CN114317390A - Seed culture medium for streptomyces lincolnensis genetic engineering bacteria and method for producing lincomycin by culture and fermentation - Google Patents
Seed culture medium for streptomyces lincolnensis genetic engineering bacteria and method for producing lincomycin by culture and fermentation Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 82
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 title claims abstract description 72
- 229960005287 lincomycin Drugs 0.000 title claims abstract description 72
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 title claims abstract description 68
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Abstract
The invention discloses a seed culture medium for streptomyces lincolnensis genetic engineering bacteria and a method for producing lincomycin by culture and fermentation. The seed culture medium comprises the following raw materials in percentage by mass: 30-40 g/L of corn steep liquor, 10-15 g/L of soybean cake powder, 15-25 g/L of starch, 5-15 g/L of glucose, 1-3 g/L of ammonium sulfate, 3-5 g/L of calcium carbonate and the balance of water. The invention starts from optimizing the seed quality of the culture medium, establishes the seed culture medium which is suitable for lincomycin genetic engineering strains, solves the problem of low matching between the existing fermentation process and the new strains transformed by genetic engineering, and the optimized seed liquid is fermented, the yield of lincomycin at the shake flask level is 3.862g/L, and the yield of lincomycin produced by fermentation is improved by more than 28 percent compared with the yield of lincomycin produced by fermentation of the original culture medium (3.009 g/L). The optimized seed liquid is utilized for fermentation, the highest yield of the lincomycin in a 15L fermentation tank is 6.56g/L, and the yield of the lincomycin produced by fermentation is improved by more than 25% compared with that of the lincomycin produced by fermentation of an original culture medium (5.17 g/L).
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a seed culture medium for streptomyces lincolnensis genetic engineering bacteria and a method for producing lincomycin by culture fermentation.
Background
Lincomycin is generated by condensation reaction of lincosamide and propyl proline, and has the action mechanism of acting on 50S subunit of sensitive bacterium ribosome, preventing the extension of peptide chain and inhibiting the protein synthesis of bacterial cells so as to play the role of antibiosis. In recent years, the total import and export amount and the total amount of lincomycin and derivatives are rising year by year, and the improvement of the yield of the lincomycin is necessary in view of the huge medicinal and economic values of the lincomycin.
At present, lincomycin is mainly produced by fermenting streptomyces lincomycin, and two effective ways of obtaining high-yield strains and optimizing a fermentation process are provided for improving the yield of lincomycin.
The microbial fermentation process is generally divided into different stages of strain preparation, seed culture, fermentation, downstream processes and the like. Most researchers have focused on the fermentation stage, and the optimal composition and physiological state of seed culture are often overlooked, which may lead to failure of the fermentation stage during microbial fermentation. And different strains have the most suitable fermentation process, and the matching of the existing fermentation process and the new strain modified by genetic engineering is a key common problem to be solved urgently. In the lincomycin fermentation process, the component proportion of the shake flask horizontal seed culture medium and the fermentation tank horizontal primary seed culture medium is consistent, and the seed culture medium is optimized to be more convenient for industrial application. However, the research on the aspect of Streptomyces lincolensis gene engineering bacteria has not been carried out at present.
Disclosure of Invention
The invention mainly aims to provide a seed culture medium for streptomyces lincolnensis genetic engineering bacteria and a method for producing lincomycin by culture and fermentation, and aims to solve the problem that the prior art does not have a seed culture medium for streptomyces lincolnensis genetic engineering bacteria.
In order to achieve the aim, the invention provides a seed culture medium for streptomyces lincolnensis genetic engineering bacteria, which comprises the following raw materials in percentage by mass: 30-40 g/L of corn steep liquor, 10-15 g/L of soybean cake powder, 15-25 g/L of starch, 5-15 g/L of glucose, 1-3 g/L of ammonium sulfate, 3-5 g/L of calcium carbonate and the balance of water.
Further, the mass concentration of each raw material is as follows: 36.357g/L of corn steep liquor, 13.441g/L of soybean cake powder, 20g/L of starch, 10g/L of glucose, 1.5g/L of ammonium sulfate, 4g/L of calcium carbonate and the balance of water.
