A method for producing guanosine by fermentation

Abstract

The invention relates to the technical field of fermentation production, and discloses a method for fermenting and producing guanosine, comprising the following steps: inoculating a guanosine-producing strain into a slant culture medium for activation culture; then inoculating into a primary seed culture medium for culture to obtain a primary seed solution; inoculating the primary seed solution into a secondary seed culture medium for culture to obtain a secondary seed solution; inoculating the secondary seed solution into a fermentation culture medium, controlling the initial air volume of the fermentation equipment to be 700-900L / h and the initial rotation speed to be 200-400rpm, increasing the air volume and rotation speed according to the OD growth during the fermentation process, and controlling the dissolved oxygen content to be ≥30%; after the air volume and rotation speed are exhausted, when the OD value is 20-25, adding a glucose solution for the first time; when the residual sugar content of the fermented liquid is 5-7%, adding a glucose solution for the second time; when the residual sugar content of the fermented liquid is ≤1.0%, the fermentation is terminated. By adding a glucose solution to dilute the fermented liquid during the peak growth period of the microorganism, the oxygen consumption rate of the microorganism can be reduced, the dissolved oxygen level can be stabilized, and the guanosine yield and conversion rate can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation production, and particularly relates to a method for producing guanosine by fermentation. Background Art

[0002] As one of the nucleoside substances, guanosine has a wide range of uses and can be used in the production and preparation of food and medicine. Due to the wide range of applications and large demand for guanosine, the production of guanosine has gradually tended to be industrialized. In order to reduce the cost of industrial production of guanosine and improve production efficiency, how to improve the yield and conversion rate of industrial production of guanosine has become the key.

[0003] At present, the production method of guanosine mainly adopts microbial fermentation. In the process of microbial fermentation, the respiration intensity of microorganisms is an important factor affecting the growth and reproduction of bacteria and the metabolism of products. Generally, the greater the respiration intensity, the higher the production level. The oxygen required in the process of microbial fermentation mainly comes from the sterile air provided by the air system of the fermentation equipment, and the oxygen supply capacity of the fermentation equipment is fixed when the fermentation equipment is completed. When the respiration intensity of microbial fermentation is greater than the oxygen supply capacity of the fermentation equipment, there will be an imbalance between the supply and demand of dissolved oxygen, thereby affecting the fermentation production level of the microorganism, and it is impossible to maximize the yield and conversion rate. If the oxygen supply capacity of the fermentation equipment is upgraded, the cost is too high, which is not conducive to the industrial production of guanosine. Therefore, it is necessary to develop a low-cost fermentation production method for maintaining the balance of dissolved oxygen supply and demand to improve the yield and conversion rate of guanosine. Summary of the invention

[0004] In order to address the deficiencies of the prior art, the present invention provides a method for producing guanosine by fermentation, wherein glucose solution is added to dilute the fermentation broth during the peak period of microbial growth to reduce the density of the bacteria in the fermentation broth, thereby reducing the oxygen consumption rate of the bacteria and achieving a balanced state of dissolved oxygen supply and demand, thereby improving the yield and conversion rate of guanosine, and the production cost is low.

[0005] The technical effects to be achieved by the present invention are achieved through the following technical aspects:

[0006] The present invention provides a method for producing guanosine by fermentation, comprising the following steps:

[0007] S1, inoculating the guanosine-producing bacteria into the slant culture medium for activation culture;

[0008] S2, inoculating the activated guanosine-producing bacteria into a primary seed culture medium for cultivation to obtain a primary seed solution;

[0009] S3, inoculating the primary seed solution into the secondary seed culture medium for culturing to obtain the secondary seed solution;

[0010] S4. Inoculate the secondary seed liquid into the fermentation medium, and ferment in the fermentation equipment, control the initial air volume of the fermentation equipment to 700-900L / h, and the initial rotation speed to 200-400rpm. During the fermentation process, increase the air volume and rotation speed according to the OD growth of the fermentation liquid, and control the dissolved oxygen content of the fermentation liquid to be ≥30%; after the air volume and rotation speed are increased, when the OD value is 20-25, add the glucose solution for the first time; when the residual sugar content of the fermented liquid is 5-7%, add the glucose solution for the second time; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation.

