A method for separating and purifying natamycin from a fermentation broth

Abstract

The application discloses a method for separating and purifying natamycin from fermentation liquor, which comprises the following steps: S1, homogenizing treatment is performed on the fermentation liquor containing the natamycin; S2, solid-liquid separation is performed on the fermentation liquor after the homogenizing treatment, the solid is dried, crushed and sieved; S3, the solid obtained through the sieving treatment in the step S2 is put into an extraction tube of a Soxhlet extractor, the natamycin is extracted by using a solvent, and enriched natamycin crystal is collected in a bottle at the bottom of the Soxhlet extractor; and S4, the enriched natamycin crystal in the step S3 is separated, collected, washed and dried to obtain the natamycin. The method is simple in process, small in solvent loss, easy in recovery, low in cost and suitable for industrial production.

Description

Technical Field

[0001] This invention relates to natamycin, and more particularly to a method for isolating and purifying natamycin from fermentation broth. Background Technology

[0002] Natamycin is a polyene macrolide antibiotic that specifically inhibits fungi without inhibiting bacteria. Its antibacterial spectrum includes almost all molds and yeasts, as well as some protozoa and algae. It is a natural antifungal preservative with advantages of safety, broad spectrum, non-toxicity, and high efficacy, and can replace chemical preservatives in many fields.

[0003] The molecular formula of natamycin is C2 33 H 47 NO 13 It is an amphoteric substance, such as Figure 1 As shown, the molecule contains one basic group and one acidic group, along with four interconnected carbon-carbon double bonds. It is slightly soluble in water and sparingly soluble in most organic solvents. At room temperature, natamycin's solubility in water ranges from 30 to 100 mg / L. Being an amphoteric substance, its solubility is lowest at neutral pH, but increases with increasing or decreasing pH, particularly at pH values ​​below 3 or above 9.

[0004] In existing technologies, natamycin is prepared by fermentation using Streptomyces such as Streptomyces nataleus and Streptomyces flavus. Due to its low solubility, most extraction methods involve adjusting the pH value to increase its solubility, and then adjusting the pH value to the isoelectric point to precipitate it for purification. However, this purification method requires high-end equipment, involves complex processes, consumes large amounts of solvent, and suffers from high losses. Furthermore, natamycin undergoes irreversible degradation when the pH is changed, resulting in poor product extraction rate and low purity. In addition, the use of large amounts of acid and alkali also poses unpredictable environmental problems.

[0005] The invention patent with publication number CN 103665074 A discloses a method for extracting and purifying natamycin from fermentation broth. It uses sodium carbonate buffer solution as a non-organic solvent to extract natamycin from fermentation broth and does not require pH adjustment during the extraction process, thus solving the problem of high organic solvent loss and saving the amount of acid and alkali used. However, the yield and purity of natamycin are not high.

[0006] Therefore, there is a need to provide a method for separating and purifying natamycin from fermentation broth, which can reduce solvent consumption while ensuring high yield and purity of natamycin. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for separating and purifying natamycin from fermentation broth. This method reduces solvent consumption while ensuring high yield and purity of natamycin.

[0008] A method for isolating and purifying natamycin from fermentation broth, comprising the following steps:

[0009] S1. Homogenize the fermentation broth containing natamycin.

[0010] S2. Perform solid-liquid separation on the homogenized fermentation broth, and dry, crush, and sieve the solids;

[0011] S3. The solid obtained by sieving in step S2 is placed into the extraction tube of a Soxhlet extractor and natamycin is extracted with solvent. The enriched natamycin crystals are obtained in the collection bottle at the bottom of the Soxhlet extractor.

[0012] S4. Separate the natamycin crystals enriched in step S3, collect, wash, and dry them to obtain natamycin.

[0013] Preferably, the solvent in step S3 is selected from one or more of acetone, tetrahydrofuran, and chloroform.

[0014] Preferably, the ratio of solvent volume to solid mass in extraction bottle in step S3 is (10-100):1.

[0015] Preferably, the temperature in step S3 is not lower than 60°C.

[0016] Preferably, the solid-liquid separation method in step S2 is centrifugation or evaporation.

[0017] Preferably, the centrifugal separation conditions are: rotation speed of 7000-9000 rpm, centrifugation for 20-40 min.

[0018] Preferably, the evaporation temperature is 35–45°C and the vacuum degree is 0.05–0.15 MPa.

[0019] Preferably, the separation method in step S4 is filtration or centrifugation.

[0020] Preferably, the washing solvent in step S4 is selected from one or more of water and ethanol.

[0021] The fermentation broth containing natamycin is homogenized using a high-pressure homogenizer to break up the bacterial cells, ensuring complete release and uniform dispersion of natamycin in the broth, facilitating subsequent solid-liquid separation. The resulting solid is then pulverized and sieved; this increases the contact area between the solid and the solvent during subsequent natamycin extraction, shortening the extraction time and improving extraction efficiency.

