Method for improving fermentation yield of natamycin by regulating biomass growth

Abstract

The invention discloses a method for improving fermentation yield of natamycin by regulating biomass growth, and belongs to the technical field of industrial biology. The method comprises the following steps: in the process of fermentation production of natamycin, controlling the concentration of residual sugar in a fermentation culture medium, promoting biomass to be converted from a growth period to a production period, and feeding branched chain amino acid in a high-speed synthesis stage of natamycin to improve the synthesis rate of natamycin, thereby improving the fermentation yield of natamycin. The growth of biomass and the synthesis time point and the synthesis speed of natamycin are regulated and controlled by controlling the residual sugar concentration in the fermentation stage of natamycin, the residual sugar concentration in the early stage of fermentation is high, rapid propagation of thalli is promoted, secondary metabolism is inhibited, the residual sugar concentration is gradually reduced after entering the middle stage, the thalli are promoted to be converted from the growth stage to the production stage, and the yield of natamycin is increased. Further, branched chain amino acid is added in the later stage of fermentation, so that the element production capacity of hyphae in the later stage of fermentation is enhanced, and the fermentation content of natamycin is remarkably increased.

Description

Technical Field

[0001] The invention relates to a method for increasing the fermentation yield of natamycin by regulating the growth of biomass, and belongs to the field of industrial biotechnology. Background Art

[0002] Natamycin is a polyene macrolide antifungal agent. Currently, its production is mainly achieved by Streptomyces (Streptomyces Chattanooga, Streptomyces chrysosporium, Streptomyces lydicus and Streptomyces natalensis) through liquid submerged fermentation. Natamycin can bind to the sterol groups on the fungal cell membrane, thereby causing the fungal cell membrane to distort and rupture, so it has a strong inhibitory effect on fungi such as molds and yeasts. In the past 30 years, natamycin has played an important role in the treatment of fungal diseases, food preservation and freshness preservation, etc. with its extremely high safety (FDA, §172.155, 1994). It has the advantages of a wide range of applications, large market demand, and green safety. Natamycin is a secondary metabolite synthesized by the fermentation of Streptomyces. At present, its fermentation yield is low, which directly pushes up its market price (800-900 yuan / kg). To reduce the cost of natamycin application and market resistance, improving its fermentation synthesis level has become the core content of natamycin research.

[0003] At present, the domestic main method for strain selection is mutagenesis technology, which can increase the yield of natamycin to a certain extent, but the degree of increase is limited by the limitations of the technology itself. Therefore, providing a simple and convenient method to increase the yield of natamycin is extremely important for the industrial production and application of natamycin. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a method for increasing the fermentation yield of natamycin by regulating the growth of biomass, thereby promoting the synthesis of natamycin and increasing the fermentation yield of natamycin.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is: A method for increasing the fermentation yield of natamycin by regulating biomass growth. The method comprises: in the process of producing natamycin by fermentation, the residual sugar concentration in the fermentation medium is controlled to promote the transformation of the biomass from the growth phase to the production phase, and then branched-chain amino acids are added during the high-speed synthesis phase of natamycin to increase the synthesis rate of natamycin, thereby increasing the fermentation yield of natamycin.

[0006] Furthermore, the residual sugar concentration in the culture medium is controlled as follows: during fermentation 0-30 hours, the residual sugar concentration in the fermentation medium is controlled to be 10-40 g / L; during the conversion of biomass from the growth phase to the production phase, the residual sugar concentration in the fermentation medium is reduced to 5-10 g / L within 15 hours; during the high-speed synthesis stage of natamycin, the residual sugar concentration in the fermentation medium is controlled to be 0-5 g / L.

[0007] Furthermore, the residual sugar content in the fermentation process is controlled as follows: during fermentation 0-30 hours, the residual sugar concentration in the fermentation medium is controlled to be 10-15 g / L; during the conversion of biomass from the growth phase to the production phase, the residual sugar concentration in the fermentation medium is reduced to 6-8 g / L within 15 hours; during the high-speed synthesis stage of natamycin, the residual sugar concentration in the fermentation medium is controlled to be 0-2 g / L.