[0011] As a further description of the technical solution of the present invention, in S4, the concentration of the glucose solution is 50-70%, the amount of glucose solution added for the first time is 3-7% of the initial volume of the fermentation liquid, and the amount of glucose solution added for the second time is 10-15% of the initial volume of the fermentation liquid.

[0012] As a further description of the technical solution of the present invention, in S4, when the glucose solution is replenished for the first time, the glucose solution is replenished within 1 to 3 hours; during the second replenishment of the glucose solution, the residual sugar content in the fermentation liquid is controlled to drop to 2 to 4%.

[0013] As a further description of the technical solution of the present invention, in S4, the inoculation amount of the secondary seed liquid is 5-15%, the fermentation temperature is 36±1° C., and the pH of the fermentation process is controlled at 6.5-6.6.

[0014] As a further description of the technical solution of the present invention, in S3, the first-level seed liquid is inoculated into the second-level seed culture medium, and the culture is carried out in a seed culture tank. The initial air volume of the seed culture tank is controlled to be 250-350L / h, and the initial rotation speed is 100-200rpm. During the culture process, the air volume and rotation speed are increased according to the OD growth of the culture solution. During the culture process, the dissolved oxygen content is controlled to be ≥30%, and the culture is terminated when the OD value of the culture solution reaches 12-15.

[0015] As a further description of the technical solution of the present invention, in S3, the inoculation amount of the primary seed solution is 0.1-0.3%, the culture temperature is 35±1° C., and the pH is controlled at 6.4-6.5 during the culture process.

[0016] As a further description of the technical solution of the present invention, in S1, the activation culture temperature is 36±1°C, and the activation culture time is 10 to 14 hours; in S2, the culture temperature is 36±1°C, and the culture time is 8 to 12 hours.

[0017] As a further description of the technical solution of the present invention, each 1L of the fermentation medium includes the following components: 200-250g of glucose, 3-5g of yeast extract, 10-15g of yeast extract powder, 15-20g of corn peptone, 3-7g of corn steep liquor powder, 1-2g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, 10-20mg of manganese sulfate, 10-20mg of ferrous sulfate, 0.3-0.7g of defoaming agent, and 0.1-0.5mg of adenosine.

[0018] As a further description of the technical solution of the present invention, each 1L of the primary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine;

[0019] Each 1L of the secondary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine.

[0020] As a further description of the technical solution of the present invention, each 1L of the slant culture medium includes the following components: 20-50g of soluble starch, 3-7g of corn peptone, 3-7g of yeast extract, 0.3-0.7g of adenine, and 15-30g of agar.

[0021] In summary, the present invention has at least the following benefits:

[0022] The method for producing guanosine by fermentation provided by the present invention increases the dissolved oxygen content in the fermentation liquid by increasing the air volume and rotation speed of the fermentation equipment in the early stage of fermentation, provides sufficient oxygen for the fermentation production of the strain, and after the air volume and rotation speed of the fermentation equipment are fully added, when the OD value of the fermentation liquid is 20-25, glucose solution is added to dilute the fermentation liquid, which can reduce the density of the bacteria in the fermentation liquid, thereby reducing the oxygen consumption rate of the bacteria, so as to maintain the balance of the supply and demand level of dissolved oxygen in the fermentation liquid, so that the metabolic products of the bacteria are stably accumulated, thereby improving the yield and conversion rate of guanosine, and the production cost is low. In the late stage of fermentation, when the residual sugar content of the fermentation liquid is 5-7%, glucose solution is added, which can provide sufficient nutrients for the bacteria to maintain the growth and metabolic level of the bacteria, which is conducive to further improving the yield and conversion rate of guanosine. DETAILED DESCRIPTION

[0023] For ease of understanding of the present invention, the present invention will be described more fully below in conjunction with specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thoroughly understood.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0025] The present invention provides a method for producing guanosine by fermentation, comprising the following steps:

[0026] S1, inoculating the guanosine-producing bacteria into the slant culture medium for activation culture;

