Fermentation method for improving yield and purity of natamycin

Abstract

The invention discloses a fermentation method for improving the yield and purity of natamycin, and belongs to the technical field of microbial application. The method comprises the following steps: in the fermentation process of streptomyces gilvosporeus, changing the type and content of fed-batch carbon sources according to the content of natamycin in fermentation liquor, namely changing the carbon sources fed into the fermentation liquor from glucose to cane sugar, and changing the fermentation pH value from 7.0 to 6.0 at the same time. According to the method disclosed by the invention, the content of the natamycin in the fermentation liquor and the final purity of the natamycin can be remarkably improved, the production cost of the natamycin and the high-purity natamycin is reduced, and the market occupancy of the natamycin is improved.

Description

Technical Field

[0001] The invention relates to a fermentation method for improving the yield and purity of natamycin, and belongs to the technical field of microbial application. Background Art

[0002] Natamycin is an odorless, tasteless, safe, and highly effective natural food preservative. It binds to sterols on microbial cell membranes, altering their permeability and allowing the release of substances such as amino acids and electrolytes, thereby inhibiting the normal growth and reproduction of molds and yeasts. Natamycin is colorless and odorless. Adding it to food has no effect on taste and is harmless to the human body. Therefore, it is a safe food preservative widely used in various areas of the food industry, such as condiments, baked goods, cheese products, fruit and vegetable juice purees, and foods prone to mold.

[0003] In recent years, the demand for natamycin has continued to grow both domestically and internationally, and production capacity has been unable to meet market demand. Natamycin is a secondary metabolite produced through microbial fermentation. Similar to the production of all antibiotics, natamycin must be isolated and purified from the fermentation broth to obtain a final product that meets product quality standards. With the widespread application of natamycin in the food and pharmaceutical industries, the development of a high-purity natamycin production process is of great significance. Many researchers at home and abroad have employed methods such as strain selection, fermentation medium optimization, and fermentation process optimization to increase the yield and purity of natamycin. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a fermentation method for increasing the yield and purity of natamycin, thereby improving the yield and purity of natamycin in industrial production.

[0005] In order to solve the problems of the prior art, the technical solution provided by the present invention is: A fermentation method for improving the yield and purity of natamycin comprises: changing the type and content of a fed carbon source and simultaneously changing the fermentation pH during the fermentation process of Streptomyces chrysoporus.

[0006] Furthermore, the Streptomyces sp. F607 KD strain was deposited in the China Center for Type Culture Collection on August 9, 2023, with a collection number of CCTCC NO: M 20231400, a classification name of Streptomyces sp. F607 KD, and a collection address of Wuhan, China.

[0007] Furthermore, the method is specifically as follows: during the fermentation process of Streptomyces chrysoporus, according to the natamycin content in the fermentation broth, the carbon source added to the fermentation broth is changed from glucose to sucrose, and the fermentation pH is changed from 7.0 to 6.0.

[0008] Furthermore, during the fermentation process, when the fed carbon source is a glucose solution, the glucose concentration in the fermentation broth is maintained at 20-30 g / L; when the fed carbon source is a sucrose solution, the sucrose concentration in the fermentation broth is maintained at 5-10 g / L.

[0009] Furthermore, the concentration of the glucose solution added is 500 g / L, and the concentration of the sucrose solution added is 500 g / L.

[0010] Furthermore, during the fermentation process, when the fed carbon source is glucose, the pH of the fermentation culture is controlled at 7.0; when the fed carbon source is sucrose, the pH of the fermentation culture is controlled at 6.0.

[0011] Furthermore, the solution used to adjust the pH is a liquid alkali with a concentration of 10 wt.%.

[0012] Furthermore, the time of changing the fed carbon source is: when the natamycin content in the fermentation broth is 3 g / L, 7 g / L, 10 g / L or 13 g / L, the fed carbon source is changed from glucose to sucrose.

