CN115216504A - Fermentation conversion method of salidroside - Google Patents
Fermentation conversion method of salidroside Download PDFInfo
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Abstract
The invention discloses a fermentation conversion method of salidroside, which comprises the following steps: fermenting and culturing recombinant escherichia coli, and after fermentation is finished, centrifugally collecting thalli; adding the thalli into a reaction solution with glucose and tyrosol as substrates to carry out biosynthesis of salidroside. The invention accumulates glucoside transferase in the fermentation stage, constructs an enzyme catalysis reaction system, and converts glucose and tyrosol as substrates to generate salidroside. By continuously optimizing the feed liquid formula, the reaction conditions and the control strategy in the fermentation and conversion stages. The method for producing salidroside can obtain higher salidroside yield in a short time, the content of salidroside in a conversion reaction system can reach 9206mg/L, and the conversion rate of tyrosol can reach more than 80%. The method for preparing salidroside is simple and easy to implement, low in cost and low in energy consumption, can be used for industrial large-scale production of salidroside, and has good economic and social benefits.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to a fermentation conversion method of salidroside.
Background
Salidroside is a phenolic compound widely existing in Salidroside, is one of main active monomers in Salidroside, has pharmacological activities of resisting radiation, resisting oxidation, enhancing immunity, promoting cancer cell apoptosis, etc., and can be widely used in pharmaceutical, cosmetic and food industries.
With the deep research on the action mechanism of salidroside and the definition of pharmacological action, the demand of salidroside is further increased, the traditional natural plant sources cannot meet the market demand due to the limitation of growth environment, content, separation and extraction processes and other conditions, and the chemical synthesis method has harsh reaction conditions, is easy to cause environmental pollution, has high treatment cost of hazardous wastes and is not beneficial to industrial production.
The method for preparing salidroside by fermentation of genetically modified microorganisms has the advantages of mild reaction conditions, low cost and wide source of production raw materials, and is the main trend of industrial production of salidroside. However, in the journal and patent literature disclosed at present, the recombinant saccharomyces cerevisiae, monascus and other fungi are mostly adopted for deep fermentation, and the problems of long fermentation period, low biosynthesis yield and the like exist.
Disclosure of Invention
The invention aims to provide a fermentation and transformation method of salidroside.
In order to achieve the purpose of the invention, the invention provides a fermentation conversion method of salidroside, which comprises the following steps:
A. culturing recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs by fermentation, and centrifuging to collect thallus after fermentation;
B. and D, adding the thalli obtained in the step A into a reaction solution taking glucose and tyrosol as substrates to carry out biosynthesis of salidroside.
The recombinant Escherichia coli can be referred to in the specification of U.S. Pat. No. 119-147 of US20190264221A 1.
In the foregoing method, step a includes the following substeps:
a1, preparing a recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs seed solution;
and A2, performing fermentation culture in a complementary mode.
In step A2, when the fermentation liquid OD 600nm A value of 2-5 (preferably OD) 600nm When the value reaches 3), the feeding of the feed medium is started continuously until the fermentation liquid OD is reached 600nm The fermentation is stopped when the value reaches 40 and the thallus content reaches 10-12% (volume percentage) by centrifugal detection.
Seed culture medium used: 10g/L of soybean peptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH7.0;
the fermentation medium used: 5g/L of glycerol, 5g/L of yeast powder, 5g/L of soybean peptone and 18g/L of dipotassium hydrogen phosphate. 6.8g/L potassium dihydrogen phosphate, 0.7g/L sodium sulfate, 0.5g/L magnesium sulfate, 3.2g/L ammonium chloride and 0.01g/L calcium chloride.
The feed medium used was: 10g/L of yeast powder, 10g/L of soytone and 500g/L of glycerol.
In step A2, the feeding rate of the feed medium is preferably 20 mL/L.multidot.h.
Further, step A1 comprises: inoculating 7 hr recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs into seed culture medium, and culturing at 37 + -1 deg.C to OD 600nm The value reaches 0.5-1.0, and the seed liquid is obtained.