[0027] S2, inoculating the activated guanosine-producing bacteria into a primary seed culture medium for cultivation to obtain a primary seed solution;

[0028] S3, inoculating the primary seed solution into the secondary seed culture medium for culturing to obtain the secondary seed solution;

[0029] S4. Inoculate the secondary seed liquid into the fermentation medium, and ferment in the fermentation equipment, control the initial air volume of the fermentation equipment to 700-900L / h, and the initial rotation speed to 200-400rpm. During the fermentation process, increase the air volume and rotation speed according to the OD growth of the fermentation liquid, and control the dissolved oxygen content of the fermentation liquid to be ≥30%; after the air volume and rotation speed are increased, when the OD value is 20-25, add the glucose solution for the first time; when the residual sugar content of the fermented liquid is 5-7%, add the glucose solution for the second time; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation.

[0030] It is understandable that fermentation equipment can dissolve oxygen in the fermentation liquid by introducing a certain amount of sterile air and performing mechanical stirring at a certain speed, thereby increasing the dissolved oxygen content in the fermentation liquid. Microorganisms need to consume oxygen for biological oxidation during their growth, reproduction and metabolism. The specific oxygen consumption rate of microorganisms refers to the oxygen consumption of a unit mass of bacterial cells per unit time, while the oxygen consumption rate of microorganisms refers to the oxygen consumption of a unit volume of culture liquid per unit time, which shows that the oxygen consumption rate is positively correlated with the bacterial concentration in the fermentation liquid.

[0031] In the early stage of fermentation, the dissolved oxygen content in the fermentation liquid is increased by increasing the air volume and rotation speed of the fermentation equipment, so as to provide sufficient oxygen for the fermentation production of the strains, so as to improve the growth, reproduction and metabolic production level of the bacteria. After the air volume and rotation speed of the fermentation equipment are fully added, when the fermentation liquid reaches the peak growth period of the bacteria with an OD value of 20 to 25, the fermentation liquid can be diluted by adding glucose solution to reduce the concentration of bacteria in the fermentation liquid, thereby reducing the oxygen consumption rate of the bacteria, so as to maintain the balance of the supply and demand level of dissolved oxygen in the fermentation liquid, so that the metabolic products of the bacteria can be accumulated stably, thereby improving the yield and conversion rate of guanosine, and the production cost is low. In the late stage of fermentation, when the residual sugar content of the fermentation liquid is 5 to 7%, glucose solution is added, which can provide sufficient nutrients for the bacteria to maintain the growth and metabolic level of the bacteria, which is conducive to further improving the yield and conversion rate of guanosine.

[0032] It should be noted that the feed used to dilute the fermentation liquid is not limited to glucose solution, and can also be sterile water or fermentation medium, etc., as long as the purpose of diluting the fermentation liquid can be achieved after supplementation and it is harmless to the bacteria. When the problem of insufficient dissolved oxygen also occurs in the fermentation production of other nucleoside substances, the method for producing guanosine by fermentation of the present invention can also be referred to.

[0033] In some embodiments, the guanosine-producing species can be Bacillus subtilis.

[0034] In S4, the concentration of the glucose solution is 50-70%, preferably 60%; the amount of glucose solution added for the first time is 3-7% of the initial volume of the fermentation liquid, preferably 5%; the amount of glucose solution added for the second time is 10-15% of the initial volume of the fermentation liquid, preferably 11%. In some embodiments, the total amount of glucose solution added for the first and second times during the fermentation process is 15-20% of the initial volume of the fermentation liquid, preferably 16%. When the glucose solution is added for the first time, the glucose solution is added within 1-3 hours, preferably 2 hours; during the second addition of the glucose solution, the residual sugar content of the fermentation liquid is controlled to slowly decrease to 2-4%, preferably 3%.

[0035] In some embodiments, the inoculation amount of the secondary seed liquid in S4 is 5-15%, preferably 10%, of the total volume of the fermentation liquid at 0h of fermentation; the fermentation temperature is 36±1°C; and the pH of the fermentation process is controlled at 6.5-6.6.

