that the mother liquor of the filtration be displaced: in a wash or a solvent composition? similar to avoid that the more soluble impurities in the water precipitate. Normally, the dry product has a purity of at least 94% on an anhydrous basis, the recovery yields of 40-70% are common. Cross flow filtration, as defined above, is a preferred method for the isolation of natamycin, providing an immediate elimination of the mother liquor and the washing liquid, ln this case, the product is a concentrated suspension of the crystals. which can be transformed into a dry solder by any of a number of processes, e.g., evaporation or spray drying. In the crystalline form, natamycin is stable at temperature (H.R. Morris, et al., OuLtured Da ry Products Journal, p 23, (Rgosto of 197B)). Drying temperatures maintained at 70oC are acceptable, as long as the humidity of the product is not below about 1 h%. Nataiiucine usually exists < orno a »r i h i dr« o. Natarrucine anhydrous is less stable than hydrate, '' because the adverse effects of excessive drying affect the purity of the product., 5 Normally dry product purities of 94 to 99% are obtained (calculated on an inhi ra) and recovery yields from 40 to 70% a of the invented recovery procedure.

ID EXAMPLES EXAMPLE 1 Rotary evaporator 1027 i of fermentation medium with a concentration of natamycin of 10.5 g / 1 to 500 ml. The pH was adjusted to 10.5 with 10 M sodium hydroxide and 500 ml of panol isopy was added. The mixture was stirred for approximately two hours, and then centrifuged. The rich dark-colored "lucon" is given off < -M "I nat ctim ci na, adjusted to H 5,5-7,5 with 1 M cloi hydropic acid and -MI was allowed to crystallize for several hours. Natarnicin crystals were added by < In the process, they were transferred to a filtration funnel with 40 rnl of water: isopropanol 1: 1, filtered and washed in 20 ml portions of water-isopropanol 1: 1 and made to weigh constant at 35 ° C in a vacuum oven, producing R g of solids of natarnicin, which had a purity of 96.2% on an anhydrous basis. The performance of the medium of b9"¿.

EXAMPLE 2 It was adjusted to pH 10, (5 by the addition of 1100 g of sodium carbonate, 140 i of 50% sodium hydroxide solution and sufficient water (1.3 1) to allow the heavy concentrate to mix-up during the pH adjustment, a portion of 10.2 L of concentration of the fermentation medium with a cream concentration of 134 g / l.R the resulting cussion, was added 6 kg of isopropanol containing 6 g of BHR and the mixture was stirred to dissolve the natarnici.The medium solids were spoiled by filtration in crossflow at 25-30o C. During this operation, when the suspension retained from the solids in the medium became thick, they were added 10 1 of 1: 1 water: to wash the residual icine cream, the filtrate was adjusted to pH 5.5 with 5 M hydrochloric acid and the analyte was allowed to crystallize for 1-2 hours. isolated the natamycin by filtration in a Buchner funnel, washed with approximately 500 rnl of isopropanol: water 40:60, sec It was mixed with approximately 500 rnl of water and dried in a laboratory hood up to 550 g of natarní solids, which had a purity greater than 97% in an anhydrous base. The yield in the concentrated medium was approximately 40%.

EXAMPLE 3 A volume of 9600 'milestones of the fermentation medium containing 1.06% cream was concentrated to a volume of 535 gallons by a 55-bOoC cross-flow filtration. During this operation, as the retained suspension became thick, 915 gallons of co-water or wash were added. When the concentration of the retained washing medium could not be further filtered, it was adjusted to pH 10.6 with sodium carbonate and sodium hydroxide, 0000 g of isopropanol were added to dissolve the natamicma and one pound (0.000 g) was added. , 4536 lg) of sodium erythorbate will protect it against oxidation. The solids in the medium were removed by cross-flow filtration at 23-28oC. During this operation, as the retained suspension of the solids in the medium became thick, 1625 gallons of isopropanol: water was added 1: 1 to wash the residual hatariucine. The filtrate containing natamycin was adjusted to pH b, 5 with hydrochloric acid. The natanucine was allowed to crystallize for 1-2 hours, then isolated by filtration, washed with water and dried, yielding approximately 40 l-g of ice cream, which was 98% pure on an anhydrous basis. The yield was approximately 60%.

Claims (7)

NOVELTY OF THE INVENTION CLAIMS

1. - A procedure for the recovery of water from a fermentation medium containing natarrpcina which comprises the steps d r. ) adjusting the pH of said medium to a greater than 10 pprox and adding a sufficient amount of < Solvent substance is soluble in water to dissolve natairncin in said medium; b) the removal of the more soluble solids from said medium with the adjusted pH; c) decreasing the pH of said medium to a level sufficient to precipitate natarnicin; and d) the removal of the na tamic i na from said medium.

2. A process according to claim 1, wherein said water-iscible solvent is selected from ethanol, propanol, isopropanol, acetone, tetrahydrofuran and combinations thereof.

3. A process according to Claim 2, wherein said solvent miscible in water is isopropanol.

4. A method according to Claim 1, in which, in step a), the pH of said medium is adjusted to between 1 and 11.

5. A method according to claim 3, wherein , in the lid a), the pH of said medium is adjusted to vire 10 and 11.

6. A procedure according to R ivin ication J, in o L that, in step c), the The pH of said medium is decreased to between 5.5 and 7.5.

7. A process according to the Rei indication 5, in which, in step c), the pH of said medium is decreased between 5.5 and 7.5. H. A process according to Claim 1, in which step b) uses cross-flow filtration. 9"- A method according to the Rei indication 7, wherein stage b) uses cross-flow filtration. 10. A method according to claim 1 comprising the preliminary stage of concentration of said medium. 11. A procedure according to the Rejection 10, wherein said medium is concentrated to a concentration of 10 to 50% in solids on a weight / volume basis. 12. A method according to claim 10, wherein said preliminary step uses filtration in flow.