[0008] By controlling the residual sugar content in the fermentation medium, the fermentation yield of natamycin can be increased. In the early stage of fermentation culture, the residual sugar concentration in the fermentation medium is controlled to be high to promote the rapid reproduction of the bacteria. In the middle stage of fermentation culture, the residual sugar concentration in the fermentation medium is controlled to be low, which can shorten the growth period of the bacteria and promote the transition of the bacteria from the growth period to the production period.

[0009] Furthermore, the residual sugar content in the fermentation medium is controlled by adjusting the flow acceleration of the glucose solution; the concentration of the glucose solution is 60wt.%.

[0010] Furthermore, the starting point of the transformation of the biomass from the growth phase to the production phase is: the absorbance value A of the fermentation liquid 600 ≥30.

[0011] Furthermore, the conditions for the conversion of the biomass from the growth phase to the production phase are as follows: the fermentation pH drops from an initial pH value of 7.5 to 6.2, and the strain OD is greater than 15. At this time, the addition of glucose solution is stopped, and the glucose solution is added again after the residual sugar concentration in the fermentation system is less than 15 g / L, and the residual sugar concentration in the fermentation system is reduced to 5-10 g / L within 15 hours.

[0012] Furthermore, the biomass is a strain used to produce natamycin, specifically Streptomyces natalensis, Streptomyces chrysoporus or Streptomyces chatanus.

[0013] Furthermore, the high-speed synthesis stage of natamycin is fermented for 60-110 hours.

[0014] Furthermore, the branched-chain amino acids are added for 72-110 hours during fermentation. The branched-chain amino acids are added to stimulate the secondary metabolism of natamycin and promote the synthesis of natamycin.

[0015] Furthermore, the branched-chain amino acid is one or more of leucine, valine or isoleucine; when there are two or more branched-chain amino acids, the mass ratio of each component is 1:1 or 1:1:1.

[0016] Furthermore, the total concentration of the branched-chain amino acids is 30-50 wt.%, and the addition rate of the branched-chain amino acids is 0.1-1.0 (g / L) / h.

[0017] Furthermore, the method for increasing the fermentation yield of natamycin by regulating biomass growth comprises the following steps: (1) Cultivation of natamycin fermentation strains: Take out Streptomyces chrysoporus from the -80℃ freezer, spread it on the solid culture medium, and place it in a constant temperature incubator at 29℃ for 48-52h. Use an inoculation loop to pick out the colonies with good growth and place them in a 500mL Erlenmeyer flask containing 100mL of liquid seed culture medium. Place it in a constant temperature shaking culture at 29℃ and 180rpm for 24-40h until A 600 ≥2.0, the strain is well cultured; (2) A natamycin fermentation medium without glucose was prepared and added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121° C. for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 8-15%. At the same time, 500 mL of a 60 wt.% glucose solution was added for fermentation. The residual sugar content was controlled during the fermentation process, and branched-chain amino acids were added. The fermentation was cultured for 130 h to obtain a fermentation broth.

[0018] Furthermore, in step (1), the components and contents of the solid culture medium are: glucose 10 g / L, yeast powder 5 g / L, peptone 5 g / L, sodium chloride 5 g / L, agar 20 g / L, pH 7.5; the components and contents of the liquid seed culture medium are: glucose 10 g / L, yeast powder 6 g / L, peptone 6 g / L, NaCl 5 g / L, pH 7.5.

[0019] Furthermore, in step (2), the components and contents of the fermentation medium are: yeast powder 10 g / L, peptone 30 g / L, NaCl 5 g / L, corn germ oil 5 g / L, ammonium sulfate 1 g / L, pH 7.5.

[0020] Furthermore, in step (2), the fermentation culture conditions are: the tank pressure is 0.08±0.005MPa, the culture temperature is 29±0.5°C, the dissolved oxygen range is 30-40%, and the pH is controlled to be ≥6.0 by adding liquid alkali with a concentration of 15wt.% throughout the process. Automatic control begins when the pH drops to 6.0.

