CN111172050B - Fermentation strategy for high yield of toxoflavin by using Burkholderia - Google Patents
Fermentation strategy for high yield of toxoflavin by using Burkholderia Download PDFInfo
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Abstract
Burkholderia plantarii: (Burkholderiasp.HDXY-02) in the fermentation process, belonging to the technical field of microbial fermentation. The bacterial strain HDXY-02 has been preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 14054. The invention determines the optimal inoculation amount to be 3 percent and the culture temperature to be 30 ℃. Through single factor experiments and statistical analysis, the optimal culture medium is determined as follows: glucose (19.24g/L), peptone (20.45g/L), phenylalanine (0.472g/L), K2HPO4（1.5 g/L）、MgSO4(0.75g/L) and pH 7.0. The yield of the toxoflavin after optimization reaches 1533mg/L, is improved by 296 percent compared with that before optimization, and a feasible fermentation strategy is provided for the high yield of the toxoflavin by a microbial fermentation method.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, in particular to a fermentation strategy for producing toxoflavin by using Burkholderia, and particularly relates to an optimization strategy for fermentation conditions for greatly improving the yield of toxoflavin by using the strain.
Background
Toxoflavin is a small molecule substance synthesized by microorganisms, and the molecular formula is C7H7N5O2The molar mass was 193 g/mol. It has been found to be useful as antibacterial and antitumor agents. The toxoflavin has antagonistic ability against various pathogenic bacteria. In addition, the compound also has strong antitumor activity and good inhibitory activity on various cancer cells such as lung cancer cells, ovarian cancer cells, gastric cancer cells and the like. Therefore, the toxoflavin has high research value in the aspect of cell apoptosis as a new antibiotic and antitumor drug, can reduce the toxicity of toxoflavin through various structural modifications in application, and has certain commercial prospect as an antibiotic and an antitumor drug.
At present, toxoflavin can be synthesized by a chemical or microbiological method, but the chemical synthesis has the defects of complex general steps, more required organic reagents, more byproducts, environmental pollution and the like. Compared with a chemical production method, the microbial method for producing toxoflavin has the advantages of mild reaction conditions, high utilization rate of raw materials, simple process, easy control and the like, is beneficial to environmental protection, and is easy to popularize and apply. But the yield of toxoflavin in microorganisms is low at present, and the yield of toxoflavin still has great potential.
The ability of the microorganism to synthesize metabolites depends not only on the metabolic characteristics of the strain itself but also on the culture conditions and medium components, such as carbon-nitrogen source, culture temperature, pH, lysogen, etc.
Statistical methods have been widely used in studies of fermentation conditions and optimization of high-yielding media. Plackett-Burman design (PBD) and response surface analysis (RSM) overcome the defects of multiple times of experiments, long experiment period and the like of the traditional single-factor method, and can quickly and effectively screen out main effect factors from a plurality of influence factors and realize the level optimization of the main effect factors. The application of statistical methods such as PBD and Box-Behnken design (BBD) greatly improves the efficiency of optimization work, and has been widely applied to the optimization work of fermentation conditions of various microorganisms.
Also, there are many factors affecting the synthesis of toxoflavin by microorganisms, including culture temperature, pH, culture time, and medium composition, in addition to the strain. By separating the high toxoflavin-producing strains in the environment and optimizing the fermentation conditions and the culture medium formula of the strains on the basis, the optimal growth conditions of the strains and the optimal culture medium components for synthesizing metabolites are maintained, so that the strains are in the optimal state for synthesizing the target metabolites, the efficiency of producing toxoflavin by the strains can be improved to the greatest extent, and the method is an important way for solving the problem of the existing toxoflavin raw material source.
Disclosure of Invention
The object of the present invention is to provide: a fermentation strategy of Burkholderia sp (HDXY-02), in particular to an optimization strategy of fermentation conditions for greatly improving the yield of toxoflavin of the strain.
The Burkholderia (classified and named Burkholderia sp.) related by the invention has the registration number of CGMCC No.14054, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation unit address is No. 3 of West Lu No.1 of Beijing Kogyo sunward area, and the preservation date is 2017, 4 months and 20 days.