[0036] As a further optimization, in S3, the primary seed liquid is inoculated into the secondary seed culture medium, and culture is carried out in a seed culture tank, the initial air volume of the seed culture tank is controlled to be 250-350 L / h, preferably 300 L / h, the initial rotation speed is 100-200 rpm, preferably 150 rpm, the air volume and rotation speed are increased according to the OD growth of the culture solution during the culture process, the dissolved oxygen content is controlled to be ≥30% during the culture process, and the culture is terminated when the OD value of the culture solution reaches 12-15, preferably 14. By regulating the air volume and rotation speed during the secondary seed culture process, a higher dissolved oxygen level in the culture solution can be maintained, thereby increasing the growth and reproduction rate of the bacteria, shortening the culture time, reducing energy consumption, reducing production costs, and being more conducive to the industrial production of guanosine.

[0037] In some embodiments, the inoculation amount of the primary seed solution in S3 is 0.1-0.3% of the volume of the secondary seed culture medium, preferably 0.1%, the culture temperature is 35±1° C., and the pH is controlled at 6.4-6.5 during the culture process.

[0038] In some embodiments, the activation culture temperature in S1 is 36±1°C, and the activation culture time is 10 to 14 hours, preferably 12 hours. The culture temperature in S2 is 36±1°C, and the culture time is 8 to 12 hours, preferably 10 hours.

[0039] Specifically, each 1L fermentation medium includes the following components: 200-250g of glucose, 3-5g of yeast extract, 10-15g of yeast extract powder, 15-20g of corn peptone, 3-7g of corn steep liquor powder, 1-2g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, 10-20mg of manganese sulfate, 10-20mg of ferrous sulfate, 0.3-0.7g of defoamer, and 0.1-0.5mg of adenosine. Preferably, each 1L fermentation medium includes the following components: 220g of glucose, 4g of yeast extract, 14g of yeast extract powder, 18g of corn peptone, 5g of corn steep liquor powder, 1.5g of potassium dihydrogen phosphate, 2g of magnesium sulfate, 15mg of manganese sulfate, 15mg of ferrous sulfate, 0.5g of defoamer, and 0.3mg of adenosine.

[0040] Specifically, each 1L of primary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine. Preferably, each 1L of primary seed culture medium includes the following components: 35g of glucose, 3g of corn peptone, 15g of yeast extract, 2g of potassium dihydrogen phosphate, 0.9g of magnesium sulfate, 10mg of manganese sulfate, 10mg of ferrous sulfate, and 0.5mg of adenosine.

[0041] Each 1L of secondary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine. Preferably, each 1L of secondary seed culture medium includes the following components: 35g of glucose, 3g of corn peptone, 15g of yeast extract, 2g of potassium dihydrogen phosphate, 0.9g of magnesium sulfate, 10mg of manganese sulfate, 10mg of ferrous sulfate, and 0.5mg of adenosine.

[0042] Specifically, each 1L slant culture medium includes the following components: 20-50g soluble starch, 3-7g corn peptone, 3-7g yeast extract, 0.3-0.7g adenine, and 15-30g agar. Preferably, each 1L slant culture medium includes the following components: 30g soluble starch, 5g corn peptone, 5g yeast extract, 0.5g adenine, and 20g agar.

[0043] Example 1

[0044] This embodiment provides a method for producing guanosine by fermentation, comprising the following steps:

[0045] Preliminary preparation:

[0046] Preparation of slant medium: Each 1L slant medium includes the following components: 30g soluble starch, 5g corn peptone, 5g yeast extract, 0.5g adenine, 20g agar;

[0047] Preparation of primary seed culture medium: Each 1L of primary seed culture medium includes the following components: 35g glucose, 3g corn peptone, 15g yeast extract, 2g potassium dihydrogen phosphate, 0.9g magnesium sulfate, 10mg manganese sulfate, 10mg ferrous sulfate, and 0.5mg adenosine;

[0048] Preparation of secondary seed culture medium: Each 1L of secondary seed culture medium includes the following components: 35g glucose, 3g corn peptone, 15g yeast extract, 2g potassium dihydrogen phosphate, 0.9g magnesium sulfate, 10mg manganese sulfate, 10mg ferrous sulfate, and 0.5mg adenosine;

[0049] Preparation of fermentation medium: Each 1L fermentation medium includes the following components: 220g glucose, 4g yeast extract, 14g yeast extract powder, 18g corn peptone, 5g corn steep liquor powder, 1.5g potassium dihydrogen phosphate, 2g magnesium sulfate, 15mg manganese sulfate, 15mg ferrous sulfate, 0.5g defoamer, 0.3mg adenosine, and citric acid is used to adjust the pH to 3.7.