Further, step A2 includes: b, inoculating the seed solution obtained in the step A1 into a fermentation culture medium according to the volume ratio of 0.02-0.04% (preferably 0.03%), and performing fermentation culture under the conditions of 35-38 ℃ and 0.05 +/-0.01 MPa; OD of the solution to be fermented 600nm When the value reaches 8-10 ℃, the temperature is reduced to 23-26 ℃ for continuous culture.
The initial dissolved oxygen of the reaction is 100 percent, the dissolved oxygen is controlled to be more than 20 percent in the fermentation process, the aeration is controlled to be 1 +/-0.3 VVM, the rotating speed is controlled to be 150-450rpm, and the pH of the fermentation system is controlled to be 6.8-7.0 in the whole fermentation process. Preferably, the pH of the system is adjusted with aqueous ammonia.
In the foregoing method, in the step B, the reaction solution is: tyrosol 0.7g/L, glucose 30g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 2.7g/L, calcium chloride 0.1g/L, formulated with water.
The step B comprises the following steps: will 10 8 -10 9 Adding 1.5kg of CFU/g thallus into a reaction tank filled with reaction liquid, wherein the liquid filling amount of the reaction tank is 30L/50L, and performing biosynthesis of salidroside at 35-38 ℃ under the pressure of 0.05 +/-0.01 MPa;
the initial dissolved oxygen of the reaction tank is 100 percent, the dissolved oxygen is controlled to be not less than 60 percent in the reaction process, and the pH value of the system is controlled to be 7.2 to 7.5 in the whole biosynthesis process. Preferably, the pH of the system is adjusted with sodium hydroxide.
Further, the step B also comprises the step of adding a glucose solution and a tyrosol solution into the reaction liquid in the biosynthesis process of the salidroside.
Preferably, the glucose solution is fed when the glucose concentration in the reaction solution is consumed to 2%, and the glucose concentration in the reaction solution is controlled to be 2-3%.
More preferably, the feeding rate of the glucose solution corresponds to a feeding of 3-6g glucose per liter of reaction solution per hour.
Preferably, the tyrosol solution is fed when the tyrosol concentration in the reaction liquid is consumed to 0.03%, and the tyrosol concentration in the reaction liquid is controlled to be 2-5mmoL/L.
More preferably, the feeding rate of the tyrosol solution corresponds to the feeding of 200-280mg tyrosol per liter of reaction solution per hour.
In the step B, the synthesis reaction is stopped when the salidroside content is increased to be less than 100 mu g/mL.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention accumulates glucoside transferase in the fermentation stage, constructs an enzyme catalysis reaction system, and converts glucose and tyrosol as substrates to generate salidroside. By continuously optimizing the feed liquid formula, the reaction conditions and the control strategy in the fermentation and conversion stages. The method for producing salidroside can obtain higher salidroside yield in a short time, the content of salidroside in a conversion reaction system can reach 9206mg/L, and the conversion rate of tyrosol can reach more than 80%. The method for preparing salidroside is simple and easy to implement, low in cost and low in energy consumption, can be used for industrial large-scale production of salidroside, and has good economic and social benefits.
The process replaces the common carbon source glucose of the escherichia coli by glycerol, and correspondingly optimizes the proportion of various materials in the fermentation liquid, so that the optimal combination is obtained to reduce the growth speed and prevent the enzyme from forming inclusion bodies, and simultaneously, the accumulation of metabolites such as acetic acid, glutamic acid and the like is reduced, and the generation of glucoside transferase which is hindered by feedback inhibition is prevented.
And (II) a control strategy of gradient cooling is adopted in the fermentation culture process, and a large number of experimental verifications are carried out on the cooling time and range in different culture media, so that the temperature control condition in the invention is finally determined. The proper temperature can effectively regulate and control the growth rate, the inclusion body formation is prevented while the higher thallus content is obtained, and in addition, the proper temperature can enable the peptide chain to be folded better so as to improve the activity of the glucosidase.