The invention also provides application of the seed culture medium in the production of lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria.
The invention also provides a method for producing lincomycin by culturing and fermenting the lincomycin streptomyces through genetic engineering bacteria, which comprises the following steps:
culturing Streptomyces lincomosus genetically engineered bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, then inoculating spores obtained by culture into the seed culture medium of claim 1 or 2, carrying out shake culture at the temperature of 30 ℃ for 48h, and then transferring the spores into a fermentation culture medium for culture and fermentation.
Further, the slant culture medium comprises the following raw materials in percentage by mass: 20g/L of soluble starch, 5g/L of soybean cake powder, 0.5g/L of sodium chloride, 1g/L of potassium nitrate, 0.01g/L of ferrous sulfate heptahydrate, 1.02g/L of magnesium sulfate heptahydrate, 0.66g/L of dipotassium hydrogen phosphate trihydrate, 18g/L of agar and the balance of water.
Further, the fermentation medium comprises the following raw materials in percentage by mass: 100g/L glucose, 20g/L soybean cake powder, 1.5g/L corn steep liquor, 8g/L sodium nitrate, 5g/L sodium chloride, 6g/L ammonium sulfate, 0.3g/L dipotassium phosphate and the balance of water;
after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after uniform mixing, 8g/L calcium carbonate is added.
The invention also provides a method for producing lincomycin by culturing and fermenting the lincomycin streptomyces through genetic engineering bacteria, which comprises the following steps:
culturing Streptomyces lincomosus genetically engineered bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, inoculating spores obtained by culture into the seed culture medium of claim 1 or 2, performing rotary culture at the temperature of 30 ℃ for 48h, transferring primary seeds obtained by culture into a secondary seed culture medium, culturing at the temperature of 30 ℃ until the pH value of the culture medium is more than 6.8, and then inoculating the primary seeds into a fermentation tank containing a tank culture medium for culture and fermentation.
Further, the secondary seed culture medium comprises the following raw materials in percentage by mass: 4g/L of starch, 10g/L of soybean cake powder, 15g/L of corn steep liquor, 28g/L of glucose, 2.5g/L of ammonium sulfate, 6g/L of sodium chloride, 0.3g/L of potassium dihydrogen phosphate and the balance of water;
after the volume is fixed, the pH value is adjusted to 7.2 by using 5mol/L NaOH solution, and after the mixture is uniformly mixed, 10g/L calcium carbonate is added.
Further, the tank culture medium comprises the following raw materials in percentage by mass: 4.5g/L of starch, 15.4g/L of soybean cake powder, 15.4g/L of corn steep liquor, 17.5g/L of glucose, 4.3g/L of ammonium sulfate, 8.5g/L of sodium chloride, 0.3g/L of monopotassium phosphate, 4.3g/L of ammonium nitrate, 4.8g/L of sodium nitrate and the balance of water;
after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after even mixing, 7.7g/L calcium carbonate and 1g/L molinate are added.
The streptomyces lincolnensis genetic engineering bacteria are new strains of streptomyces lincolnensis which are transformed by genetic engineering.
Further, the Streptomyces linkensis genetically engineered bacterium is a strain LA219X delta 4846-2919 disclosed in the Chinese invention patent application with the application number CN 202011140715.9.
The invention has the beneficial effects that:
the invention sets up the seed culture medium adaptive to lincomycin gene engineering strain and the optimization method thereof from the aspect of optimizing the seed quality of the culture medium, solves the problem of low matching between the existing fermentation process and the new strain modified by gene engineering, and particularly further improves the yield of lincomycin by optimizing the seed culture conditions of the lincomycin gene engineering strain.
The optimized seed liquid is fermented, the yield of lincomycin in a shake flask horizontal state is 3.862g/L, and the yield of lincomycin produced by fermentation is improved by more than 28% compared with that produced by fermentation of an original culture medium (3.009 g/L).
The optimized seed liquid is utilized for fermentation, the highest yield of the lincomycin in a 15L fermentation tank is 6.56g/L, and the yield of the lincomycin produced by fermentation is improved by more than 25% compared with that of the lincomycin produced by fermentation of an original culture medium (5.17 g/L).