[0021] Furthermore, in step (2), during the fermentation process, the initial residual sugar concentration of the fermentation is 40 g / L; when the absorbance value A 600 When the temperature is ≥30, the residual sugar content in the culture medium is ≤10g / L, the natamycin strains begin to grow slowly, the pH is stabilized at around 6.0, and the residual sugar concentration in the culture medium is regulated to 0-5g / L from 60h to the end of fermentation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention regulates the growth of biomass, the synthesis time point and the synthesis rate of natamycin by controlling the residual sugar concentration in the natamycin fermentation stage. The residual sugar concentration is high in the early stage of fermentation, which promotes the rapid reproduction of the bacteria and inhibits secondary metabolism. After entering the middle stage, the residual sugar concentration is gradually reduced to promote the transformation of the bacteria from the growth stage to the production stage. Further, branched-chain amino acids are added in the late stage of fermentation to enhance the ability of mycelium to produce natamycin in the late stage of fermentation, thereby significantly improving the fermentation content of natamycin.

[0023] (2) The method of the present invention is simple and easy to operate, which reduces the fermentation cost, improves the production efficiency, and is conducive to the large-scale industrial production of natamycin.

[0024] (3) The method of the present invention increases the fermentation yield of natamycin by regulating the growth of biomass. Compared with the existing method for producing natamycin by fermentation, the yield of natamycin can be increased by 45%. DETAILED DESCRIPTION

[0025] Example 1 A method for increasing the fermentation yield of natamycin by regulating biomass growth comprises the following steps: (1) Cultivation of natamycin fermentation strains: Take out Streptomyces chrysoporus from the -80℃ freezer, spread it on the solid culture medium, and place it in a constant temperature incubator at 29℃ for 50 hours. Use an inoculation loop to pick out the colonies with good growth and place them in a 500mL Erlenmeyer flask containing 100mL of liquid seed culture medium. Place it in a constant temperature shaking culture at 29℃ and 180rpm for 32 hours until A 600 ≥2.0, the strain is well cultured; The formula of the solid culture medium, calculated on a 1L basis, includes the following components: 10g glucose, 5g yeast powder, 5g peptone, 5g sodium chloride, 20g agar, pH 7.5; the formula of the liquid seed culture medium, calculated on a 1L basis, includes the following components: 10g glucose, 6g yeast powder, 6g peptone, 5g NaCl, pH 7.5; (2) A natamycin fermentation medium without glucose was prepared. The formula of the fermentation medium was 1 L, including the following components: 10 g yeast powder, 30 g peptone, 5 g NaCl, 5 g corn germ oil, and 1 g ammonium sulfate. The medium was added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121° C. for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 8%. At the same time, 500 mL of a 60 wt.% glucose solution was added to start fermentation. The residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20 g / L, glucose solution was not added and fermentation was continued. After 30 hours of fermentation, the residual sugar concentration dropped to 10-15g / L, and then the residual sugar concentration of the fermentation medium was reduced to 6-8g / L within 15 hours. After 60 hours of fermentation, the residual sugar concentration was reduced to below 5g / L and controlled at 0-2g / L. To ensure this glucose concentration, the sampling frequency can be increased until the end of fermentation. At 72-110 hours of fermentation, a mixed solution of leucine, valine and isoleucine with a concentration of 30wt.% was added at a flow rate of 1.0 (g / L) / h. The fermentation culture was carried out for 130 hours to obtain the fermentation liquid.

[0026] Example 2 Step (1) is the same as step (1) in Example 1; (2) A natamycin fermentation medium without glucose was prepared. The formula of the fermentation medium was calculated based on 1 L, including the following components: 10 g yeast powder, 30 g peptone, 5 g NaCl, 5 g corn germ oil, and 1 g ammonium sulfate. The medium was added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121°C for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 15%. At the same time, 500 mL of a 60 wt.% glucose solution was added to start fermentation. The residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20 g / L, glucose solution was not added and fermentation was continued. After 30 hours of fermentation, the residual sugar concentration dropped to 10-15g / L, and then the residual sugar concentration of the fermentation medium was reduced to 6-8g / L within 15 hours. After 60 hours of fermentation, the residual sugar concentration was reduced to below 5g / L and controlled at 0-2g / L. To ensure the glucose concentration, the sampling frequency could be increased until the end of fermentation. At 72-110 hours of fermentation, a mixed solution of leucine and valine with a concentration of 50wt.% was added at a flow rate of 0.5 (g / L) / h. The fermentation was cultured for 130 hours to obtain the fermentation liquid.