[0022] A Soxhlet extractor mainly consists of three parts: an extraction flask (also called a collection flask) at the bottom, an extraction tube in the middle, and a condenser at the top. A siphon tube and a connecting tube are installed on both sides of the extraction tube, connecting it to the extraction flask. The Soxhlet extractor utilizes the principles of solvent reflux and siphoning to continuously extract the desired component from a solid mixture. During use, the collection flask is heated, causing the solvent inside to boil. The vapor rises through the connecting tube and enters the condenser, where it is condensed and drips into the extraction tube. The solvent then comes into contact with the natamycin solid for extraction. When the level of the refluxed solvent in the extraction tube exceeds the siphon tube of the Soxhlet extractor, the solvent in the extraction tube flows back into the collection flask, i.e., a siphon occurs. As the temperature rises, reflux begins again. Before each siphon, the natamycin solid is extracted by the pure hot solvent and concentrated in the collection flask. Compared to other extraction methods, using a Soxhlet extractor to extract natamycin allows for repeated solvent reuse, shortens the extraction time, and improves extraction efficiency.

[0023] Furthermore, this invention also determined the optimal conditions for extracting natamycin using a Soxhlet extractor, namely, using chloroform as the extraction solvent, with an extraction temperature of 65°C and a mass ratio of the natamycin solid mixture to the extraction solvent of 1:50, the extraction rate of natamycin reached the highest, reaching 94.26%, and the purity also reached 98.216%.

[0024] The present invention has the following beneficial effects:

[0025] 1. This invention uses a Soxhlet extractor to extract natamycin, which is simple in process. Because it is a cyclic extraction, solvent loss is small and easy to recover, saving costs and making it suitable for industrial production.

[0026] 2. The method of the present invention does not require pH adjustment in the extraction process, which not only saves the amount of acid and alkali used and is environmentally friendly, but also protects the stability of natamycin, resulting in high yield and high purity of extracted natamycin. Attached Figure Description

[0027] Figure 1 The chemical structural formula of natamycin;

[0028] Figure 2 A schematic diagram of a Soxhlet extractor;

[0029] Figure 3 The relationship between extraction rate and time for different solvents at 65℃;

[0030] Figure 4 To illustrate the relationship between extraction rate and time when using chloroform as the extraction solvent at different temperatures;

[0031] Figure 5 The relationship between the extraction rate at 65℃ and different solid-liquid ratios when using chloroform as the extraction solvent;

[0032] Figure 6 The HPLC chromatogram of natamycin crystals in Example 6 is shown below.

[0033] Figure 7 This is the HPLC chromatogram of natamycin crystals from Example 7. Detailed Implementation

[0034] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. Any changes made by those skilled in the art after reading the specification of the present invention, as long as they are within the scope of the claims, will be protected by patent law.

[0035] Example 1

[0036] The natamycin fermentation broth obtained after fermentation was subjected to high-pressure homogenization to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. The homogenized natamycin fermentation broth was then subjected to vacuum rotary evaporation to obtain a natamycin solid mixture, which was dried in a vacuum drying oven at 45°C for 24 hours. The solid was then ground into powder using a mortar and pestle, and sieved through a 200-mesh sieve.

[0037] Three identical Soxhlet extractors, labeled No. 1, No. 2, and No. 3, were used. 20 g of pulverized and sieved natamycin mixture was added to the extraction tube of each Soxhlet extractor. 1 L of chloroform, acetone, and tetrahydrofuran solvent were added to the bottom collection flasks (round-bottom flasks) of Soxhlet extractors No. 1, No. 2, and No. 3, respectively. Extraction was performed by reflux in a 65°C water bath. Data were collected and analyzed at regular intervals, and recovery versus time curves were plotted and recorded. Figure 3 After reflux extraction for 10 hours, natamycin was enriched in the round-bottom flask, accompanied by the formation of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. The mass of natamycin was weighed and the recovery rate was calculated. The results are as follows: Figure 3 As shown, under the same extraction conditions, natamycin yield was highest when chloroform was used as the solvent.

[0038] Example 2

[0039] The natamycin fermentation broth obtained after fermentation was subjected to high-pressure homogenization to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. The homogenized natamycin fermentation broth was then subjected to vacuum rotary evaporation to obtain a natamycin solid mixture, which was dried in a vacuum drying oven at 45°C for 24 hours. The solid was then ground into powder using a mortar and pestle, and sieved through a 200-mesh sieve.

