A method for extracting and purifying natamycin from fermentation broth

Abstract

The application discloses a kind of extraction and purification method of natamycin in fermentation broth, belong to the field of bioengineering. Including the following steps: in brown yellow spore chain streptomyces fermentation broth, add filter aid perlite, after stirring and mixing, suction filtration;Add water in filter residue and adjust pH, after sufficient stirring, suction filtration, collect filtrate;After macroporous resin wet column is loaded, make natamycin filtrate flow through resin column and carry out dynamic adsorption;Water flow is used to wash the resin column of adsorption completion, it is placed in conical flask and desorption liquid is added, and static desorption is carried out;After static desorption, macroporous resin and desorption liquid are loaded together and desorption is carried out dynamically, and effluent is collected;Desorption liquid is adjusted pH, vacuum drying is obtained, and crude product powder is obtained;Wet powder is obtained by washing and centrifugation, and natamycin finished product is obtained by vacuum drying. The product obtained by the application can reach more than 98%, can effectively improve product quality, improve yield, reduce production cost, can meet the needs of industrial production, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of industrial microbial technology and relates to a method for preparing high-purity natamycin. Background Technology

[0002] Natamycin is a natural polyene macrolide antibiotic with the chemical formula C2. 33 H 47 NO 13 It is mainly composed of several types of Streptomyces (such as Streptomyces natalum). Streptomyces natalensis Streptomyces chatanuga Streptomyces chatanoogensis and Streptomyces chrysogenum Streptomyces gilvosporeus Natamycin's unique four-conjugated double-bonded 26-membered lactone ring structure enables it to effectively bind to ergosterol in fungal cell membranes at low concentrations, exhibiting potent broad-spectrum antifungal activity. Furthermore, natamycin is stable in powder form, readily soluble in organic solvents, and shows no loss of activity when stored in the dark. Due to its low toxicity to mammalian cells and lack of significant side effects, natamycin has been approved for food preservation and sterilization in over 40 countries. In addition, in the medical field, this antibiotic is widely used to treat fungal infections and in cancer treatment and agriculture as a crop protectant to prevent fungal contamination. Currently, the industrial production of natamycin still faces challenges such as difficult separation and extraction, low product purity, poor quality, and low yield; therefore, the efficiency of natamycin's industrial production needs further improvement.

[0003] The search revealed the following published documents related to this invention's patent application: 1. The patent "A method for preparing high-purity natamycin" (CN 117777220 A) proposes to use chitosan to precipitate and separate natamycin from the natamycin suspension obtained after pretreatment. Then, the pH of the solution is adjusted according to the gradient to precipitate natamycin crystals. After separation and drying, the finished product is obtained. The purity of the finished product is above 98%, but the yield of natamycin has not been studied in depth.

[0004] 2. The patent "A Method for Preparing High-Purity Natamycin" (CN 103665074 A) involves extracting solid natamycin from fermentation broth using sodium carbonate solution, then adjusting the pH to 5.0-6.0 to allow crystals to precipitate, followed by separation and drying to obtain the finished product. The yield is over 65%, and the purity is over 95%. However, the crystallization time is relatively long, ranging from 10 to 15 hours, increasing the time cost in industrial production.

[0005] 3. The patent "Preparation Method of Natamycin Flake Crystals" (CN 102286044 A) uses methanol to extract natamycin from dried mycelium, then acidifies the concentrated natamycin extract in stages to obtain a crystallizing solution of natamycin flake crystals, which is then separated and dried to obtain natamycin flake crystals. However, this method uses the organic solvent methanol, which has a greater environmental impact and increases the cost in industrial production.

[0006] Based on the above-mentioned situation, this invention provides a method for extracting natamycin from Streptomyces fermentation broth using macroporous resin. This method uses a 60%–80% ethanol solution as the desorption solvent to extract natamycin. Compared to organic solvents such as methanol, ethanol has lower pollution control costs and a lower environmental impact. Furthermore, natamycin is widely used in food preservation and pharmaceutical preparations. Currently, the chemical and pharmaceutical industries restrict the use of highly toxic organic solvents; using ethanol as the desorption solvent avoids the introduction of toxic solvents into the product from the raw material stage, achieving a green and environmentally friendly approach. Natamycin crystallization is performed under vacuum drying at 50°C and a vacuum degree of less than 0.08 MPa for approximately 6 hours, achieving a final yield of 52.12% and a purity of over 98%. By comparison, this invention patent application differs fundamentally from the aforementioned patent publications. Summary of the Invention

[0007] To address the high production costs associated with traditional natamycin extraction and purification processes, this invention provides a high-yield natamycin strain and its preparation method.