[0027] Example 3 Step (1) is the same as step (1) in Example 1; (2) Prepare a natamycin fermentation medium without glucose. The fermentation medium formula is based on 1L, including the following components: 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, and 1g ammonium sulfate. Add it to a 15L fermentation tank, with a total liquid volume of 10L. Sterilize it at 115-121℃ for 20-30min. After sterilization, adjust the pH to 7.5. Inoculate the cultured bacteria at a 15% inoculum size, and add 500mL of Fermentation culture was started with a glucose solution having a concentration of 60wt.%, and the residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20g / L, glucose solution was not added and fermentation culture was continued. The residual sugar concentration dropped to 10-15g / L after 30h of fermentation, and then the residual sugar concentration of the fermentation medium was reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration dropped to below 5g / L and was controlled at 0-2g / L. To ensure this glucose concentration, the sampling frequency could be increased until the end of fermentation. At 72-110h of fermentation, a mixed solution of leucine and isoleucine with a concentration of 50wt.% was added at a flow rate of 1.0 (g / L) / h. The fermentation culture was carried out for 130h to obtain a fermentation liquid.

[0028] Example 4 Step (1) is the same as step (1) in Example 1; (2) A natamycin fermentation medium without glucose was prepared. The formula of the fermentation medium was calculated based on 1 L, including the following components: 10 g yeast powder, 30 g peptone, 5 g NaCl, 5 g corn germ oil, and 1 g ammonium sulfate. The medium was added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121°C for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 8%. At the same time, 500 mL of a 60 wt.% glucose solution was added to start fermentation. The residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20 g / L, glucose solution was not added and the fermentation was continued. Fermentation culture, the residual sugar concentration dropped to 10-15g / L after 30h of fermentation, and then reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration dropped to below 5g / L and was controlled at 0-2g / L. To ensure the glucose concentration, the sampling frequency was increased until the end of fermentation. At 72-110h of fermentation, a 30wt.% leucine solution was added at a rate of 0.5 (g / L) / h. The fermentation culture was continued for 130h to obtain the fermentation liquid.

[0029] Example 5 Step (1) is the same as step (1) in Example 1; (2) A natamycin fermentation medium without glucose was prepared. The formula of the fermentation medium was calculated based on 1 L, including the following components: 10 g yeast powder, 30 g peptone, 5 g NaCl, 5 g corn germ oil, and 1 g ammonium sulfate. The medium was added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121°C for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 8%. At the same time, 500 mL of a 60 wt.% glucose solution was added to start fermentation. The residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20 g / L, glucose solution was not added and the fermentation was continued. Fermentation culture, the residual sugar concentration dropped to 10-15g / L after 30h of fermentation, and then reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration dropped to below 5g / L and was controlled at 0-2g / L. To ensure the glucose concentration, the sampling frequency was increased until the end of fermentation. At 72-110h of fermentation, a 30wt.% valine solution was added at a rate of 0.3 (g / L) / h. The fermentation culture was continued for 130h to obtain the fermentation liquid.

[0030] Example 6 Step (1) is the same as step (1) in Example 1; (2) A natamycin fermentation medium without glucose was prepared. The formula of the fermentation medium was calculated based on 1 L, including the following components: 10 g yeast powder, 30 g peptone, 5 g NaCl, 5 g corn germ oil, and 1 g ammonium sulfate. The medium was added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121°C for 20-30 min. After sterilization, the pH was adjusted to 7.5. The cultured bacteria were inoculated at an inoculum rate of 8%. At the same time, 500 mL of a 60 wt.% glucose solution was added to start fermentation. The residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20 g / L, glucose solution was not added and fermentation was continued. Fermentation culture, the residual sugar concentration dropped to 10-15g / L after 30h of fermentation, and then reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration dropped to below 5g / L and was controlled at 0-2g / L. To ensure the glucose concentration, the sampling frequency could be increased until the end of fermentation. At 72-110h of fermentation, a 30wt.% isoleucine solution was added at a rate of 0.7 (g / L) / h. The fermentation culture was continued for 130h to obtain the fermentation liquid.