[0040] Three identical Soxhlet extractors, labeled No. 1, No. 2, and No. 3, were used. 20 g of pulverized and sieved natamycin mixture was added to the extraction tube of each Soxhlet extractor. 1 L of chloroform solvent was added to the bottom collection flasks (round-bottom flasks) of Soxhlet extractors No. 1, No. 2, and No. 3, respectively. Extraction was performed by reflux in a water bath at 60℃, 65℃, and 70℃. Data were collected and analyzed at regular intervals, and recovery versus time curves were plotted and recorded. Figure 4 After 18 hours of reflux extraction, natamycin was enriched in the round-bottom flask, accompanied by the formation of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. The mass of natamycin was weighed and the recovery rate was calculated. The results are as follows: Figure 4 As shown, within the range of 60-75℃, the higher the extraction temperature, the shorter the extraction time. However, the extraction time is not significantly different at 65℃ and 70℃. Considering both energy consumption and safety, 65℃ is considered the optimal extraction temperature.

[0041] Example 3

[0042] The natamycin fermentation broth obtained after fermentation was subjected to high-pressure homogenization to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. The homogenized natamycin fermentation broth was then subjected to vacuum rotary evaporation to obtain a natamycin solid mixture, which was dried in a vacuum drying oven at 45°C for 24 hours. The solid was then ground into powder using a mortar and pestle, and sieved through a 200-mesh sieve.

[0043] Three identical Soxhlet extractors, labeled No. 1, No. 2, and No. 3, were used. 20 g of pulverized and sieved natamycin mixture was added to the extraction tube of each Soxhlet extractor. 200 mL, 1 L, and 2 L of chloroform solvent were added to the bottom collection flasks (round-bottom flasks) of Soxhlet extractors No. 1, No. 2, and No. 3, respectively. Extraction was performed by reflux in a 65°C water bath. Data were collected and analyzed at regular intervals, and recovery versus time curves were plotted and recorded. Figure 5 After reflux extraction for 10 hours, natamycin was enriched in the round-bottom flask, accompanied by the formation of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. The mass of natamycin was weighed and the recovery rate was calculated. The results are as follows: Figure 5As shown, the extraction rate of natamycin was lowest when the ratio of solid natamycin mixture to solvent was 1:10; the extraction rate of natamycin was highest when the ratio of solid natamycin mixture to solvent was 1:100; however, the extraction rates of natamycin were not significantly different when the ratio of solid natamycin mixture to solvent was 1:100 and 1:50, and the extraction rates of natamycin were the same when the extraction time was 10 h.

[0044] Example 4

[0045] The natamycin fermentation broth obtained after fermentation was subjected to high-pressure homogenization to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. The homogenized natamycin fermentation broth was then subjected to vacuum rotary evaporation to obtain a natamycin solid mixture, which was dried in a vacuum drying oven at 45°C for 24 hours. The solid was then ground into powder using a mortar and pestle, and sieved through a 200-mesh sieve.

[0046] Three identical Soxhlet extractors, labeled No. 1, No. 2, and No. 3, were used. 20 g of pulverized and sieved natamycin mixture was added to the extraction tube of each Soxhlet extractor. 400 mL, 600 mL, and 800 mL of chloroform solvent were added to the bottom collection flasks (round-bottom flasks) of Soxhlet extractors No. 1, No. 2, and No. 3, respectively, and the extraction was performed by reflux extraction in a 65°C water bath. Data were collected and analyzed at regular intervals, and a table of recovery rate versus time was created and recorded in Table 1. After 10 hours of reflux extraction, natamycin was enriched in the round-bottom flasks, accompanied by the appearance of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. The mass of natamycin was weighed and the recovery rate was calculated.

[0047] Table 1: Natamycin Recovery Rate and Recovery Time at Different Feed-to-Liquid Ratios

[0048]

[0049]

[0050] Example 5

[0051] The natamycin fermentation broth obtained after fermentation was subjected to high-pressure homogenization to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. The homogenized natamycin fermentation broth was then subjected to vacuum rotary evaporation to obtain a natamycin solid mixture, which was dried in a vacuum drying oven at 45°C for 24 hours. The solid was then ground into powder using a mortar and pestle, and sieved through a 200-mesh sieve.

[0052] Four identical Soxhlet extractors, labeled No. 1, 2, 3, and 4, were used. 20 g of pulverized and sieved natamycin mixture was added to the extraction tube of each Soxhlet extractor. 240 mL, 280 mL, 320 mL, and 360 mL of chloroform solvent were added to the bottom collection flasks (round-bottom flasks) of Soxhlet extractors No. 1, 2, 3, and 4, respectively, and the mixture was subjected to reflux extraction in a 65°C water bath. Data were collected and analyzed at regular intervals, and a table of recovery rate versus time was created and recorded in Table 2. After 10 hours of reflux extraction, natamycin was enriched in the round-bottom flasks, accompanied by the appearance of crystals. The crystals were separated from the mother liquor by filtration, and the sample was washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. The mass of natamycin was measured, and the recovery rate was calculated.