And thirdly, the invention also synergistically optimizes the reaction concentration of glucose and tyrosol and the use amount of the centrifugal thalli in the reaction stage. A large amount of exploration experiments are carried out on centrifugal thalli, glucose concentration and tyrosol concentration by combining with the conditions of industrial production equipment, so that the reaction rate is maintained at the optimal level, and the yield of salidroside is improved. In the catalytic reaction process, the reaction speed is reduced when the concentration of the substrate is too low, but escherichia coli enters a vigorous metabolic state when the concentration of the substrate is too high, and a large amount of byproducts are generated to influence the synthesis rate of salidroside. Too little thallus dosage can also reduce the conversion efficiency, and too much thallus dosage can destroy the stability of the enzyme catalysis reaction system conditions such as dissolved oxygen content, temperature uniformity and the like due to too high growth and metabolism rate. Therefore, the feeding rate in the reaction phase needs to be strictly controlled according to the optimal conditions obtained by a large amount of experiments to improve the yield of salidroside.
Detailed Description
The invention provides a fermentation conversion method of salidroside, which comprises the following steps:
(1) Adding seed liquid of the fermentation strain into a fermentation culture medium; the initial dissolved oxygen of the fermentation tank is 100 percent, the dissolved oxygen is controlled to be more than 20 percent in the fermentation process, and ammonia water is adopted to adjust the pH value of the fermentation liquor in the whole process;
(2) When OD is reached 600nm A value of 2-5 (preferably OD) 600nm When the value is 3), continuously adding a carbon-nitrogen source mixed solution (supplemented culture medium) in a flowing manner, and continuously culturing;
(3) When fermentation broth OD 600nm When the value reaches 8-10, reducing the culture temperature (to 23-26 ℃);
(4) Fermenting for 15-17h, when OD is 600nm The fermentation is stopped when the value reaches 40 and the content of the thalli reaches 10-12% by centrifugal detection.
Wherein, the step (1) is to add the seed liquid of the fermentation strain into the fermentation culture medium according to the volume ratio of 0.02 percent to 0.04 percent (preferably 0.03 percent) of the seed liquid to the fermentation culture medium.
The fermentation strain is recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs disclosed in US20190264221A1, and the capacity of biosynthesizing salidroside is obtained by over-expressing exogenous 4-hydroxybenzaldehyde reductase genes and glucoside transferase genes.
In the fermentation process of the method, the initial temperature is controlled to be 35-38 ℃ (preferably 36-38 ℃), the temperature is reduced to be 23-26 ℃ (preferably 24-26 ℃), the pressure is 0.05 +/-0.01 MPa, and ammonia water is fed in the whole fermentation process to adjust the pH value to be not less than 6.8.
In the method, the formula of the fermentation medium in the step (1) is as follows: 5g/L of glycerol, 5g/L of yeast powder, 5g/L of soybean peptone, 18g/L of dipotassium hydrogen phosphate, 6.8g/L of potassium dihydrogen phosphate, 0.7g/L of sodium sulfate, 0.5g/L of magnesium sulfate, 3.2g/L of ammonium chloride and 0.01g/L of calcium chloride, and the components are prepared by water.
In the method, the method for feeding the carbon-nitrogen source mixed solution in the step (2) is to supplement 10-15g of glycerol, 0.4-0.6g of yeast powder and 0.4-0.6g of soytone per liter of fermentation liquor per hour, and the mass of the glycerol, the yeast powder and the soytone is calculated according to the mass of pure substances. The mixed solution for feeding is prepared separately, and the concentration of glycerol can be 40-70%.
Further, the invention provides a method for synthesizing salidroside by converting tyrosol and glucose through constructing a glucoside transferase catalytic reaction system through the fermentation product, which comprises the following steps:
(5) Adding thalli obtained by centrifuging fermentation liquor into reaction liquid; the initial dissolved oxygen of the reaction tank is 100 percent, the dissolved oxygen is controlled to be not less than 60 percent in the reaction process, and the pH value of the fermentation liquor is adjusted by adopting sodium hydroxide in the whole process;
(6) When the glucose concentration in the reaction solution is consumed to 2%, feeding a glucose solution;
(7) When the tyrosol concentration in the reaction liquid is consumed to 0.03%, feeding tyrosol solution, and controlling the tyrosol concentration in the reaction liquid to be 2-5mmoL/L according to the actual conversion condition.
(8) The reaction was stopped when the salidroside content increased less than 100. Mu.g/mL.
In the reaction process of the method, the temperature is controlled to be 35-38 ℃ (preferably 36-38 ℃), the pressure is 0.05 +/-0.01 MPa, and sodium hydroxide is fed in the whole reaction process to adjust the pH value to be not less than 7.2.
