KR100461577B1 - Purification process of a natamycin - Google Patents

Abstract

The present invention comprises the steps of recovering the precipitate after centrifuging the culture medium containing at least 2.5 g / L, preferably 2.5 to 10 g / L natamycin per 1 L culture medium; Extracting by adding 0.5-1.2 L of methanol to 1 g of natamycin contained in the precipitate and suspending it; Increasing the solubility of natamycin by adding hydrochloric acid to the suspension containing natamycin to lower the pH to 2.5 to 3.5; Centrifuging the suspension to collect only the supernatant; Adding NaOH to the supernatant to raise the pH to about 7.0 to 8.0 and stirring for 10 to 20 minutes, followed by centrifugation to remove the precipitates; Concentrating the supernatant from which the precipitate was removed to 1/5 to 1/6 of the original volume by using a vacuum pressure concentrating device; Binding the concentrate to silica gel 60 using 15-30 g of silica gel 60 per 100 mg natamycin; Eluting impurities using distilled water and 30% (v / v) methanol sequentially in the natamycin extract column bound to silica gel; Eluting natamycin bound to silica gel 60 with 60-80% (v / v) methanol using 300 mL for 100 mg of natamycin in the amount of 60-80% (v / v) methanol used; It relates to a method for purifying natamycin, characterized in that the eluate containing natamycin is obtained by obtaining a natamycin crystal using a vacuum decompression device.

Description

Purification process of natamycin {Purification process of a natamycin}

The present invention relates to a method for extracting, purifying and recovering natamycin from a natamycin culture. More specifically, the present invention comprises the steps of recovering the precipitate after centrifuging the culture medium containing at least 2.5 g / L, preferably 2.5 to 10 g / L natamycin per 1 L culture medium; Extracting by adding 0.5-1.2 L of methanol to 1 g of natamycin contained in the precipitate and suspending it; Increasing the solubility of natamycin by adding hydrochloric acid to the suspension containing natamycin to lower the pH to 2.5 to 3.5; Centrifuging the suspension to collect only the supernatant; Adding NaOH to the supernatant to raise the pH to about 7.0 to 8.0 and stirring for 10 to 20 minutes, followed by centrifugation to remove the precipitates; Concentrating the supernatant from which the precipitate was removed to 1/5 to 1/6 of the original volume by using a vacuum pressure concentrating device; Binding the concentrate to silica gel 60 using 15-30 g of silica gel 60 per 100 mg natamycin; Eluting impurities using distilled water and 30% (v / v) methanol sequentially in the natamycin extract column bound to silica gel; Eluting natamycin bound to silica gel 60 with 60-80% (v / v) methanol using 300 mL for 100 mg of natamycin in the amount of 60-80% (v / v) methanol used; It relates to a method for purifying natamycin, characterized in that the eluate containing natamycin is obtained by obtaining a natamycin crystal using a vacuum decompression device.

Natamycin (fimaricin) is a glycoside that is an amino sugar bound to a polycyclic lactone having four conjugated double bonds. It is an antifungal substance belonging to the polyene macrolide system in chemical structure and has one base and one acidic group. (See Figure 1)

Commercially available natamycin is known to be white or yellow with little odor and taste and a very stable crystalline form (REF). It is also known that natamycin is generally poorly soluble in water and polar organic solvents and hardly soluble in nonpolar solvents (REF). Due to these characteristics, various researches on recovery processes have been conducted, but the reported methods are not available because they are protected by intellectual property rights, and there are few reports on the method of purifying natamycin. Therefore, an object of the present invention is to develop a method for efficiently recovering and purifying natamycin.