[0031] Example 7 Step (1) is the same as step (1) in Example 1; (2) Prepare a natamycin fermentation medium without glucose. The fermentation medium formula is based on 1L, including the following components: 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, and 1g ammonium sulfate. Add it to a 15L fermentation tank, with a total liquid volume of 10L. Sterilize it at 115-121℃ for 20-30min. After sterilization, adjust the pH to 7.5. Inoculate the cultured bacteria at a 15% inoculum size, and add 500mL of Fermentation culture was started with a glucose solution with a concentration of 60wt.%, and the residual sugar concentration was controlled during the fermentation process. Sugar samples were taken every 5 hours during the entire fermentation process. When the residual sugar concentration was lower than 20g / L, glucose solution was not added and fermentation culture was continued. The residual sugar concentration dropped to 10-15g / L after 30h of fermentation, and then the residual sugar concentration of the fermentation medium was reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration dropped to below 5g / L and was controlled at 0-2g / L. To ensure this glucose concentration, the sampling frequency could be increased until the end of fermentation. At 72-110h of fermentation, a mixed solution of leucine, valine and isoleucine with a concentration of 30wt.% was added at a flow rate of 1.0 (g / L) / h. The fermentation culture was carried out for 130h to obtain a fermentation liquid.

[0032] Comparative Example 1 Step (1) is the same as step (1) in Example 1; (2) The fermentation medium formula is as follows (based on 1L medium): 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, 1g ammonium sulfate, and 10L volume is added to a 15L fermentation tank, sterilized at 115-121℃ for 20-30min, and the volume after sterilization is 10L. After sterilization, the pH is adjusted to 7.5, and the cultured bacteria are inoculated at an inoculum of 8%, and 500mL of 60wt.% glucose aqueous solution is inoculated to start fermentation. During the entire fermentation process, samples are taken every 5h to measure sugar, and glucose is added to control the residual sugar concentration to above 30g / L. Glucose is no longer added until 110h of fermentation. The fermentation culture is cultured for 130h to obtain the fermentation liquid.

[0033] Comparative Example 2 Step (1) is the same as step (1) in Example 1; (2) The formula of the fermentation medium is as follows (based on 1L of medium): 10g of yeast powder, 30g of peptone, 5g of NaCl, 5g of corn germ oil, and 1g of ammonium sulfate. The mixture is added to a 15L fermenter according to a volume of 10L and sterilized at 115-121°C for 20-30min. The volume after sterilization is 10L. The pH is adjusted to 7.5 after sterilization. The cultured bacteria are inoculated at an inoculum rate of 8%. At the same time, 500mL of a 60wt.% glucose solution is added to start fermentation. During the entire fermentation process, sugar samples are taken every 5h for measurement. When the residual sugar content is lower than 20g / L, glucose solution is not added and fermentation is continued. After 30h of fermentation, the residual sugar concentration drops to 10-15g / L. Then, the residual sugar concentration of the fermentation medium is reduced to 6-8g / L within 15h. After 60h of fermentation, the residual sugar concentration drops to below 5g / L. To ensure this glucose concentration, the sampling frequency can be increased until the fermentation is completed. After 130h of fermentation, the fermentation liquid is obtained.

[0034] Comparative Example 3 Step (1) is the same as step (1) in Example 1; (2) The formula of the fermentation medium is as follows (based on 1L of medium): 10g of glucose, 10g of yeast powder, 30g of peptone, 5g of NaCl, 5g of corn germ oil, and 1g of ammonium sulfate. The mixture is added to a 15L fermenter according to a volume of 10L and sterilized at 115-121°C for 20-30min. The volume after sterilization is 10L. The pH is adjusted to 7.5 after sterilization. The cultured bacteria are inoculated at an inoculum rate of 8%. At the same time, 500mL of a 60wt.% glucose solution is added to start fermentation. During the entire fermentation process, sugar samples are taken every 5h for measurement. When the residual sugar content is higher than 5g / L, glucose is not added. When the residual sugar content is lower than 5g / L, glucose is added and the residual sugar concentration is maintained below 5g / L. To ensure this glucose concentration, the sampling frequency can be increased until the fermentation is completed. The fermentation culture lasts for 130h to obtain the fermentation liquid.