In the method, the formula of the reaction solution in the step (5) is as follows: tyrosol 0.7g/L, glucose 30g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 2.7g/L, calcium chloride 0.1g/L, formulated with water.
In the method, the method for feeding glucose in the step (6) is to supplement 3-6g of glucose per liter of fermentation liquor per hour, and the glucose is calculated according to the solid mass of the glucose. The supplementary material is prepared with glucose solution alone in concentration of 40-70%.
In the method, the method for feeding tyrosol in the step (7) is to supplement 200-280mg of tyrosol per liter of fermentation liquor per hour, and the tyrosol is calculated according to the solid mass of the tyrosol. The supplementary material is prepared by tyrosol solution alone, and the concentration is not higher than 5%.
Recombinant E.coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs used in the present invention was provided by the white head institute for biological research, cambridge, mass.
The reagents used in the following examples are as follows:
seed culture medium: 10g/L of soybean peptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH7.0.
Fermentation medium: 5g/L of glycerol, 5g/L of yeast powder, 5g/L of soybean peptone, 18g/L of dipotassium hydrogen phosphate, 6.8g/L of potassium dihydrogen phosphate, 0.7g/L of sodium sulfate, 0.5g/L of magnesium sulfate, 3.2g/L of ammonium chloride and 0.01g/L of calcium chloride.
Reaction solution: tyrosol 0.7g/L, glucose 30g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 2.7g/L, calcium chloride 0.1g/L
And (3) a feed culture medium: 10g/L of yeast powder, 10g/L of soytone and 500g/L of glycerol.
600g/L glucose solution and 300g/L sodium hydroxide solution are prepared and sterilized separately for feeding, and 42g/L tyrosol solution is filtered and sterilized for later use. In addition, 25% -28% ammonia water is prepared to be added in a flowing way for use.
Example 1 fermentative conversion of Salidroside (50L bench test)
Preparing 3L seed culture medium in 5L seed tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, inoculating recombinant Escherichia coli cultured in shake flask seed culture medium at 0.3% (volume ratio), culturing at 37 + -1 deg.C for 7 hr until OD is reached 600nm The value reaches 0.6, and the seed liquid is obtained.
Preparing 30L fermentation medium in a 50L fermentation tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, adjusting pH to 6.8 with ammonia water, inoculating the cultured seed liquid at 0.03% (volume ratio), controlling temperature at 37 + -1 deg.C and pressure at 0.05 + -0.01 MPa, controlling aeration and rotation speed according to dissolved oxygen condition to ensure dissolved oxygen is above 20% (setting initial dissolved oxygen of fermentation tank as 100%), controlling aeration at 1 + -0.3 VVM, controlling rotation speed at 150-450rpm, and adding ammonia water to control pH not less than 6.8.
Monitoring of fermentation broth OD 600nm Value, when OD 600nm When the value reaches 3, feeding the supplemented medium at a fixed speed, wherein the specific supplement amount is 20mL of mixed solution per liter of fermentation liquor per hour, namely 20 mL/L.h. When fermentation broth OD 600nm When the value reached 10, the temperature was lowered to 22 ℃ to continue the culture. When fermentation broth OD is obtained 600nm When the value reaches 40 and the cell content exceeds 10% (volume ratio), the fermentation is stopped. And (4) quickly centrifuging the fermentation liquor, removing supernatant, and collecting thalli.
Preparing 30L of reaction solution in a 50L reaction tank, sterilizing at 121 ℃ for 25min, cooling to 37 ℃, and adjusting the pH to 7.2 by using a sodium hydroxide solution.
And (3) putting all the thalli obtained by centrifugation into the reaction liquid, controlling the temperature at 37 +/-1 ℃ and the pressure at 0.07 +/-0.01 MPa in the reaction process, controlling the aeration and the rotating speed according to the dissolved oxygen condition to ensure that the dissolved oxygen is more than 60 percent (setting the initial dissolved oxygen of the reaction tank as 100 percent), controlling the aeration to be 1 +/-0.3 VVM and the rotating speed to be 200-600rpm, and adding sodium hydroxide in the whole process to control the pH to be not less than 7.2.