[0053] Table 2: Natamycin Recovery Rate and Recovery Time at Different Feed-to-Liquid Ratios

[0054]

[0055]

[0056] Example 6

[0057] The natamycin fermentation broth obtained after fermentation was homogenized using a high-pressure homogenizer to break up the bacterial cells, ensuring uniform dispersion of natamycin crystals and bacterial fragments. 200 mL of the homogenized natamycin fermentation broth was centrifuged at 8000 rpm for 30 min using a Beckman centrifuge to obtain 43.4 g of natamycin solid mixture, which was then dried in a vacuum drying oven at 45℃ for 24 h. The solid was then pulverized using a ball mill and sieved through a 200-mesh sieve. HPLC analysis showed that the natamycin content in the solid powder was 75.63%. 20.03 g of the sieved natamycin mixture was added to the extraction tube of a Soxhlet extractor. 1 L of chloroform solvent was added to the collection flask of the Soxhlet extractor, and the mixture was refluxed in a 65℃ water bath. As the natamycin dissolved and entered the collection flask, natamycin crystals gradually precipitated, increasing the natamycin content and decreasing the volume of boiling chloroform. After 8 hours of reflux extraction, natamycin was enriched in the round-bottom flask, accompanied by the formation of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. Finally, 14.28 g of off-white natamycin powder was obtained, with a yield of 94.26%. HPLC analysis showed a purity of 98.216%, with a maximum single impurity of <0.5%, conforming to the national standard GB 25532—2010, and a weight content of 97.24%.

[0058] Example 7

[0059] The natamycin fermentation broth obtained after fermentation was used to break up the bacterial cells using a high-pressure homogenizer to ensure uniform dispersion of natamycin crystals and bacterial fragments. 200 mL of the homogenized natamycin fermentation broth was taken and evaporated under reduced pressure to obtain 37.43 g of natamycin solid mixture, which was then dried in a vacuum drying oven at 45°C for 24 h. The solid was then ground into a mortar and pestle and sieved through a 200-mesh sieve. HPLC analysis showed that the natamycin content in the solid powder was 77.45%. 20.24 g of the sieved natamycin mixture was added to the extraction tube of a Soxhlet extractor. 400 mL of chloroform solvent was added to the collection flask of the Soxhlet extractor, and the mixture was subjected to reflux extraction in a 65°C water bath. As the natamycin dissolved and entered the collection flask, the natamycin content increased, and as the volume of boiling chloroform decreased, natamycin crystals gradually precipitated. After 11.5 hours of reflux extraction, natamycin was enriched in the round-bottom flask, accompanied by the formation of crystals. The crystals were separated from the mother liquor by filtration, and then washed three times alternately with deionized water and 95% ethanol, for a total of six washes, to obtain wet natamycin. The wet natamycin was dried in a vacuum drying oven at 45°C for 24 hours. Finally, 14.54 g of off-white natamycin powder was obtained, with a yield of 92.75%. HPLC analysis showed a purity of 98.771%, with a maximum single impurity of <0.5%, conforming to the national standard GB 25532—2010, and a weight content of 98.44%.

Claims

1. A method for separating and purifying natamycin from fermentation broth, characterized in that, Includes the following steps: S1. Homogenize the fermentation broth containing natamycin. S2. Perform solid-liquid separation on the homogenized fermentation broth, and dry, crush, and sieve the solids; S3. The solid obtained after sieving in step S2 is placed into the extraction tube of a Soxhlet extractor, and natamycin is extracted with a solvent. The enriched natamycin crystals are collected in the bottom collection bottle of the Soxhlet extractor. The solvent is selected from one or more of acetone, tetrahydrofuran, and chloroform. S4. Separate the natamycin crystals enriched in step S3, collect, wash, and dry them to obtain natamycin.

2. The method for separating and purifying natamycin from fermentation broth according to claim 1, characterized in that, In step S3, the ratio of solvent volume to solid mass in the extraction bottle is (10-100):

1.

3. The method for separating and purifying natamycin from fermentation broth according to claim 1, characterized in that, The temperature in step S3 shall not be lower than 60°C.

4. The method for separating and purifying natamycin from fermentation broth according to claim 1, characterized in that, In step S2, the solid-liquid separation method is centrifugation or evaporation.

5. The method for separating and purifying natamycin from fermentation broth according to claim 4, characterized in that, The centrifugation conditions are: 7000-9000 rpm for 20-40 min.

6. The method for separating and purifying natamycin from fermentation broth according to claim 4, characterized in that, The evaporation temperature is 35–45°C, and the vacuum degree is 0.05–0.15 MPa.

7. The method for separating and purifying natamycin from fermentation broth according to claim 1, characterized in that, In step S4, the separation method is either filtration or centrifugation.

8. The method for separating and purifying natamycin from fermentation broth according to claim 1, characterized in that, The washing solvent in step S4 is selected from one or more of water and ethanol.

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