[0035] Comparative Example 4 Step (1) is the same as step (1) in Example 1; (2) The fermentation medium formula is as follows (based on 1L medium): 10g glucose, 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, 1g ammonium sulfate, and 10L volume is added to a 15L fermenter. Sterilize at 115-121℃ for 20-30min. The sterilized volume is 10L. After sterilization, the pH is adjusted to 7.5. The cultured bacteria are inoculated at an inoculum of 8%, and 500mL of 60wt.% glucose aqueous solution is inoculated to start fermentation. During the entire fermentation process, samples are taken every 5h to measure sugar, and glucose is added to control the residual sugar concentration to above 30g / L. No more glucose is added until 110h of fermentation. At 72-110h of fermentation, a 30wt.% leucine solution is added at a flow rate of 1.0 (g / L) / h. The fermentation culture is cultured for 130h to obtain the fermentation liquid.

[0036] Comparative Example 5 Step (1) is the same as step (1) in Example 1; (2) The fermentation medium formula is as follows (based on 1L medium): 10g glucose, 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, 1g ammonium sulfate, and 10L volume is added to a 15L fermenter. Sterilize at 115-121℃ for 20-30min. The sterilized volume is 10L. After sterilization, the pH is adjusted to 7.5. The cultured bacteria are inoculated at an inoculum of 8%, and 500mL of 60wt.% glucose aqueous solution is inoculated to start fermentation. During the entire fermentation process, samples are taken every 5h to measure sugar, and glucose is added to control the residual sugar concentration to above 30g / L. No more glucose is added until 110h of fermentation. At 72-110h of fermentation, valine solution with a concentration of 30wt.% is added at a flow rate of 1.0 (g / L) / h. Fermentation is cultured for 130h to obtain fermentation liquid.

[0037] Comparative Example 6 Step (1) is the same as step (1) in Example 1; (2) The fermentation medium formula is as follows (based on 1L medium): 10g glucose, 10g yeast powder, 30g peptone, 5g NaCl, 5g corn germ oil, 1g ammonium sulfate, and 10L volume is added to a 15L fermenter. Sterilize at 115-121℃ for 20-30min. The volume after sterilization is 10L. After sterilization, the pH is adjusted to 7.5. The cultured bacteria are inoculated at an inoculum of 8%, and 500mL of 60wt.% glucose aqueous solution is inoculated to start fermentation. During the entire fermentation process, samples are taken every 5h to measure sugar, and glucose is added to control the residual sugar concentration to above 30g / L. No more glucose is added until 110h of fermentation. At 72-110h of fermentation, a 30wt.% isoleucine solution is added at a flow rate of 1.0 (g / L) / h. Fermentation is cultured for 130h to obtain fermentation liquid.

[0038] Comparative Example 7 Step (1) is the same as step (1) in Example 1; (2) The fermentation medium formula is as follows (based on 1L of medium): 10g of glucose, 10g of yeast powder, 30g of peptone, 5g of NaCl, 5g of corn germ oil, and 1g of ammonium sulfate. Add 10L of the medium into a 15L fermenter and sterilize at 115-121℃ for 20-30min. The volume after sterilization is 10L. After sterilization, adjust the pH to 7.5, inoculate the cultured bacteria at an inoculum rate of 8%, and at the same time inoculate 500mL of a 60wt.% glucose aqueous solution to start fermentation. During the entire fermentation process, sugar samples were taken every 5 hours for measurement, and glucose was added to above 30 g / L to control the residual sugar concentration at above 20 g / L. No more glucose was added until 110 hours of fermentation. From 72 to 110 hours of fermentation, a mixed solution of 10 wt.% isoleucine, 10 wt.% valine and 10 wt.% isoleucine was added at a rate of 1.0 (g / L) / h. The fermentation was cultured for 130 hours to obtain the fermentation broth.

[0039] Experimental Example 1 The contents of natamycin in the fermentation broths of Examples 1-7 were determined and are listed in Table 1.

[0040] Table 1. Content of Natamycin As can be seen from Table 1, the content of natamycin in the fermentation broth obtained by the method of the present invention is higher than 13.0 g / L. It can be seen that the content of natamycin in the fermentation broth obtained by the method of the present invention is high, and the fermentation content of natamycin can be significantly improved.

[0041] Experimental Example 2 The contents of natamycin in the fermentation broths of Example 1 and Comparative Examples 1-7 were determined and listed in Table 2.