And monitoring the glucose concentration, starting to feed a glucose solution when the content is lower than 2%, and feeding 5g of glucose (calculated according to the solid mass of the glucose) per liter of reaction solution per hour, namely 5 g/L.multidot.h. After the reaction is started for 2 hours, the tyrosol solution is fed, and 220mg of tyrosol (calculated according to the solid mass of tyrosol) is fed into each liter of reaction solution every hour, namely 220 mg/L.h. After the start of the feeding, the glucose concentration in the reaction mixture was controlled to 2% and the tyrosol concentration was controlled to 5mM.
Stopping reaction when the salidroside content increases less than 100 mg/L.h, and determining the final salidroside content to be 9206mg/L.
Example 2 fermentative conversion of Salidroside (5000L pilot plant experiment)
Preparing 300L of seed culture medium in a 500L seeding tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, inoculating recombinant Escherichia coli cultured in the seed culture medium in a shake flask in an amount of 0.3% (volume ratio) for 7 hr, and culturing at 37 + -1 deg.C for 7 hr.
Preparing 3000L fermentation medium in 5000L fermentation tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, adjusting pH to 6.8 with ammonia water, inoculating the cultured seed solution at 0.03% (volume ratio), controlling temperature at 37 + -1 deg.C and pressure at 0.05 + -0.01 MPa, controlling aeration and rotation speed according to dissolved oxygen condition to ensure dissolved oxygen above 20% (setting initial dissolved oxygen position of fermentation tank at 100%), controlling aeration at 0.5-1VVM, controlling rotation speed at 50-150rpm, and controlling pH not lower than 6.8 by adding ammonia water in whole course.
Monitoring of fermentation broth OD 600nm Value, when OD 600nm When the value reaches 3, feeding the supplemented medium at a fixed speed, wherein the specific addition amount is that 200mL of mixed solution is supplemented to each liter of fermentation liquor every hour, namely 20 mL/L.h. When fermentation broth OD 600nm When the value reached 10, the culture temperature was lowered to 22 ℃ to continue the culture. When fermentation broth OD 600nm The value reaches 40, the thallus content exceeds 10 percent (volume ratio)When the fermentation is completed, the fermentation is stopped. And (4) quickly centrifuging the fermentation liquor, removing supernatant, and collecting thalli.
3000L of reaction solution is prepared in a 5000L reaction tank, sterilized at 121 ℃ for 25min, cooled to 37 ℃, and adjusted to pH 7.2 by sodium hydroxide solution.
Putting all the thalli obtained by centrifugation into the reaction solution, controlling the temperature at 37 +/-1 ℃ and the pressure at 0.07 +/-0.01 MPa in the reaction process, controlling the aeration and the rotating speed according to the dissolved oxygen condition to ensure that the dissolved oxygen is more than 60 percent (setting the initial dissolved oxygen of a reaction tank as 100 percent), controlling the aeration at 1 +/-0.3 VVM and the rotating speed at 50-150rpm, and feeding sodium hydroxide in the whole process to control the pH value to be not less than 7.2.
Monitoring the glucose concentration, feeding a glucose solution when the content is lower than 2%, and feeding 5g of glucose (calculated according to the solid mass of glucose) per liter of reaction solution per hour, namely 5 g/L.h. After the reaction is started for 2 hours, the tyrosol solution is fed, and 220mg of tyrosol (calculated according to the solid mass of tyrosol) is fed into each liter of reaction solution every hour, namely 220 mg/L.h. After the start of the feeding, the glucose concentration in the reaction mixture was controlled to 2% and the tyrosol concentration was controlled to 5mM.
Stopping reaction when the content of salidroside is increased to less than 100 mg/L.h, and determining the final content of salidroside to be 9097mg/L.
Example 3 control experiment (50L bench test)
In preliminary experiments, it was verified that the fermentation stage and the reaction conversion stage use the same carbon source, i.e. glucose is used as the main carbon source in the fermentation stage.
The addition amount of glucose is adjusted on the basis of the LB culture medium formula, and the inorganic salt ratio is synergistically optimized to be used as a fermentation culture medium.