The technical scheme of the invention is as follows: firstly, through a single factor experiment, the fermentation conditions in the process of producing toxoflavin by Burkholderia sp.HDXY-02 fermentation are optimized, wherein the fermentation conditions comprise three aspects of initial pH of fermentation liquor, fermentation temperature and inoculation quantity. Secondly, a fermentation KMB culture medium obtained by early screening is used as a starting culture medium, and culture medium components such as carbon and nitrogen source types and metal ions are screened by a single-factor optimization method. Again, the Plackett-Burman design was used to screen for media components that had a significant effect on Burkholderia sp.HDXY-02 toxoflavin production. And finally, optimizing the level of 3 factors which obviously influence the yield of the toxoflavin by a response surface analysis method, thereby obtaining the fermentation culture medium formula for the high yield of the toxoflavin of the strain Burkholderia sp. The invention successfully obtains an optimization strategy for producing toxoflavin by Burkholderia sp.HDXY-02 fermentation, the yield of the toxoflavin is greatly improved to 1533mg/L compared with that before optimization, and is improved by 296% compared with 387mg/L before optimization.
The toxoflavin standard was purchased from SIGMA, USA.
Seed culture medium:
LB medium (g/L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0.
Basic fermentation medium:
king's B (KMB) medium (g/L): peptone 20, K2HPO41.5, 15mL of glycerol, MgSO40.75。
Initial culture conditions:
the seed age is 14h, the temperature is 30 ℃, the inoculum size is 4%, the rotating speed of a shaking table is 200rpm, the liquid loading amount of a triangular flask is 75/250mL, and the culture is carried out for 48 h.
And (3) determining the content of toxoflavin:
the ultraviolet detection method comprises the following steps: centrifuging cultured Burkholderia sp.HDXY-02 bacterial liquid 1mL at 4 deg.C and 8000g for 10min, removing thallus, extracting supernatant with chloroform of the same volume, dissolving extract with 80% methanol (v/v) 1mL, detecting with ultraviolet, and determining OD393. Diluting toxoflavin standard substance with 80% methanol gradient, and drawing standard curve for calculating fermentation liquidToxoflavin concentration.
Optimization of strain fermentation strategy:
optimization of culture conditions for Burkholderia sp.HDXY-02 toxoflavin Synthesis
(1) Effect of inoculum size on Burkholderia sp.HDXY-02 toxoflavin production
The invention inspects the influence of different inoculation amounts on the yield of the toxoflavin, respectively sets the inoculation amounts to 1%, 2%, 3%, 4%, 5% and 6%, and carries out the determination of the content of the toxoflavin under the culture conditions as described above so as to determine the optimal inoculation amount which is beneficial to the synthesis of the Burkholderia sp.
(2) Effect of initial pH on Burkholderia sp.HDXY-02 toxoflavin production
According to the invention, on the basis of determining the inoculation amount, the influence of the initial pH on the yield of the toxoflavin is considered, the initial pH of the culture medium is respectively set to be pH5.0, pH6.0, pH7.0, pH8.0 and pH9.0, the culture conditions are as described above, and the content of the toxoflavin in the fermentation supernatant is determined so as to determine the optimal initial pH of the culture medium which is favorable for synthesis of Burkholderia sp.HDXY-02 toxoflavin.
(3) Effect of culture temperature on Burkholderia sp.HDXY-02 toxoflavin production
According to the invention, on the basis of determining the inoculation amount and the initial pH value, the influence of the culture temperature on the yield of the toxoflavin is considered, the culture temperature is respectively set to be 25 ℃, 28 ℃, 30, 33, 37 and 40 ℃, the content of the toxoflavin in the fermentation supernatant is determined under the culture conditions as described above, so as to determine the optimal culture temperature of the culture medium which is favorable for synthesis of Burkholderia sp.
2. Medium composition optimization for biosynthesis of toxoflavin by the strain Burkholderia sp
(1) Influence of carbon Source on Burkholderia sp.HDXY-02 toxoflavin production
The invention inspects the influence of carbon sources, namely glucose, glycerol, sucrose, mannose, succinic acid, citric acid and lactose, on the synthesis amount of Burkholderia sp.HDXY-02 toxoflavin, wherein the concentration of the carbon source is 1%.
(2) Influence of organic Nitrogen Source on Burkholderia sp.HDXY-02 toxoflavin production
The present invention investigates the influence of organic nitrogen sources, peptone, soybean meal, yeast extract, beef extract, peptone on Burkholderia sp.HDXY-02 toxoflavin synthesis. The selected nitrogen source is used as the only nitrogen source to be added, and the concentration is 0.5 percent.
(3) Effect of inorganic Nitrogen sources and amino acids on Burkholderia sp.HDXY-02 toxoflavin production
The present invention examines inorganic nitrogen source- (NH)4)2SO4、KNO3、NaNO3And the amino acids tyrosine, phenylalanine, aspartic acid, on Burkholderia sp.HDXY-02 toxoflavin production, the inorganic nitrogen source concentration was set at 0.2%, the amino acid was set at 0.01%.