[0042] Table 2. Content of Natamycin Comparative Example 1 controls the entire fermentation process according to the prior art. Examples 2-3, based on Comparative Example 1, control the residual sugar concentration in the fermentation medium to control the growth and fermentation of the bacteria. Comparative Examples 4-7, based on Comparative Example 1, add one or more branched-chain amino acids in the late fermentation process.

[0043] It can be seen from Table 2 that, compared with Comparative Example 1, Comparative Examples 2-3 can control the growth of bacteria and increase the content of natamycin by controlling the residual sugar concentration in the fermentation process, and Comparative Examples 4-7 can increase the content of natamycin by adding branched-chain amino acids in the late fermentation period. The content of natamycin in Comparative Examples 2-7 is increased by more than 10%, while the content of natamycin obtained in Example 1 is increased by more than 45%. It can be seen that the method of the present invention can significantly increase the fermentation content of natamycin by controlling the residual sugar concentration in the fermentation medium and adding branched-chain amino acids.

Claims

1. A method for increasing the fermentation yield of natamycin by regulating biomass growth, characterized in that: In the process of fermentation production of natamycin, the residual sugar concentration in the fermentation medium is controlled to promote the transformation of biomass from the growth phase to the production phase, and then branched-chain amino acids are added during the high-speed synthesis stage of natamycin to increase the synthesis rate of natamycin, thereby increasing the fermentation yield of natamycin.

2. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The residual sugar concentration in the culture medium is controlled as follows: at 0-30h of fermentation, the residual sugar concentration in the fermentation medium is controlled to be 10-40g / L; during the conversion of biomass from the growth phase to the production phase, the residual sugar concentration in the fermentation medium is reduced to 5-10g / L within 15h; in the high-speed synthesis stage of natamycin, the residual sugar concentration in the fermentation medium is controlled to be 0-5g / L.

3. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The starting point of the transformation of the biomass from the growth phase to the production phase is: the absorbance value A of the fermentation liquid 600 ≥30.

4. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to any one of claims 1 to 3, characterized in that: The conditions for the conversion of the biomass from the growth phase to the production phase are as follows: the fermentation pH drops from an initial pH value of 7.5 to 6.2, and the strain OD is greater than 15. At this time, the addition of glucose solution is stopped, and the glucose solution is added again after the residual sugar concentration in the fermentation system is less than 15 g / L, and the residual sugar concentration in the fermentation system is reduced to 5-10 g / L within 15 hours.

5. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The natamycin high-speed synthesis stage is fermented for 60-110 hours.

6. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The time for adding branched-chain amino acids is 72-110 hours of fermentation.

7. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The branched-chain amino acids are one or more of leucine, valine or isoleucine.

8. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 6 or 7, characterized in that: The total concentration of the branched-chain amino acids is 30-50 wt.%, and the addition amount of the branched-chain amino acids is 0.1-1.0 (g / L) / h.

9. A method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 1, characterized in that: The following steps are involved: (1) Cultivation of natamycin fermentation strains: Take out Streptomyces chrysoporus from the -80℃ freezer, spread it on the solid culture medium, and place it in a constant temperature incubator at 29℃ for 48-52h. Use an inoculation loop to pick out the colonies with good growth and place them in a 500mL Erlenmeyer flask containing 100mL of liquid seed culture medium. Place it in a constant temperature shaking culture at 29℃ and 180rpm for 24-40h until A 600 ≥3.0, the strain is well cultured; (2) A natamycin fermentation medium without glucose was prepared and added to a 15 L fermentation tank with a total liquid volume of 10 L. The medium was sterilized at 115-121° C. for 20-30 min. After sterilization, the pH was adjusted to 7.

5. The cultured bacteria were inoculated at an inoculum rate of 8-15%. At the same time, 500 mL of a 60 wt.% glucose solution was added for fermentation. The residual sugar content was controlled during the fermentation process, and branched-chain amino acids were added. The fermentation was cultured for 130 h to obtain a fermentation broth.

10. The method for increasing the fermentation yield of natamycin by regulating biomass growth according to claim 9, characterized in that: In step (2), the fermentation culture conditions are: tank pressure of 0.08±0.005MPa, culture temperature of 29±0.5°C, dissolved oxygen range of 30-40%, and pH ≥ 6.0 by adding 15wt.% liquid caustic soda throughout the process, and automatic control begins when the pH drops to 6.0.

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