The fermentation medium (carbon source is glucose) formula is as follows: 20g/L of glucose, 10g/L of soybean peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 18g/L of dipotassium phosphate, 6.8g/L of potassium dihydrogen phosphate, 0.35g/L of sodium sulfate, 0.6g/L of magnesium sulfate and 0.01g/L of calcium chloride.
Feed medium 1: 10g/L of yeast powder and 10g/L of soytone;
feed medium 2: glucose 500g/L.
The fermentative conversion was carried out in the same manner as in example 1, specifically as follows:
preparing 3L seed culture medium in 5L seed tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, inoculating recombinant Escherichia coli cultured in shake flask seed culture medium at 0.3% (volume ratio), culturing at 37 + -1 deg.C for 7 hr until OD is reached 600nm The value reaches 0.6, and the seed liquid is obtained.
Preparing 30L fermentation medium in a 50L fermentation tank, sterilizing at 121 deg.C for 25min, cooling to 37 deg.C, adjusting pH to 6.8 with ammonia water, inoculating the cultured seed liquid at 0.03% (volume ratio), controlling temperature at 37 + -1 deg.C and pressure at 0.05 + -0.01 MPa, controlling aeration and rotation speed according to dissolved oxygen condition to ensure dissolved oxygen is above 20% (setting initial dissolved oxygen of fermentation tank as 100%), controlling aeration at 1 + -0.3 VVM, controlling rotation speed at 150-450rpm, and adding ammonia water to control pH not less than 6.8.
Monitoring of fermentation broth OD 600nm Value, when OD 600nm When the value reaches 3, feeding the supplemented medium 1 and the supplemented medium 2 at a fixed speed, wherein the specific addition amount is 20mL of supplemented medium 1 mixed solution added to each liter of fermentation liquor per hour, namely 20 mL/L.h; 8mL of glucose solution of a feed culture medium 2 is added to each liter of fermentation liquor every hour (each liter of reaction liquid is added with 4g of glucose every hour according to the solid mass of the glucose), namely 8 mL/L.h; . When fermentation broth OD 600nm When the value reached 10, the culture temperature was lowered to 22 ℃ to continue the culture. When fermentation broth OD 600nm When the value reaches 40 and the thallus content exceeds 10% (volume ratio), the fermentation is stopped. And (4) quickly centrifuging the fermentation liquor, removing supernatant, and collecting thalli.
Preparing 30L of reaction solution in a 50L reaction tank, sterilizing at 121 ℃ for 25min, cooling to 37 ℃, and adjusting the pH value to 7.2 by using a sodium hydroxide solution.
And (3) putting all the thalli obtained by centrifugation into the reaction liquid, controlling the temperature at 37 +/-1 ℃ and the pressure at 0.07 +/-0.01 MPa in the reaction process, controlling the aeration and the rotating speed according to the dissolved oxygen condition to ensure that the dissolved oxygen is more than 60 percent (setting the initial dissolved oxygen of the reaction tank as 100 percent), controlling the aeration to be 1 +/-0.3 VVM and the rotating speed to be 200-600rpm, and adding sodium hydroxide in the whole process to control the pH to be not less than 7.2.
Monitoring the glucose concentration, feeding a glucose solution when the content is lower than 2%, and feeding 5g of glucose (calculated according to the solid mass of glucose) per liter of reaction solution per hour, namely 5 g/L.h. After the reaction is started for 2 hours, the tyrosol solution is fed, and 130mg of tyrosol is fed into each liter of reaction solution every hour (calculated according to the solid mass of the tyrosol), namely 130 mg/L.h (the enzyme activity is poor, the substrate consumption speed is low, and the feeding speed is reduced). After the start of the feeding, the glucose concentration in the reaction mixture was controlled to 2% and the tyrosol concentration was controlled to 5mM.
Stopping reaction when the content of salidroside is increased to less than 100 mg/L.h, and determining that the content of salidroside is 4816mg/L and the conversion rate is about 50%.
The comparison experiment shows that the production process can be optimized according to the method to greatly improve the output of salidroside and the conversion rate of tyrosol. In a 50L-scale fermentation conversion experiment, the yield is improved by nearly 90 percent, and the conversion rate of the tyrosol is improved by about 60 percent.