In the present invention, when the natamycin culture solution was stored at 4 and room temperature for 14 days, the activity of natamycin was maintained at 80% or more at 4, but rapidly decreased to 27% at room temperature. In addition, when comparing the residual activity of natamycin during the heat treatment of the culture solution at 60, 70, 80 and 100 for 10, 30, and 60 minutes, respectively, the ratio of the reduction of the natamycin residual activity in the culture medium that was not heat treated was decreased. It was low, and the highest residual activity of natamycin was maintained when the culture solution was heat-treated at 60 to 10 minutes. Investigation of the amount of extraction solvent and resin used to develop an efficient natamycin purification process showed that 1 L of methanol was the most efficient method for extracting 1 g of natamycin and 100 mg of natamycin in column chromatography with silica gel 60. When using 30g of resin was found to be the most efficient. 2 g of natamycin was obtained from 1,790 mL of the culture solution containing 4.2 g of natamycin by applying methanol amount required for the established natamycin extraction and column chromatography using silica gel 60. Purification process developed in the present invention was able to obtain natamycin with a purity of 96% and a recovery of 47%.

Figure 1 shows the structural formula of natamycin.

Figure 2 shows the purification process of natamycin.

Figure 3 shows the HPLC profile of the commercial natamycin and purified natamycin.

Figure 4 compares the antimicrobial activity of Natamycin purified using Natamycin Saccharomyces cerevisiae commercially available.

The present invention relates to a method for extracting, purifying and recovering natamycin from a natamycin culture. The present invention relates to a method for extracting natamycin from a culture solution containing natamycin and a method for removing and recovering impurities from extracted natamycin.

The present invention comprises the steps of recovering the precipitate after centrifuging the culture medium containing at least 2.5 g / L, preferably 2.5 to 10 g / L natamycin per 1 L culture medium; Extracting by adding 0.5-1.2 L of methanol to 1 g of natamycin contained in the precipitate and suspending it; Increasing the solubility of natamycin by adding hydrochloric acid to the suspension containing natamycin to lower the pH to 2.5 to 3.5; Centrifuging the suspension to collect only the supernatant; Adding NaOH to the supernatant to raise the pH to about 7.0 to 8.0 and stirring for 10 to 20 minutes, followed by centrifugation to remove the precipitates; Concentrating the supernatant from which the precipitate was removed to 1/5 to 1/6 of the original volume by using a vacuum pressure concentrating device; Binding the concentrate to silica gel 60 using 15-30 g of silica gel 60 per 100 mg natamycin; Eluting impurities using distilled water and 30% (v / v) methanol sequentially in the natamycin extract column bound to silica gel; Eluting natamycin bound to silica gel 60 with 60-80% (v / v) methanol using 300 mL for 100 mg of natamycin in the amount of 60-80% (v / v) methanol used; It provides a method for purifying natamycin, characterized in that the eluate containing natamycin is obtained by obtaining a natamycin crystal using a vacuum decompression device.

When the natamycin culture was stored at 4 and room temperature for 14 days, the activity of natamycin was maintained at 80% or higher at 4, but decreased rapidly to 27% at room temperature. In addition, when comparing the residual activity of natamycin during the heat treatment of the culture solution at 60, 70, 80 and 100 for 10, 30, and 60 minutes, respectively, the ratio of the reduction of the natamycin residual activity in the culture medium that was not heat treated was decreased. It was low, and the highest residual activity of natamycin was maintained when the culture solution was heat-treated at 60 to 10 minutes. Investigation of the amount of extraction solvent and resin used to develop an efficient natamycin purification process showed that 1 L of methanol was the most efficient method for extracting 1 g of natamycin and 100 mg of natamycin in column chromatography with silica gel 60. When using 30g of resin was found to be the most efficient. 2 g of natamycin was obtained from 1,790 mL of the culture solution containing 4.2 g of natamycin by applying methanol amount required for the established natamycin extraction and column chromatography using silica gel 60. Purification process developed in the present invention was able to obtain natamycin with a purity of 96% and a recovery of 47%.

Experimental Example 1: Production of Strains Used and Natamycin

The strain used for the production of natamycin is Streptomyces natalensis ATCC 27448, and the medium composition for producing the material is 1.4% (w / v) soy flour, 1% (w / v) glucose, 3% ( w / v) lactose and 0.3% (w / v) yeast extract (pH 7.0). Purification of natamycin was used by storing 4 cultures obtained by culturing for 7-8 days at a stirring speed of 30 to 300 rpm using a 5L fermenter (Best Korea Co. Ltd., KOREA).