[0013] Furthermore, the method of changing the fed carbon source is specifically as follows: when the natamycin content in the fermentation broth is 3 g / L, 7 g / L, 10 g / L or 13 g / L, stopping the fed glucose solution; when the glucose concentration in the fermentation broth is lower than 3 g / L, feeding the sucrose solution until the sucrose concentration in the fermentation broth is 5-10 g / L.

[0014] Furthermore, the fermentation method for improving the yield and purity of natamycin is used in the fermentation production of natamycin, comprising the following steps: fermenting natamycin using Streptomyces chrysospora in a fermentation medium; when the natamycin content in the fermentation broth is 3 g / L, 7 g / L, 10 g / L, or 13 g / L, stopping the flow-feeding of glucose until the glucose concentration in the fermentation broth is lower than 3 g / L, flow-feeding a sucrose solution until the sucrose concentration in the fermentation broth is 5-10 g / L, and changing the pH control parameter from 7.0 to 6.0; and fermenting and culturing for 110-120 hours at a temperature of 30° C., an air volume of 1 vvm, and a rotation speed of 400 r / min to obtain a fermentation broth containing natamycin; and then separating and purifying the fermentation broth to obtain natamycin.

[0015] Furthermore, the fermentation medium formula is: 20 g / L glucose, 6 g / L yeast powder, 20 g / L peptone, 2 g / L sodium chloride, 1 g / L magnesium sulfate, and the rest is water, and the pH is adjusted to 7.0 with liquid alkali (10 wt.%).

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Based on the characteristics of natamycin synthesis and metabolism by Streptomyces chrysoporus, the present invention changes the metabolic pathway of Streptomyces chrysoporus by changing the carbon source and pH during the fermentation culture process, thereby increasing the fermentation content and purity of natamycin and reducing the generation of related impurities during the fermentation process.

[0017] (2) When the content of natamycin in the fermentation broth reaches a specific value, the present invention stops adding glucose until the glucose concentration in the fermentation broth is lower than 3 g / L, and then adds sucrose solution. This can significantly increase the fermentation content and purity of natamycin. The fermentation content is 21.3 g / L and the purity is 98.3%. This significantly reduces the production cost of natamycin and high-purity natamycin, and increases the market share of natamycin. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] In the embodiment, the seed culture medium formula is as follows: 10 g / L glucose, 6 g / L yeast powder, 6 g / L peptone, 5 g / L sodium chloride, and the rest is water, and the pH is adjusted to 7.0 with liquid caustic soda (10 wt.%).

[0021] Seed fermentation culture: inoculate the -20℃ frozen strain into the shake flask seed (200 / 500mL) culture medium, culture at 30℃, rotation speed 250r / min, when the shake flask seed A 600 When the concentration reaches 1.8-1.9, if there are no foreign bacteria under microscopic examination, the 10L fermentation tank can be planted.

[0022] Fermentation medium formula: glucose 20 g / L, yeast powder 6 g / L, peptone 20 g / L, sodium chloride 2 g / L, magnesium sulfate 1 g / L, and the rest is water. Liquid alkali (10 wt.%) is used to adjust the pH to 7.0.

[0023] 10L fermentation tank control: culture at 30℃, ventilation volume 1 vvm and rotation speed 400 r / min, control glucose concentration at 20-30g / L, use liquid caustic soda (10wt.%) to control pH at 7.0 during fermentation, or control sucrose concentration at 5-10g / L, use liquid caustic soda (10wt.%) to control pH at 6.0 during fermentation.

[0024] Isolation and purification of natamycin: After fermentation stops, add composite protease to the fermentation broth for enzymatic hydrolysis, raise the temperature to 50°C, and keep warm for 4 hours; remove the supernatant by centrifugation to collect the precipitate, add alcohol to the precipitate, and control the alcohol content between 55-60%; adjust the pH to 10.40-10.60 with 30wt.% liquid alkali, maintain for 1 hour, slowly add HCl solution to adjust to pH 6.5, and let it stand for 4-6 hours for crystallization; remove the supernatant by centrifugation to collect the crystals, dry at 55-60°C, and test the purity.