Drawings
FIG. 1 is a flowchart of the experiment for verifying the effect of the seed culture medium of the present invention in the production of lincomycin by culturing and fermenting Streptomyces lincomosus engineering bacteria.
Fig. 2 is a pareto chart of the effect of various factors provided by the embodiment of the present invention.
FIG. 3 is a normal diagram of the effect of various factors provided by an embodiment of the present invention.
Fig. 4 is a climbing experiment yield graph provided by the embodiment of the present invention.
FIG. 5 is a graph of the response of the interaction of corn steep liquor and soybean meal provided in accordance with an embodiment of the present invention.
FIG. 6 is a graph of yield before and after optimization of shake flask horizontal seed culture conditions provided by embodiments of the present invention.
FIG. 7 is a high performance liquid chromatogram before and after optimization of shake flask horizontal seed culture conditions provided by embodiments of the present invention.
FIG. 8 is a graph showing the yield before and after optimization of the conditions for horizontal seed culture in a fermentor according to an embodiment of the present invention.
FIG. 9 is a high performance liquid chromatogram before and after optimization of the horizontal seed culture conditions of the fermentor provided by the embodiment of the invention.
Detailed Description
The invention is further described below with reference to the following examples:
the various starting materials used in the following examples are all commercially available products known in the art unless otherwise specified. The streptomyces lincosus genetic engineering bacteria used below is the strain LA219X delta 4846-2919 disclosed by the Chinese invention patent application with the application number CN 202011140715.9; the composition of each medium used below was as follows:
slant culture medium: 20g/L of soluble starch, 5g/L of soybean cake powder, 0.5g/L of sodium chloride, 1g/L of potassium nitrate, 0.01g/L of ferrous sulfate heptahydrate, 1.02g/L of magnesium sulfate heptahydrate, 0.66g/L of dipotassium hydrogen phosphate trihydrate, 18g/L of agar and the balance of water. Sterilizing at 121 deg.C for 20 min.
Fermentation medium: 100g/L glucose, 20g/L soybean cake powder, 1.5g/L corn steep liquor, 8g/L sodium nitrate, 5g/L sodium chloride, 6g/L ammonium sulfate, 0.3g/L dipotassium phosphate and the balance of water; after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after uniform mixing, 8g/L calcium carbonate is added. Sterilizing at 121 deg.C for 20 min.
Secondary seed culture medium: 4g/L of starch, 10g/L of soybean cake powder, 15g/L of corn steep liquor, 28g/L of glucose, 2.5g/L of ammonium sulfate, 6g/L of sodium chloride, 0.3g/L of potassium dihydrogen phosphate and the balance of water; after the volume is fixed, the pH value is adjusted to 7.2 by using 5mol/L NaOH solution, and after the mixture is uniformly mixed, 10g/L calcium carbonate is added. Sterilizing at 121 deg.C for 20 min.
Tank culture medium: 4.5g/L of starch, 15.4g/L of soybean cake powder, 15.4g/L of corn steep liquor, 17.5g/L of glucose, 4.3g/L of ammonium sulfate, 8.5g/L of sodium chloride, 0.3g/L of monopotassium phosphate, 4.3g/L of ammonium nitrate, 4.8g/L of sodium nitrate and the balance of water; after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after even mixing, 7.7g/L calcium carbonate and 1g/L molinate are added. Sterilizing at 121 deg.C for 20-25 min.
The invention relates to a seed culture medium for streptomyces lincolnensis genetic engineering bacteria, which comprises the following raw materials in percentage by mass: 30-40 g/L of corn steep liquor, 10-15 g/L of soybean cake powder, 15-25 g/L of starch, 5-15 g/L of glucose, 1-3 g/L of ammonium sulfate, 3-5 g/L of calcium carbonate and the balance of water.