Experimental Example 2: Quantitative Analysis of Natamycin

Quantitative analysis of all natamycin used in the present invention used HPLC (High Performance Liquid Chromatography) under the following conditions. All samples were extracted with methanol (for HPLC, JT Baker, USA) and properly diluted, centrifuged at 4 to 12,000 rpm for 10 minutes (MICRO 17R, Hanil Science Industrial Co. Ltd., KOREA) and quantitated by HPLC. . HPLC used in the present invention is equipped with a HPLC pump K-1001 and U. V. detector of KNAUER, Germany, and used a column of Φ 4.6x150 mm of TOSOH (JAPAN). 80% (v / v) of methanol was used as the solvent, and the temperature of the column was 40, the flow rate was 1 mL per minute, and the injection amount was 20 μl.

For quantitative analysis of natamycin by HPLC, a standard curve was prepared as follows using natamycin standard (Minimum 95%, Sigma, USA). 5 mg of natamycin is completely dissolved in 5 mL of methanol, diluted with methanol to a concentration of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μg / mL, and then centrifuged. A standard curve was prepared by quantitative analysis through HPLC.

Experimental Example 3: Biological Antibacterial Activity Test of Natamycin

The antibacterial activity of Natamycin produced by Streptomyces natalensis ATCC 27448 was measured using the Agarr diffusion method (Ref). Saccharomyces cerevisiae was assayed for YPD (1% (w / v) yeast extract, 2% (w / v) bactopeptone, 2% (w / v) glucose, 1.5% (w / v). v) agar) mixed with agar medium was used as a plate to measure the antimicrobial activity. In order to confirm the production of natamycin produced through the culture, the culture solution was diluted with methanol and absorbed 20 µl in 6 mm paper disk, placed on the prepared plate, and incubated for 30 to 24 hours to incubate the size of the transparent ring around the paper disk. Was measured. At this time, each experiment was repeated three times.

Experimental Example 4: Storage Stability and Thermal Stability Experiment

Storage stability and thermal stability tests were performed to examine changes in the activity of natamycin that may occur during the preservation and purification of the culture. For storage stability testing, the culture solution containing natamycin and the precipitate obtained by centrifugation of the culture solution at 12,000 rpm and 4 minutes for 30 minutes were stored at 4 and room temperature, respectively, at 0, 3, 8, 14, 21, 28, and 35 days. Quantitative analysis of natamycin was performed.

For thermostability testing, the culture solution containing natamycin was heat-treated at 60, 70, 80, and 100 minutes for 10, 30, and 60 minutes, and then stored at room temperature, and quantitative analysis of natamycin was performed every 0.3, 6, 9 and 17 days. The experiment was performed without heat treatment as a control.

Example 1 Extraction and Purification of Natamycin

300 mL of natamycin culture was centrifuged (Hitachi CR21F, JAPAN) for 30 minutes at 4 to 12,000 rpm. The supernatant was discarded and 300 mL of methanol was added to the remaining precipitate. After the precipitate was completely suspended in methanol, Conc. The pH was adjusted to 3.0 with HCl, and extracted by stirring for 10 minutes.

The methanol solution extracted from natamycin was properly vacuum concentrated (EYELA, Rotary evaporator N-1000, JAPAN) to prevent crystals from forming at 45.

50% (v / v) acetone was added for equilibration of Silicagel 60 by silica gel 60 column chromatography, and the mixture was slowly stirred for 10 minutes and then left for 4 to 24 hours. 50% (v / v) acetone was removed from the equilibrated Silicagel 60, washed with distilled water, and then slowly charged into the column by adding an appropriate amount of distilled water. After vacuum-loaded the concentrated sample in a column filled with silica gel 60, 600 mL of distilled water, 300 mL of 30% (v / v) methanol and 300 mL of 50% (v / v) methanol were removed to remove impurities. Sequentially injected into the column eluted slowly. Natamycin was eluted with 80% (v / v) 600 mL methanol in a column free of impurities. Purification yield and purity were investigated using the natamycin crystals produced by vacuum evaporation under reduced pressure.