[0008] (1) Add 6%~8% perlite to the fermentation liquid, stir for 10 min to mix it thoroughly, filter and obtain filter residue.

[0009] (2) Add an equal volume of water to the filter residue, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min to ensure natamycin dissolves, then filter and collect the natamycin filtrate. Dissolve the natamycin in the filter residue again.

[0010] (3) The macroporous resin HZ841 was loaded onto the column using wet packing technology at a rate of 1~3 BV / h. The concentration of natamycin in the effluent was measured in time, and the column loading was stopped immediately when the leak point was reached.

[0011] (4) Pack the macroporous resin into the column and start collecting the effluent. Control the desorption flow rate to 1~3 BV / h and collect the desorption liquid in real time.

[0012] (5) The collected natamycin eluent was adjusted to pH 6.5-7.5 with 4-6 mol / L HCl solution and then transferred to a container. It was then vacuum dried at 50-60℃ and a vacuum degree of less than 0.08 MPa until the ethanol was completely evaporated and the dried crude powder was collected.

[0013] (6) Wash the crude powder with water, centrifuge at 4000 r / min to obtain natamycin wet product, wash with water 1-3 times again until the washing liquid is colorless, and then vacuum dry the wet product at 50-60℃ and vacuum degree less than 0.08 MPa to finally obtain purified natamycin dry powder.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a method for extracting natamycin from Streptomyces fermentation broth using macroporous resin, which can produce high-quality natamycin product with a crystallization time of about 6 hours, a final yield of 52.12%, and an extraction purity of over 98%.

[0015] (2) In the extraction and purification process of natamycin, this invention uses only ethanol and does not involve other organic reagents, which effectively reduces production costs and environmental pollution, thus achieving green production. The process is simple and feasible, which is conducive to the promotion and application of industrial extraction of natamycin. Attached Figure Description

[0016] Figure 1 Extract finished product image Figure 2 HPLC chromatogram of natamycin standard Figure 3 HPLC chromatogram of natamycin extract. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0018] Example 1: Initial Macroporous Resin Extraction Process (1) Take 200 mL of fermentation broth, which is natamycin fermentation broth obtained by culturing and fermenting Streptomyces chrysogenum with a natamycin content of 10.09 g / L. Add 6%~8% perlite to it, stir for 10 min to mix it thoroughly, filter it, and obtain filter residue.

[0019] (2) Add water to the filter residue, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min to ensure natamycin dissolves, filter and collect 180 mL of natamycin filtrate.

[0020] (3) The macroporous resin HZ841 is wet-packed into the column. The filtrate containing natamycin is loaded onto the column at a rate of 1~3 BV / h. The concentration of natamycin in the effluent is measured in time. The column loading is stopped immediately when the leak point is reached.

[0021] (4) Pack the macroporous resin into the column and start collecting the effluent. Use a peristaltic pump to control the desorption flow rate to 1~3 BV / h and use an automatic step collector to collect the desorption liquid in real time.

[0022] (5) The collected 250 mL natamycin eluent was adjusted to pH 6.5-7.5 with 4-6 mol / L HCl solution and then transferred to a glass petri dish. It was dried at a constant temperature of 50-60℃ until the ethanol was completely evaporated and the dried crude powder was collected.

[0023] (6) Wash the crude powder with water, centrifuge at 4000 r / min to obtain natamycin wet product, wash with water 1 to 3 times again until the washing liquid is colorless, and then dry the wet product at a constant temperature of 50 to 60℃ to finally obtain 0.468 g of purified natamycin dry powder, with a yield of 23.19%.

[0024] Example 2: Optimization of Dissolution Times in Macroporous Resin Extraction Process (1) Take 100 mL of fermentation broth, which is natamycin fermentation broth obtained by culturing and fermenting Streptomyces chrysogenum with a natamycin content of 16.18 g / L. Add 6%~8% perlite to it, stir for 10 min to mix it thoroughly, filter it, and obtain filter residue.

[0025] (2) Add water to the filter residue, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min to ensure natamycin dissolves, filter and collect 120 mL of natamycin filtrate. Add an equal volume of water to the filter residue again, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min, filter and collect 80 mL of natamycin filtrate.

[0026] (3) The macroporous resin HZ841 is wet-packed into the column. The filtrate containing natamycin is loaded onto the column at a rate of 1~3 BV / h. The concentration of natamycin in the effluent is measured in time. The column loading is stopped immediately when the leak point is reached.