[0025] Detection of Natamycin: refer to the determination method in GB 5009.286-2022 "National Food Safety Standard - Determination of Natamycin in Food" for detection.

[0026] Example 1 Production and fermentation of natamycin (10L fermenter): When natamycin is produced by fermentation of Streptomyces chrysoporus in a fermentation medium, when the natamycin content in the fermentation broth is 3 g / L, the addition of glucose is stopped until the glucose concentration in the fermentation broth is lower than 3 g / L, and sucrose solution is added until the sucrose concentration in the fermentation broth is 5-10 g / L. The pH control parameter is changed from 7.0 to 6.0. The fermentation is cultured for 110 hours to obtain a fermentation broth containing natamycin. The fermentation broth is separated and purified to obtain natamycin.

[0027] The concentration of natamycin in the fermentation broth was 17.8 g / L, and the purity of natamycin after separation and purification was 96.1%.

[0028] Example 2 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, when the natamycin content in the fermentation broth is 7g / L, stop adding glucose until the glucose concentration in the fermentation broth is lower than 3g / L, add sucrose solution until the sucrose concentration in the fermentation broth is 5-10g / L, and change the pH control parameter from 7.0 to 6.0. Ferment and culture for 110 hours to obtain a fermentation broth containing natamycin. The fermentation broth is separated and purified to obtain natamycin.

[0029] The concentration of natamycin in the fermentation broth was 21.3 g / L, and the purity of natamycin after separation and purification was 98.3%.

[0030] Example 3 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, when the natamycin content in the fermentation broth is 10 g / L, stop adding glucose until the glucose concentration in the fermentation broth is lower than 3 g / L, add sucrose solution until the sucrose concentration in the fermentation broth is 5-10 g / L, and change the pH control parameter from 7.0 to 6.0. Ferment and culture for 110 hours to obtain a fermentation broth containing natamycin. The fermentation broth is separated and purified to obtain natamycin.

[0031] The concentration of natamycin in the fermentation broth was 18.9 g / L, and the purity of natamycin after separation and purification was 98.2%.

[0032] Example 4 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, when the natamycin content in the fermentation broth is 13 g / L, stop adding glucose until the glucose concentration in the fermentation broth is lower than 3 g / L, add sucrose solution until the sucrose concentration in the fermentation broth is 5-10 g / L, and change the pH control parameter from 7.0 to 6.0. Ferment and culture for 110 hours to obtain a fermentation broth containing natamycin. The fermentation broth is separated and purified to obtain natamycin.

[0033] The concentration of natamycin in the fermentation broth was 16.2 g / L, and the purity of natamycin after separation and purification was 94.5%.

[0034] Comparative Example 1 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, the glucose concentration of the fermentation liquid is controlled at 20-30 g / L, the pH is controlled at 7.0, and the fermentation culture is carried out for 110 hours to obtain a fermentation liquid containing natamycin. The fermentation liquid is separated and purified to obtain natamycin.

[0035] The concentration of natamycin in the fermentation broth was 15.8 g / L, and the purity of natamycin after separation and purification was 93.2%.

[0036] Comparative Example 2 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, the sucrose concentration of the fermentation liquid is controlled at 5-10 g / L and the pH is controlled at 7.0. The fermentation culture is carried out for 110 hours to obtain a fermentation liquid containing natamycin. The fermentation liquid is separated and purified to obtain natamycin.

[0037] The concentration of natamycin in the fermentation broth was 15.2 g / L, and the purity of natamycin after separation and purification was 94.3%.

[0038] Comparative Example 3 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, the glucose concentration of the fermentation liquid is controlled at 20-30 g / L, the pH is controlled at 6.0, and the fermentation culture is carried out for 110 hours to obtain a fermentation liquid containing natamycin. The fermentation liquid is then separated and purified to obtain natamycin.