Example 2: Purification Yield and Purity of Natamycin

Purification yield of natamycin was investigated by HPLC analysis for each purification process and the yield was calculated by weighing the finally obtained natamycin crystals. The purity of the finally obtained natamycin crystals was determined by HPLC analysis after extracting an appropriate amount of natamycin, properly diluting it in methanol, and centrifuging.

Example 3: Storage Stability of Natamycin

In order to examine the changes in natamycin activity that may occur during the preservation and purification of the cultures, the storage stability test was carried out by storing the culture medium and the precipitate obtained by centrifugation of the culture medium at 4 and room temperature, respectively. As a result, as shown in Table 1, when the culture solution and the precipitate were stored at 4, the activity of natamycin was maintained at 80% or more until 14 days, but the naamycin activity of the precipitate was rapidly decreased to 43% at the 21st day of storage. On the other hand, natamycin activity in culture at 4 was maintained at about 70% until 35 days of storage. When stored at room temperature, the natamycin activity of the culture solution and the precipitate rapidly decreased to 60% and 35%, respectively, on the 8th day of storage. Natamycin decreased activity faster at room temperature than at 4, and higher activity was maintained in the culture state than in the sediment state.

Example 4 Thermal Stability of Natamycin

In order to minimize the loss of natamycin that may occur during the storage and purification of cultures containing natamycin, the thermostability test is used to examine the thermal stability of natamycin and the causes of decreased activity of natamycin when the culture medium is stored for a long time. It was. As a result, as shown in Table 2, all the heat-treated cultures had a lower rate of decreasing the activity of natamycin over time during the storage period compared to the non-heat-treated cultures (control). When the culture medium was heat-treated at 60 and 70 minutes for 60 minutes, the activity was maintained at 83% or more compared to the natamycin activity of the non-heat-treated culture medium. 86, 74, and 60% were maintained, respectively, and when the culture was heat-treated in boiling water for 10, 30, 60 minutes, the activity was maintained to 81, 54, and 35%, respectively. The above results suggest that the decrease in the activity of natamycin during the heat treatment may be due to the decrease in the factor of reducing the activity of natamycin by the heat treatment. The heat treatment of the culture medium has the advantage of lowering the rate of decrease in the activity of natamycin, but when compared to the control of the natamycin activity of the control and the heat-treated culture on day 6 storage, most of the control group showed higher activity of natamycin. Therefore, if the culture medium is stored and used within 6 days, it is more economical to store the culture solution as it is, and to heat the culture solution for 60 to 10 minutes if long term storage is required for more than 17 days.

Example 5 Selection of Extraction Solvent for Natamycin

Solubility was measured for 50% (v / v) acetic acid, methanol, butanol, distilled water, isopropanol and ethanol to select a solvent from which the natamycin could be economically extracted from the culture. Solubility in natamycin was 50% acetic acid>methanol>ethanol> H ₂ O>isopropanol> butanol as in Table 3. 50% acetic acid and methanol, which are much more soluble in natamycin than other solvents, were selected as extraction solvents and examined the efficiency of the purification process. As a result, the 50% acetic acid was excellent in solubility, but the stability of natamycin was lower than methanol and impurities were increased during the purification process. Therefore, in the present invention, methanol was extracted with much less of the above problems compared to 50% acetic acid in extracting natamycin from the culture.

Example 6: Determination of the amount of natamycin extractant

In order to examine the most efficient extraction of natamycin using methanol from the culture solution (300 ml), 100 mL of methanol was added from 100 mL to 800 mL in order to investigate the content of natamycin in each step. As a result, as shown in Table 4, extraction with 300 mL of methanol showed recovery of 83% natamycin, and when more methanol was used, it showed similar or rather low recovery. Therefore, in consideration of economics, 300 mL of methanol was determined as the amount of methanol for natamycin extraction. Olsen et al. Reported that when natamycin is present in methanol, natamycin and methanol form methyl esters, resulting in a decrease in natamycin content. (REF) Therefore, in order to reduce the loss of activity of natamycin against methanol used in all purification processes, it is necessary to minimize the time that natamycin is dissolved in methanol and store it at a low temperature of about 4 degrees. In addition, in the extraction of natamycin, it was most efficient to extract 1 L of methanol twice per 1 g of natamycin and extracted by countercurrent distribution.