The seed culture medium for the streptomyces lincolnensis genetic engineering bacteria can be used for producing lincomycin by culturing and fermenting the streptomyces lincolnensis genetic engineering bacteria. Specifically, based on the seed culture medium, the invention designs two application methods for producing lincomycin by culturing and fermenting streptomyces lincomosus genetic engineering bacteria, and the application method comprises the following steps:
culturing Streptomyces lincolnensis genetic engineering bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, inoculating spores obtained by culture into the seed culture medium for Streptomyces lincolnensis genetic engineering bacteria, performing shake culture at the temperature of 30 ℃ for 48h, and transferring to a fermentation culture medium for culture and fermentation.
Another application method comprises the following steps:
culturing Streptomyces lincolnensis genetic engineering bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, inoculating spores obtained by culture into a seed culture medium for Streptomyces lincolnensis genetic engineering bacteria (the seed culture medium is used as a primary seed culture medium), performing rotary culture at the temperature of 30 ℃ for 48h, transferring the primary seeds obtained by culture into a secondary seed culture medium, culturing at the temperature of 30 ℃ until the pH of the culture medium is more than 6.8, and then inoculating the primary seeds into a fermentation tank containing a tank culture medium for culture and fermentation.
Based on the two application methods, shake flask horizontal verification and 15L fermentation tank horizontal verification are provided below, and the effect of the seed culture medium in the production of lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria is verified.
And (3) shake flask horizontal verification:
culturing Streptomyces lincolnensis gene engineering bacteria on slant culture medium at 30 deg.C for 7d, digging 1cm2Inoculating the cultured spores into the seed culture medium, carrying out shake culture at the temperature of 30 ℃ for 48 hours, transferring the spores into a fermentation culture medium, carrying out shake culture at the temperature of 30 ℃ for 7 days, and centrifuging 2ml of bacterial liquid at 12000rpm for 10 min; then 200 mul of supernatant is added with 800 mul of ethanol for even mixing, and the mixture is centrifuged at 12000rpm for 10 minutes; and finally, injecting the supernatant into a detection bottle through a filtering membrane for yield detection.
Horizontal verification of a 15L fermentation tank:
coating Streptomyces lincolnensis gene engineering bacteria on slant culture medium, culturing at 30 deg.C for 7 days, and collecting the cultured surface area of 1cm2Transferring the spores into a 500mL shake flask filled with 30mL of primary seed culture medium (namely the seed culture medium of the invention), culturing for 48h at the temperature of 30 ℃ and the rotation speed of 240rpm, then transferring 30mL of primary seeds into a 2L shake flask filled with 200mL of secondary seed culture medium, and inoculating into a 15L fermentation tank containing the tank culture medium when the culture medium is cultured at the temperature of 30 ℃ until the pH value of the culture medium reaches above 6.8. Taking 2ml of bacterial liquid every 24 hours and centrifuging at 12000rpm for 10 min; then 200 mul of supernatant is added with 800 mul of ethanol for even mixing, and the mixture is centrifuged at 12000rpm for 10 minutes; and finally, injecting the supernatant into a detection bottle through a filtering membrane for yield detection.
Example 1
Plackett-Burman design results and analysis to screen for significant influencing factors:
according to the influence factors on the response value in the culture conditions, the raw components of a seed culture medium (corn starch, glucose, soybean cake powder, corn steep liquor, ammonium sulfate and calcium carbonate) and the optimization of fermentation process parameters (seed age, rotating speed and inoculation amount) are selected, 2 virtual factors are additionally added, the Plackett-Burman design of 12 times of experiments is selected, and the primary effects of the main effects and the interaction of the factors are examined, so that the factors which obviously influence the yield are screened out.
The factors and levels of the variables are shown in Table 1, and the response value is the yield of lincomycin A.
TABLE 1 Plackett-Burman Experimental design
The two-level factor design and experimental results are shown in tables 1 and 2. Results of the anova analysis by software analysis with lincomycin a production as a response value are shown in table 3.
TABLE 2 Plackett-Burman test results
TABLE 3 Effect of factors in dependent variables
Regression analysis is carried out by taking the yield of the lincomycin A as a dependent variable, the result is shown in table 3, the data in table 3 and the results shown in fig. 2 and fig. 3 are obtained, and three significant factors are obtained by screening by taking the yield of the lincomycin A as an index: the P values of the corn steep liquor (D), the rotating speed (G) and the soybean cake powder (C) are all less than 0.01, the factors are very significant factors, and the P value of the model item is less than 0.05, so that the model item is significant and has statistical significance. The steepest climbing experiment was carried out by selecting corn steep liquor (D) and soybean cake flour (C).