[0050] Preparation of glucose solution: The concentration of glucose solution is 60%.

[0051] S1. Take out the glycerol tube containing Bacillus subtilis from the ultra-low temperature refrigerator, take the strain and evenly spread it on the slant culture medium for activation culture, and culture it at 36°C for 12 hours;

[0052] S2, take a loop of activated Bacillus subtilis and inoculate it into the primary seed culture medium, and culture it on a shaking table at 36°C for 10 hours to obtain the primary seed solution;

[0053] S3, take 10ml of the first-level seed solution (inoculation volume of 0.1%) and inoculate it into the second-level seed culture medium, and culture it in a seed culture tank. The culture temperature is 35.5°C, and ammonia water is added to control the pH to 6.45. The initial air volume of the seed culture tank is controlled to be 300L / h, and the initial speed is 150rpm. The dissolved oxygen control is calibrated before transplanting. The air volume and speed are appropriately increased according to the OD growth of the culture solution. The dissolved oxygen content is controlled to be ≥30% during the culture process. When the OD value of the culture solution reaches 14, the culture is terminated to obtain the second-level seed solution;

[0054] S4, add 33.3L fermentation medium to a 50L fermenter, take 3.7L secondary seed solution (inoculation amount is 10%) and inoculate it into the fermentation medium, the total volume of the fermentation liquid after 0h of fermentation is 37L; control the temperature in the fermenter to 36°C, add ammonia water to control the initial pH of the fermentation liquid to 6.5, and control the pH of the fermentation liquid to 6.6 after 15h of fermentation; control the initial air volume of the fermenter to 800L / h and the initial speed to 300rpm, and adjust the temperature according to the OD growth of the fermentation liquid during the fermentation process. Increase the air volume and rotation speed appropriately according to the situation, and control the dissolved oxygen ≥30% during the fermentation process; after the air volume and rotation speed of the fermentation tank are fully added, when the OD value of the fermentation liquid reaches 20-25, quickly add 1.8L of glucose solution for the first time, and complete the replenishment within 2h; when the residual sugar content of the fermented liquid is 5%, add 4.2L of glucose solution for the second time, and control the residual sugar content of the fermentation liquid to slowly decrease from 5% to 3% during the replenishment process; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation, and the fermentation time is 126h.

[0055] During the fermentation process, at the peak growth period of the bacteria, the dissolved oxygen content of the fermentation liquid is between 15% and 25%. By adding glucose solution, the dissolved oxygen content of the fermentation liquid can be controlled to increase to 30% to 45%.

[0056] Example 2

[0057] This embodiment provides a method for producing guanosine by fermentation, comprising the following steps:

[0058] Preliminary preparation: The preparation of slant culture medium, primary seed culture medium, secondary seed culture medium, fermentation medium and glucose solution is the same as that in Example 1.

[0059] S1. Take out the glycerol tube containing Bacillus subtilis from the ultra-low temperature refrigerator, take the strain and evenly spread it on the slant culture medium for activation culture, and culture it at 36°C for 12 hours;

[0060] S2, take a loop of activated Bacillus subtilis and inoculate it into the primary seed culture medium, and culture it on a shaking table at 36°C for 10 hours to obtain the primary seed solution;

[0061] S3, take 10ml of the first-level seed solution (inoculation volume of 0.1%) and inoculate it into the second-level seed culture medium, and culture it in a seed culture tank. The culture temperature is 35.5°C, and ammonia water is added to control the pH to 6.45. The initial air volume of the seed culture tank is controlled to be 300L / h, and the initial speed is 150rpm. The dissolved oxygen control is calibrated before transplanting. The air volume and speed are appropriately increased according to the OD growth of the culture solution. The dissolved oxygen content is controlled to be ≥30% during the culture process. When the OD value of the culture solution reaches 14, the culture is terminated to obtain the second-level seed solution;