Although the invention has been described in detail with respect to the general description and the specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The fermentation conversion method of salidroside is characterized by comprising the following steps:
A. culturing recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs by fermentation, and centrifuging to collect thallus after fermentation;
B. and D, adding the thalli obtained in the step A into a reaction solution taking glucose and tyrosol as substrates to carry out biosynthesis of salidroside.
2. The method according to claim 1, characterized in that step a comprises the following sub-steps:
a1, preparing a recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs seed solution;
a2, fermentation culture is carried out in a supplementary mode;
in step A2, when the fermentation liquid OD 600nm When the value reaches 2-5, the feeding of the feed medium is started continuously until the fermentation liquor OD 600nm Stopping fermentation when the value reaches 40 and the content of the thalli reaches 10-12% by centrifugal detection;
seed culture medium used: 10g/L of soybean peptone, 5g/L of yeast extract, 10g/L of sodium chloride and pH7.0;
the fermentation medium used: 5g/L of glycerol, 5g/L of yeast powder, 5g/L of soybean peptone, 18g/L of dipotassium phosphate, 6.8g/L of potassium dihydrogen phosphate, 0.7g/L of sodium sulfate, 0.5g/L of magnesium sulfate, 3.2g/L of ammonium chloride and 0.01g/L of calcium chloride;
the feed medium used was: 10g/L of yeast powder, 10g/L of soytone and 500g/L of glycerol.
3. The method according to claim 2, wherein the feed medium is fed at a feed rate of 20 mL/L-h in step A2.
4. The method according to claim 3, wherein step A1 comprises: inoculating 7 hr recombinant Escherichia coli BL21 (DE 3) E.coli- (pHhis 8-4) -UGTs into seed culture medium, and culturing at 37 + -1 deg.C to OD 600nm The value reaches 0.5-1, namely the seed liquid;
the step A2 comprises the following steps: b, inoculating the seed solution obtained in the step A1 into a fermentation culture medium according to the volume ratio of 0.02-0.04%, and performing fermentation culture under the conditions of 35-38 ℃ and the pressure of 0.05 +/-0.01 MPa; OD of the solution to be fermented 600nm When the value reaches 8-10 ℃, reducing the temperature to 23-26 ℃ and continuing to culture;
the initial dissolved oxygen of the reaction is 100 percent, the dissolved oxygen is controlled to be more than 20 percent in the fermentation process, the aeration is controlled to be 1 +/-0.3 VVM, the rotating speed is controlled to be 150-450rpm, and the pH value of the fermentation system is controlled to be 6.8-7.0 in the whole fermentation process.
5. The method according to any one of claims 1 to 4, wherein the reaction solution in step B is: tyrosol 0.7g/L, glucose 30g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 2.7g/L, calcium chloride 0.1g/L, formulated with water.
6. The method of claim 5, wherein step B comprises: will 10 8 -10 9 Adding 1.5kg of CFU/g thallus into a reaction tank filled with reaction liquid, wherein the liquid filling amount of the reaction tank is 30L/50L, and performing biosynthesis of salidroside at 35-38 ℃ under the pressure of 0.05 +/-0.01 MPa;
the initial dissolved oxygen of the reaction tank is 100 percent, the dissolved oxygen is controlled to be not less than 60 percent in the reaction process, and the pH value of the system is controlled to be 7.2 to 7.5 in the whole biosynthesis process.
7. The method according to claim 6, wherein the step B further comprises the step of feeding a glucose solution and a tyrosol solution to the reaction solution during biosynthesis of salidroside.
8. The method according to claim 7, wherein in the step B, the glucose solution is fed after the glucose concentration in the reaction solution is consumed to 2%, and the glucose concentration in the reaction solution is controlled to 2-3%.
Preferably, the feeding rate of the glucose solution corresponds to a feeding of 3-6g glucose per liter of reaction solution per hour.
9. The method of claim 7, wherein in step B, the tyrosol solution is fed when the tyrosol concentration in the reaction solution is consumed to 0.03%, and the tyrosol concentration in the reaction solution is controlled to 2-5mmoL/L;
preferably, the feeding rate of the tyrosol solution corresponds to the feeding of 200-280mg tyrosol per liter of reaction solution per hour.
10. The method according to any one of claims 6 to 9, wherein in step B, the synthesis reaction is stopped when the salidroside content increases by less than 100 μ g/mL.
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