Example 2
Determining the result and analysis of the central point of the response surface by a steepest climbing experiment:
estimating the positive and negative coefficient values of the coefficients according to the two factors of the corn steep liquor (D) and the soybean cake meal (C) in the table 3, and increasing or decreasing the positive and negative coefficient values in sequence, wherein the soybean cake meal is positive and should be increased; corn steep liquor is negative and should be decreased.
TABLE 4 design and results of steepest climb experiments
The results of the climbing experiment show that: with the gradual increase of the concentration of the soybean cake powder and the gradual decrease of the concentration of the corn steep liquor, the yield of the lincomycin is changed from increasing to decreasing, as shown in figure 4. When the soybean meal (C) is 12.2g/L and the corn steep liquor (D) is 35g/L, the yield reaches the maximum and is in a three-factor maximum response value area. Therefore, each factor level of experiment number 2 is a central value for designing a subsequent response surface experiment.
Example 3
Designing a response surface optimization result and analyzing by a central composite experiment:
the factors and levels of the central recombination experiment are shown in table 5.
TABLE 5 Central composite design factor level
According to the above experimental results, the corn steep liquor (D) and the soybean cake meal (C) were selected to design a center composite experiment, and the experimental design and results are shown in Table 6.
TABLE 6 center composite design and results
Through software analysis, multiple regression fitting is carried out on the data in the table 6, and the regression equation of the lincomycin A yield to the corn steep liquor and the soybean cake powder is obtained as follows:
Y＝-14.215+0.5669D+1.152C-0.007135D*D-0.03791C*C-0.00364D*C
y is the predicted yield of lincomycin, D is corn steep liquor and C is soybean cake powder. The results of the experiment were subjected to significance test and analysis of variance, and the results are shown in table 7.
TABLE 7 response surface model regression coefficients and their significance
TABLE 8 analysis of variance of regression models
And analyzing the regression coefficient and the significance of the response surface model, and identifying the influence of each factor on the lincomycin yield by using T test and P value as an index as shown in Table 7. Wherein a model term p-value of less than 0.05 indicates that the model term is significant. At the moment, secondary terms of the corn steep liquor (D), the soybean cake powder (C) and the corn steep liquor (D) and the soybean cake powder (C) have a significant influence (P is less than 0.05) on the yield of the lincomycin, and an interaction term of the corn steep liquor (D) and the soybean cake powder (C) also has a significant influence (P is less than 0.05) on the yield of the lincomycin, so that certain interaction exists between the corn steep liquor (D) and the soybean cake powder (C).
Analysis of the variance of the response surface quadratic model, as shown in table 8, also demonstrated that the regression was statistically significant at a 95% confidence level (P < 0.0001). The mismatching term for the model is also not significant (P0.444), so the model is considered sufficient to predict over the range of variables employed.
TABLE 9 model confidence analysis
The feasibility of the response surface model was analyzed as shown in table 9. By determining the coefficient R2The fitting degree of the model is tested, the result is 0.9963, the 99.63 percent of sample variation is caused by variables, and only the total variance of less than 0.4 percent cannot be explained by the model. Because R is2Regression models with values greater than 0.9 are considered to have very high correlations. Thus, the current R2The values reflect a good fit between the observed response and the prediction, indicating that the model is reliable. The adjusted coefficient of determination (R-Sq ═ 99.36%) also proves to be betterThe significance of the model is realized.
To better understand the effect of variables on lincomycin production, the predictive model was represented as a 3D response surface plot, as shown in fig. 5. The model is used for predicting the maximum yield of the lincomycin, when the D encoding level is 0.2714 and the C encoding level is 1.1285, the corn steep liquor and the soybean meal are 36.357g/L and 13.441g/L respectively, and the maximum yield of the lincomycin is 3.825 g/L.