[0062] S4, add 31.5L fermentation medium to a 50L fermenter, take 3.5L secondary seed solution (inoculation amount is 10%) and inoculate it into the fermentation medium, the total volume of the fermentation liquid after 0h of fermentation is 35L; control the temperature in the fermenter to 36°C, add ammonia water to control the initial pH of the fermentation liquid to 6.5, and control the pH of the fermentation liquid to 6.6 after 15h of fermentation; control the initial air volume of the fermenter to 800L / h and the initial speed to 300rpm, and increase the OD value of the fermentation liquid during the fermentation process; Increase the air volume and rotation speed appropriately under long conditions, and control the dissolved oxygen ≥30% during the fermentation process; after the air volume and rotation speed of the fermentation tank are fully added, when the OD value of the fermentation liquid reaches 20-25, quickly add 2L of glucose solution for the first time, and complete the replenishment within 2h; when the residual sugar content of the fermented liquid is 5%, add 4L of glucose solution for the second time, and control the residual sugar content of the fermentation liquid to slowly decrease from 5% to 3% during the replenishment process; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation, and the fermentation time is 124h.

[0063] During the fermentation process, at the peak growth period of the bacteria, the dissolved oxygen content of the fermentation liquid is between 15% and 25%. By adding glucose solution, the dissolved oxygen content of the fermentation liquid can be controlled to increase to 30% to 45%.

[0064] Example 3

[0065] This embodiment provides a method for producing guanosine by fermentation, comprising the following steps:

[0066] Preliminary preparation: The preparation of slant culture medium, primary seed culture medium, secondary seed culture medium, fermentation medium and glucose solution is the same as that in Example 1.

[0067] S1. Take out the glycerol tube containing Bacillus subtilis from the ultra-low temperature refrigerator, take the strain and evenly spread it on the slant culture medium for activation culture, and culture it at 36°C for 12 hours;

[0068] S2, take a loop of activated Bacillus subtilis and inoculate it into the primary seed culture medium, and culture it on a shaking table at 36°C for 10 hours to obtain the primary seed solution;

[0069] S3, take 10ml of the first-level seed solution (inoculation volume of 0.1%) and inoculate it into the second-level seed culture medium, and culture it in a seed culture tank. The culture temperature is 35.5°C, and ammonia water is added to control the pH to 6.45. The initial air volume of the seed culture tank is controlled to be 300L / h, and the initial speed is 150rpm. The dissolved oxygen control is calibrated before transplanting. The air volume and speed are appropriately increased according to the OD growth of the culture solution. The dissolved oxygen content is controlled to be ≥30% during the culture process. When the OD value of the culture solution reaches 14, the culture is terminated to obtain the second-level seed solution;

[0070] S4, add 33.3L fermentation medium to a 50L fermenter, take 3.7L secondary seed solution (inoculation amount is 10%) and inoculate it into the fermentation medium, the total volume of the fermentation liquid after 0h of fermentation is 37L; control the temperature in the fermenter to 36°C, add ammonia water to control the initial pH of the fermentation liquid to 6.5, and control the pH of the fermentation liquid to 6.6 after 15h of fermentation; control the initial air volume of the fermenter to 800L / h and the initial speed to 300rpm, and adjust the temperature according to the OD growth of the fermentation liquid during the fermentation process. Increase the air volume and rotation speed appropriately according to the situation, and control the dissolved oxygen ≥30% during the fermentation process; after the air volume and rotation speed of the fermentation tank are fully added, when the OD value of the fermentation liquid reaches 20-25, quickly add 1.8L of glucose solution for the first time, and complete the replenishment within 1 hour; when the residual sugar content of the fermented liquid is 5%, add 4.2L of glucose solution for the second time, and control the residual sugar content of the fermentation liquid to slowly decrease from 5% to 3% during the replenishment process; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation, and the fermentation time is 126h.