[0027] (4) Pack the macroporous resin into the column and start collecting the effluent. Use a peristaltic pump to control the desorption flow rate to 1~3 BV / h and use an automatic step collector to collect the desorption liquid in real time.

[0028] (5) The collected 250 mL natamycin eluent was adjusted to pH 6.5-7.5 with 4-6 mol / L HCl solution and then transferred to a glass petri dish. It was dried at a constant temperature of 50-60℃ until the ethanol was completely evaporated and the dried crude powder was collected.

[0029] (6) Wash the crude powder with water, centrifuge at 4000 r / min to obtain natamycin wet product, wash with water 1 to 3 times again until the washing liquid is colorless, and then dry the wet product at a constant temperature of 50 to 60℃ to finally obtain 0.645 g of purified natamycin dry powder, with a yield of 39.87%.

[0030] Example 3: Optimization of Drying Method in Macroporous Resin Extraction Process (1) Take 100 mL of fermentation broth, which is natamycin fermentation broth obtained by culturing and fermenting Streptomyces chrysogenum with a natamycin content of 15.23 g / L. Add 6%~8% perlite to it, stir for 10 min to mix it thoroughly, filter it, and obtain filter residue.

[0031] (2) Add water to the filter residue, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min to ensure natamycin dissolves, filter and collect 100 mL of natamycin filtrate. Add an equal volume of water to the filter residue again, adjust the pH to 11-12 with 4-6 mol / L NaOH solution, stir for 10 min, filter and collect 100 mL of natamycin filtrate.

[0032] (3) The macroporous resin HZ841 is wet-packed into the column. The filtrate containing natamycin is loaded onto the column at a rate of 1~3 BV / h. The concentration of natamycin in the effluent is measured in time. The column loading is stopped immediately when the leak point is reached.

[0033] (4) Pack the macroporous resin into the column and start collecting the effluent. Use a peristaltic pump to control the desorption flow rate to 1~3 BV / h and use an automatic step collector to collect the desorption liquid in real time.

[0034] (5) The collected 250 mL natamycin eluent was adjusted to pH 6.5-7.5 with 4-6 mol / L HCl solution and then transferred to a glass petri dish. It was then vacuum dried at 50-60℃ and a vacuum degree of less than 0.08 MPa until the ethanol was completely evaporated and the dried crude powder was collected.

[0035] (6) Wash the crude powder with water, centrifuge at 4000 r / min to obtain natamycin wet product, wash with water 1 to 3 times again until the washing liquid is colorless, and then vacuum dry the wet product at 50 to 60°C and vacuum degree less than 0.08 MPa to finally obtain 0.794 g of purified natamycin dry powder, with a yield of 52.12%.

Claims

1. A method for extracting and purifying natamycin from fermentation broth, characterized in that, Includes the following steps: (1) Add 6%~8% of perlite as a filter aid to the fermentation broth of Streptomyces chrysogenum, stir to mix thoroughly, and filter. (2) Add an equal volume of water to the filter residue, adjust the pH to 11-12, then stir to ensure that natamycin dissolves, then filter and collect the natamycin filtrate. Dissolve the natamycin in the filter residue again. (3) The macroporous resin is packed with a wet column and the natamycin filtrate is loaded onto the column for dynamic adsorption. The concentration of natamycin in the effluent is monitored simultaneously. The process is terminated immediately if sample leakage is detected. (4) Pack the macroporous resin into a column for dynamic desorption and start collecting the effluent. (5) Adjust the pH of the collected natamycin eluent to 6.5-7.5, transfer it to a glass petri dish and dry it, ensuring that the crude product powder is collected after the ethanol has completely evaporated. (6) The wet product of natamycin was obtained by washing with water and centrifugation and then vacuum drying to prepare natamycin dry powder.

2. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In step (2), the solution used to adjust the pH to 11-12 is a 4-6 mol / L NaOH solution.

3. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In step (3), the macroporous resin is HZ841.

4. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In step (4), the desorption solution used is a 60% to 80% ethanol-water solution with pH = 11 to 12.

5. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In step (4), the dynamic desorption rate is 1~3 BV / h.

6. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In step (5), the solution used to adjust the pH to 6.5~7.5 is a 4~6 mol / L HCl solution.

7. The method for extracting and purifying natamycin from fermentation broth according to claim 1, characterized in that: In steps (5) and (6), the vacuum drying conditions are: a set temperature of 50~60℃ and a vacuum degree of less than 0.08 MPa.

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