LA219X Delta4846-2919 was constructed on the basis of the starting strain LA219X, obtained by traceless knockout of the Lrp family regulatory gene SLCG-4846 and the TetR family regulatory gene SLCG-2919 in LA 219X. The microorganism can change the metabolic mode to adapt to the change of the environment through gene regulation, the regulation gene knockout can cause the change of the metabolic pathway, and the absorption and utilization of components such as nitrogen source, carbon source, nitrate, phosphate and the like in the culture environment can be correspondingly changed. Since LA219X delta 4846-2919 is the modification of regulatory genes in LA219X, the optimization of the culture medium is of great significance for improving the lincomycin fermentation potential of engineering bacteria.
On the basis of an original culture medium, a seed culture medium of streptomyces lincolnensis is optimized through experimental design methods such as Plackett-Burman experimental design, steepest climbing experiment, response surface experimental design and the like, the optimal concentrations of corn steep liquor and soybean cake powder are 36.357g/L and 13.441g/L respectively after response surface optimization, the optimized seed liquid is utilized for fermentation, the yield of lincomycin at the shake flask level reaches 3.862g/L, and is improved by more than 28% compared with the yield of lincomycin produced by fermentation of the original culture medium (3.009g/L), as shown in figure 6, and a high performance liquid phase analysis map thereof is as shown in figure 7. The highest yield of the lincomycin in the horizontal state of the 15L fermentation tank is 6.56g/L, which is improved by more than 25 percent compared with the yield of the lincomycin produced by fermentation under the original culture condition (5.17g/L), as shown in figure 8, and as shown in figure 9, a high performance liquid phase analysis map is shown.
In conclusion, the present invention verifies that:
1. according to the influence factors on the lincomycin yield in the culture conditions, the raw components of a seed culture medium (starch, glucose, soybean cake powder, corn steep liquor, ammonium sulfate and calcium carbonate) and the parameters of the fermentation process are optimized (the seed age, the rotating speed and the inoculation amount), 2 virtual factors are additionally added, the Plackett-Burman design with 12 times of experiments is selected, the primary effects of the main effects and the interaction of the factors are examined, and the significant factors are screened.
2. According to the experiment result of Plackett-Burman, significant factors are selected, the direction and the step length of the steepest climbing experiment are determined, other factors are all low in level, the change trend of the lincomycin yield is researched, the optimal concentration range of important factors is determined, and the central point of a central composite design experiment is determined.
3. And (3) further optimizing the main factors screened by the Plackett-Burman experiment and the optimal concentration determined by the steepest climbing experiment by using central composite design, running the experiment to obtain a result, fitting the obtained mathematical model with experimental data, determining the model by using graph and variance analysis, and finally determining the culture conditions of the streptomyces lincolnensis seeds.
According to the invention, on the basis of an original culture medium, through experimental design methods such as a Plackett-Burman experiment, a steepest climbing experiment, a central compound experiment and the like, the seed culture conditions of the streptomyces lincolnensis are optimized, the optimal concentrations of the obtained corn steep liquor and soybean cake powder are 36.357g/L and 13.441g/L respectively, the optimized seed liquid is utilized for fermentation, the yield of lincomycin at the shake flask level is 3.862g/L, and the yield of the lincomycin produced by fermentation is improved by more than 28% compared with that of the lincomycin produced by fermentation of the original culture medium (3.009 g/L). The highest yield of the lincomycin in the horizontal state of a 15L fermentation tank is 6.56g/L, which is improved by more than 25 percent compared with the yield of the lincomycin produced by fermentation under the original culture condition (5.17 g/L). The optimized seed culture medium is used for obviously improving the ability of producing lincomycin by fermenting the lincomycin gene engineering bacteria, and the result lays a foundation for the application of the lincomycin gene engineering bacteria in industrialization.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims; meanwhile, any modification, equivalent replacement and improvement of the above embodiment according to the essential technology of the present invention still fall within the technical scope of the present invention.
Claims (9)
1. A seed culture medium for streptomyces lincolnensis genetic engineering bacteria is characterized in that: according to mass concentration, the raw materials comprise: 30-40 g/L of corn steep liquor, 10-15 g/L of soybean cake powder, 15-25 g/L of starch, 5-15 g/L of glucose, 1-3 g/L of ammonium sulfate, 3-5 g/L of calcium carbonate and the balance of water.