Example 7: Efficient Determination of Silica Gel 60

In order to determine the most efficient use of silica gel 60, 100 mg of natamycin concentrate was used, and column chromatography was performed using 10, 15, 30, and 40 g of Silicagel 60, respectively, to determine the purification yield and purity. saw. As shown in Table 5, when column chromatography was performed by varying the amount of Silicagel 60, all showed the highest activity of natamycin when eluted with 80% methanol. Therefore, the purification yield and purity of each of the compartments eluted with 80% methanol were ascertained. As a result, the purified yield and purity of natamycin were the highest at 99.2% and 96.4%, respectively. Therefore it was most efficient to use 30 g of Silicagel 60 for 100 mg of natamycin.

Example 8: Purification of Natamycin via Scale up

Based on the purification process of natamycin, scale up was conducted to examine the possibility of industrial application. After recovering the precipitate by centrifugation, 1,790 mL of the culture solution containing about 4.2 g of natamycin was extracted twice with 1 L of methanol, and then concentrated appropriately. Silicagel 60 column chromatography was performed after filtering the concentrated sample using cotton wool to remove foreign matters in column chromatography. As a result, as shown in Table 6, about 2.0 g of natamycin having a purity of 96% and a purification yield of 47% was obtained.

Example 9: Antimicrobial Activity of Purified Natamycin

The biological antimicrobial activity of the purified natamycin and the product of Sigma Co., Ltd. used as a label was compared using the agar diffusion method using a paper disk. Two samples were diluted with methanol at a concentration of 100 μg / mL, and 20 μl of each sample was absorbed into a 6 mm paper disc. A YPD agar medium containing Saccharomyces cerevisiae, a test bacterium, was mixed. After incubation for 24 hours, the size of the clear ring on the plate was measured. As shown in FIG. 3, both samples had similar antimicrobial activity against Saccharomyces cerevisiae.

According to the present invention, it was found that 1L of methanol was most effective to extract 1 g of natamycin, and 30 g of resin was used for 100 mg of natamycin in column chromatography using silica gel 60. Developed an efficient natamycin purification process and obtained 2 g of natamycin from 1,790 mL of culture solution containing 4.2 g of natamycin by applying the amount of methanol required for the established natamycin extraction and column chromatography using silica gel 60. Purification process yields natamycin with 96% purity and 47% recovery to improve the productivity of natamycin, making it safe for food, pharmaceutical, and bio pesticides. Very useful invention that can be widely used for .

Claims (1)

Centrifuging the culture solution containing 2.5 to 10 g / L natamycin per culture medium and recovering the precipitate; Extracting by adding 0.5-1.2 L of methanol to 1 g of natamycin contained in the precipitate and suspending it; Increasing the solubility of natamycin by adding hydrochloric acid to the suspension containing natamycin to lower the pH to 2.5 to 3.5; Centrifuging the suspension to collect only the supernatant; Adding NaOH to the supernatant to raise the pH to about 7.0 to 8.0 and stirring for 10 to 20 minutes, followed by centrifugation to remove the precipitates; Concentrating the supernatant from which the precipitate was removed to 1/5 to 1/6 of the original volume by using a vacuum pressure concentrating device; Binding the concentrate to silica gel 60 using 15-30 g of silica gel 60 per 100 mg natamycin; Eluting impurities using distilled water and 30% (v / v) methanol sequentially in the natamycin extract column bound to silica gel; Eluting natamycin bound to silica gel 60 with 60-80% (v / v) methanol using 300 mL for 100 mg of natamycin in the amount of 60-80% (v / v) methanol used; A method of purifying natamycin, characterized in that the eluate containing natamycin is obtained by obtaining a natamycin crystal using a vacuum decompression device.