[0071] During the fermentation process, at the peak growth period of the bacteria, the dissolved oxygen content of the fermentation liquid is between 15% and 25%. By adding glucose solution, the dissolved oxygen content of the fermentation liquid can be controlled to increase to 30% to 45%.

[0072] Comparative Example 1

[0073] This comparative example provides a method for producing guanosine by fermentation, comprising the following steps:

[0074] Preliminary preparation: The preparation of slant culture medium, primary seed culture medium, secondary seed culture medium, fermentation medium and glucose solution is the same as that in Example 1.

[0075] S1. Take out the glycerol tube containing Bacillus subtilis from the ultra-low temperature refrigerator, take the strain and evenly spread it on the slant culture medium for activation culture, and culture it at 36°C for 12 hours;

[0076] S2, take a loop of activated Bacillus subtilis and inoculate it into the primary seed culture medium, and culture it on a shaking table at 36°C for 10 hours to obtain the primary seed solution;

[0077] S3, take 10ml of the first-level seed solution (inoculation volume of 0.1%) and inoculate it into the second-level seed culture medium, and culture it in a seed culture tank. The culture temperature is 35.5°C, and ammonia water is added to control the pH to 6.45. The initial air volume of the seed culture tank is controlled to be 300L / h, and the initial speed is 150rpm. The dissolved oxygen control is calibrated before transplanting. The air volume and speed are appropriately increased according to the OD growth of the culture solution. The dissolved oxygen content is controlled to be ≥30% during the culture process. When the OD value of the culture solution reaches 14, the culture is terminated to obtain the second-level seed solution;

[0078] S4, add 33.3L fermentation medium to a 50L fermentor, take 3.7L secondary seed liquid (inoculation amount is 10%) and inoculate it into the fermentation medium, and the total volume of the fermentation liquid after 0h of fermentation is 37L; control the temperature in the fermenter to 36°C, add ammonia water to control the initial pH of the fermentation liquid to 6.5, and control the pH of the fermentation liquid to 6.6 after 15h of fermentation; control the initial air volume of the fermenter to 800L / h and the initial speed to 300rpm, increase the air volume and speed appropriately according to the OD growth of the fermentation liquid during the fermentation process, and control the dissolved oxygen to be ≥30% during the fermentation process; when the residual sugar content of the fermented liquid is 5%, add 6L of glucose solution, and control the residual sugar content of the fermented liquid to slowly decrease from 5% to 3% during the addition process; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation, and the fermentation time is 122h.

[0079] During the fermentation process, at the peak growth period of the bacteria, the dissolved oxygen content of the fermentation liquid is between 15% and 25%. By increasing the air volume and rotation speed of the fermentation tank, the dissolved oxygen content of the fermentation liquid can be controlled to increase to 15% to 30%.

[0080] Test Example 1

[0081] In this test example, the guanosine yield and conversion rate were measured and calculated for the fermentation broths of Examples 1 to 3 and Comparative Example 1. The results are shown in Table 1.

[0082] Table 1 Guanosine yield and conversion rate results of Examples 1 to 3 and Comparative Example 1

[0083]

[0084]

[0085] By comparing the results of Examples 1 to 3 with Comparative Example 1 in Table 1, it can be seen that the guanosine yield and conversion rate of the fermentation broth in Examples 1 to 3 are significantly higher than those in Comparative Example 1, which proves that by adding glucose solution to dilute the bacterial concentration of the fermentation broth when the air volume and rotation speed of the fermentation equipment are maximized, the oxygen consumption rate of the bacteria can be reduced, so that the dissolved oxygen supply and demand level of the fermentation broth continues to maintain a balanced state, so that the metabolic products of the bacteria can be stably accumulated, thereby improving the yield and conversion rate of guanosine, and the method for producing guanosine by fermentation of the present invention has low cost and is conducive to the industrial production of guanosine.

[0086] The above contents are merely examples and illustrations of the structure of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and these obvious replacement forms all belong to the protection scope of the present invention.