[0039] The concentration of natamycin in the fermentation broth was 16.1 g / L, and the purity of natamycin after separation and purification was 94.8%.

[0040] Comparative Example 4 Production and fermentation of natamycin (10L fermenter): When producing natamycin by microbial fermentation in a fermentation medium, the sucrose concentration of the fermentation liquid is controlled at 5-10 g / L, the pH is controlled at 6.0, and the fermentation culture is carried out for 110 hours to obtain a fermentation liquid containing natamycin. The fermentation liquid is separated and purified to obtain natamycin.

[0041] The concentration of natamycin in the fermentation broth was 15.3 g / L, and the purity of natamycin after separation and purification was 95.1%.

[0042] Example 1 The concentrations of natamycin in the fermentation broths of Examples 1-4 and Comparative Examples 1-4 and the purity of natamycin after separation and purification are listed in Table 1.

[0043] Table 1. Concentration of natamycin in fermentation broth and purity of natamycin after separation and purification As shown in Table 1, the natamycin content in the fermentation broth was 7 g / L. When the carbon source fed to the fermentation broth was changed from glucose to sucrose and the pH was changed from 7.0 to 6.0, the natamycin content reached a maximum of 21.3 g / L, and the purity reached a maximum of 98.3%. This demonstrates that the fermentation method for increasing the yield and purity of natamycin according to the present invention can significantly increase the natamycin content and purity in the fermentation broth by changing the metabolic pathway of Streptomyces chrysospora by changing the carbon source and pH during the fermentation culture process.

Claims

1. A fermentation method for increasing the yield and purity of natamycin, characterized in that: The method comprises: changing the type and content of the carbon source fed during the fermentation of Streptomyces chrysoporus, and changing the fermentation pH at the same time.

2. The fermentation method for increasing the yield and purity of natamycin according to claim 1, characterized in that: The method specifically comprises: during the fermentation process of Streptomyces chrysoporus, according to the content of natamycin in the fermentation broth, the carbon source added to the fermentation broth is changed from glucose to sucrose, and the fermentation pH is changed from 7.0 to 6.

0.

3. The fermentation method for increasing the yield and purity of natamycin according to claim 1 or 2, characterized in that: The Streptomyces sp. F607-KD strain was deposited in the China Center for Type Culture Collection on August 9, 2023, with a collection number of CCTCC NO:M 20231400, a classification name of Streptomyces sp. F607-KD, and a collection address of Wuhan, China.

4. The fermentation method for increasing the yield and purity of natamycin according to claim 1 or 2, characterized in that: During the fermentation process, when the carbon source added is glucose, the glucose concentration in the fermentation liquid is maintained at 20-30 g / L; when the carbon source added is sucrose, the sucrose concentration in the fermentation liquid is maintained at 5-10 g / L.

5. The fermentation method for increasing the yield and purity of natamycin according to claim 4, characterized in that: When the carbon source added during the fermentation process was glucose, the pH of the fermentation culture was controlled at 7.0; when the carbon source added during the fermentation process was sucrose, the pH of the fermentation culture was controlled at 6.

0.

6. The fermentation method for increasing the yield and purity of natamycin according to claim 1, characterized in that: The time for changing the carbon source is: when the content of natamycin in the fermentation broth is 3 g / L, 7 g / L, 10 g / L or 13 g / L, the carbon source is changed from glucose to sucrose.

7. The fermentation method for increasing the yield and purity of natamycin according to claim 6, characterized in that: The method for changing the carbon source is as follows: when the natamycin content in the fermentation broth is 3g / L, 7g / L, 10 / L or 13g / L, the glucose solution is stopped from being added, and when the glucose concentration in the fermentation broth is lower than 3g / L, sucrose solution is added until the sucrose concentration in the fermentation broth is 5-10g / L.

Images