2. The seed culture medium for streptomyces lincolnensis genetic engineering bacteria as claimed in claim 1, wherein the mass concentration of each raw material is as follows: 36.357g/L of corn steep liquor, 13.441g/L of soybean cake powder, 20g/L of starch, 10g/L of glucose, 1.5g/L of ammonium sulfate, 4g/L of calcium carbonate and the balance of water.
3. The use of the seed culture medium of claim 1 or 2 for the production of lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria.
4. A method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria is characterized by comprising the following steps:
culturing Streptomyces lincomosus genetically engineered bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, then inoculating spores obtained by culture into the seed culture medium of claim 1 or 2, carrying out shake culture at the temperature of 30 ℃ for 48h, and then transferring the spores into a fermentation culture medium for culture and fermentation.
5. The method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria as claimed in claim 4, wherein the slant culture medium comprises the following raw materials in mass concentration: 20g/L of soluble starch, 5g/L of soybean cake powder, 0.5g/L of sodium chloride, 1g/L of potassium nitrate, 0.01g/L of ferrous sulfate heptahydrate, 1.02g/L of magnesium sulfate heptahydrate, 0.66g/L of dipotassium hydrogen phosphate trihydrate, 18g/L of agar and the balance of water.
6. The method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria as claimed in claim 4, wherein the fermentation medium comprises the following raw materials in mass concentration: 100g/L glucose, 20g/L soybean cake powder, 1.5g/L corn steep liquor, 8g/L sodium nitrate, 5g/L sodium chloride, 6g/L ammonium sulfate, 0.3g/L dipotassium phosphate and the balance of water;
after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after uniform mixing, 8g/L calcium carbonate is added.
7. A method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria is characterized by comprising the following steps:
culturing Streptomyces lincomosus genetically engineered bacteria on a slant culture medium at the temperature of 30 ℃ for 7d, inoculating spores obtained by culture into the seed culture medium of claim 1 or 2, performing rotary culture at the temperature of 30 ℃ for 48h, transferring primary seeds obtained by culture into a secondary seed culture medium, culturing at the temperature of 30 ℃ until the pH value of the culture medium is more than 6.8, and then inoculating the primary seeds into a fermentation tank containing a tank culture medium for culture and fermentation.
8. The method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria as claimed in claim 7, wherein the secondary seed culture medium comprises the following raw materials in mass concentration: 4g/L of starch, 10g/L of soybean cake powder, 15g/L of corn steep liquor, 28g/L of glucose, 2.5g/L of ammonium sulfate, 6g/L of sodium chloride, 0.3g/L of potassium dihydrogen phosphate and the balance of water;
after the volume is fixed, the pH value is adjusted to 7.2 by using 5mol/L NaOH solution, and after the mixture is uniformly mixed, 10g/L calcium carbonate is added.
9. The method for producing lincomycin by culturing and fermenting streptomyces lincolnensis genetic engineering bacteria as claimed in claim 7, wherein the tank culture medium comprises the following raw materials in mass concentration: 4.5g/L of starch, 15.4g/L of soybean cake powder, 15.4g/L of corn steep liquor, 17.5g/L of glucose, 4.3g/L of ammonium sulfate, 8.5g/L of sodium chloride, 0.3g/L of monopotassium phosphate, 4.3g/L of ammonium nitrate, 4.8g/L of sodium nitrate and the balance of water;
after constant volume, 5mol/L NaOH solution is used for adjusting the pH value to 7.45-7.5, and after even mixing, 7.7g/L calcium carbonate and 1g/L molinate are added.
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| CN101220343A (en) * | 2008-01-23 | 2008-07-16 | 华东理工大学 | Seed culture medium produced with lincomycin ferment optimized with response surface method |
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| US20140030242A1 (en) * | 2011-01-04 | 2014-01-30 | Won Gon Kim | Antifungal composition comprising polycyclic peptide compound and method for preparing the same |
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