Claims

1. A method for producing guanosine by fermentation, characterized in that: The following steps are involved: S1, inoculating the guanosine-producing bacteria into the slant culture medium for activation culture; S2, inoculating the activated guanosine-producing bacteria into a primary seed culture medium for cultivation to obtain a primary seed solution; S3, inoculating the primary seed solution into the secondary seed culture medium for culturing to obtain the secondary seed solution; S4. Inoculate the secondary seed liquid into the fermentation medium, and ferment in the fermentation equipment, control the initial air volume of the fermentation equipment to 700-900L / h, and the initial rotation speed to 200-400rpm. During the fermentation process, increase the air volume and rotation speed according to the OD growth of the fermentation liquid, and control the dissolved oxygen content of the fermentation liquid to be ≥30%; after the air volume and rotation speed are increased, when the OD value is 20-25, add the glucose solution for the first time; when the residual sugar content of the fermented liquid is 5-7%, add the glucose solution for the second time; when the residual sugar content of the fermented liquid is ≤1.0%, end the fermentation.

2. The method for producing guanosine by fermentation according to claim 1, characterized in that: In S4, the concentration of the glucose solution is 50-70%, the amount of the glucose solution added for the first time is 3-7% of the initial volume of the fermentation liquid, and the amount of the glucose solution added for the second time is 10-15% of the initial volume of the fermentation liquid.

3. The method for producing guanosine by fermentation according to claim 2, characterized in that: In S4, when the glucose solution is replenished for the first time, the glucose solution is replenished within 1 to 3 hours; during the second replenishment of the glucose solution, the residual sugar content of the fermentation liquid is controlled to decrease to 2 to 4%.

4. The method for producing guanosine by fermentation according to claim 1, characterized in that: In S4, the inoculation amount of the secondary seed liquid is 5-15%, the fermentation temperature is 36±1° C., and the pH value during the fermentation process is controlled to be 6.5-6.

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5. The method for producing guanosine by fermentation according to claim 1, characterized in that: In S3, the first-level seed liquid is inoculated into the second-level seed culture medium, and the culture is carried out in a seed culture tank. The initial air volume of the seed culture tank is controlled to be 250-350 L / h, and the initial rotation speed is 100-200 rpm. During the culture process, the air volume and the rotation speed are increased according to the OD growth of the culture solution. During the culture process, the dissolved oxygen content is controlled to be ≥30%, and the culture is terminated when the OD value of the culture solution reaches 12-15.

6. The method for producing guanosine by fermentation according to claim 5, characterized in that: In the S3, the inoculation amount of the primary seed solution is 0.1-0.3%, the culture temperature is 35±1° C., and the pH is controlled at 6.4-6.5 during the culture process.

7. The method for producing guanosine by fermentation according to claim 1, characterized in that: In the S1, the activation culture temperature is 36±1° C., and the activation culture time is 10 to 14 h; in the S2, the culture temperature is 36±1° C., and the culture time is 8 to 12 h.

8. The method for producing guanosine by fermentation according to claim 1, characterized in that: The fermentation medium comprises the following components per 1L: 200-250g of glucose, 3-5g of yeast extract, 10-15g of yeast extract powder, 15-20g of corn peptone, 3-7g of corn steep liquor powder, 1-2g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, 10-20mg of manganese sulfate, 10-20mg of ferrous sulfate, 0.3-0.7g of defoaming agent and 0.1-0.5mg of adenosine.

9. The method for producing guanosine by fermentation according to claim 1, characterized in that: Each 1L of the primary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine; Each 1L of the secondary seed culture medium includes the following components: 30-40g of glucose, 1-5g of corn peptone, 10-20g of yeast extract, 1-3g of potassium dihydrogen phosphate, 0.5-1.2g of magnesium sulfate, 8-12mg of manganese sulfate, 8-12mg of ferrous sulfate, and 0.3-0.7mg of adenosine.

10. The method for producing guanosine by fermentation according to claim 1, characterized in that: Each 1L of the slant culture medium comprises the following components: 20-50g of soluble starch, 3-7g of corn peptone, 3-7g of yeast extract, 0.3-0.7g of adenine, and 15-30g of agar.

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