(4) Effect of inorganic salts on Burkholderia sp.HDXY-02 toxoflavin production
The invention inspects inorganic salt-NaCl, MgSO4、KH2PO4、K2HPO4、Na2SO4And KCl on burkholderia sp.hdxy-02 toxoflavin synthesis, at a concentration of 0.2%.
(5) Effect of Metal ions on Burkholderia sp.HDXY-02 toxoflavin production
The invention investigates the influence of metal ions, Fe, Ca, Cd, Mn, Zn and Cu, on the synthesis of Burkholderia sp.HDXY-02 toxoflavin, with the concentration of 0.01%.
(6) Statistical method for optimizing Burkholderia sp.HDXY-02 toxoflavin-producing medium components
On the basis of the single factors, the invention further screens factors which obviously influence the synthesis of Burkholderia sp.HDXY-02 toxoflavin by adopting a Plackett-Burman design. Response surface analysis was performed according to the major influencing factors screened by the Plackett-Burman design and the levels of the components of the fermentation medium for the production of toxoflavin by the strain Burkholderia sp.
The invention has the beneficial effects that: through single factor experiments and statistical methods, fermentation strategy research is carried out on the strain Burkholderia sp.HDXY-02, and the culture conditions and the fermentation medium components of the toxoflavin high-yield strain are optimized and fermentation experiments are verified. Burkholder under shaking bottle conditionOptimal culture conditions for toxoflavin production by ia sp.HDXY-02 are: the inoculum size was 3%, the incubation temperature was 30 ℃ and the initial pH was 7.0. The most preferred medium is glucose (19.24g/L), peptone (20.45g/L), phenylalanine (0.472g/L), K2HPO4(1.5g/L)、MgSO4(0.75 g/L). Successfully obtains a fermentation strategy for synthesizing the toxoflavin by the strain Burkholderia sp.HDXY-02, and the yield of the toxoflavin is greatly improved compared with that before optimization. The synthesis amount of the toxoflavin after optimization reaches 1533mg/L, the toxoflavin producing capacity is greatly improved, the yield is improved by 296% compared with 387mg/L before optimization and is about 4 times before optimization, an ideal result is obtained, and a beneficial guidance is provided for industrialization of toxoflavin production by microbial fermentation.
Drawings
FIG. 1 Effect of initial pH on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 2 Effect of culture temperature on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 3 Effect of carbon sources on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 4 Effect of organic Nitrogen sources on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 5 Effect of inorganic Nitrogen sources and amino acids on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 6 Effect of inorganic salts on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 7 Effect of Metal ions on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 8 pareto chart analysis of the effect of various factors on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 9 Main Effect graph analysis of the Effect of factors on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 10 Effect of interaction of factors on Burkholderia sp.HDXY-02 toxoflavin production
FIG. 11 response surface analysis of the Effect of major factors on Burkholderia sp.HDXY-02 toxoflavin production
Detailed Description
Example 1 Effect of inoculum size on Burkholderia sp.HDXY-02 toxoflavin production
Too large or too small an amount of inoculation is detrimental to the synthesis of the toxoflavin product. Therefore, the amount of the inoculum should be controlled within a suitable range during the fermentation process. The content of the flavin in the fermentation liquor increases along with the increase of the inoculation amount, when the inoculation amount is higher than 3%, the content of the flavin in the fermentation liquor reaches the highest, and the content of the flavin in the fermentation liquor gradually decreases along with the continuous increase of the inoculation amount. The optimal inoculum size for toxoflavin production by Burkholderia sp.HDXY-02 fermentation was determined to be 3%, and therefore 3% was selected for subsequent testing.
Example 2 Effect of initial pH on Burkholderia sp.HDXY-02 toxoflavin production
pH is an important factor affecting cell growth and product metabolism. The effect of toxoflavin synthesis at different initial pH values was investigated by shake flask culture. The results are shown in FIG. 1. At a pH in the range of 5.0-7.0, the toxoflavin production gradually increases with increasing pH, reaches a maximum of 387mg/L at a pH of 7.0, and starts to decrease with increasing pH after a pH of more than 7.0. The optimum initial pH for toxoflavin production by Burkholderia sp.HDXY-02 fermentation is in the neutral range, with pH7.0 being the optimum. Thus, the initial pH of 7.0 was selected for subsequent experiments.
Example 3 Effect of culture temperature on Burkholderia sp.HDXY-02 toxoflavin production
Temperature affects the biological processes of microorganisms by affecting the structure of microbial membranes, the synthesis and activity of enzymes and proteins. As can be seen from FIG. 2, the optimum temperature for the production of toxoflavin by Burkholderia sp.HDXY-02 fermentation is 30 ℃, and the synthesis of toxoflavin is more favored at 30 ℃, when the content of toxoflavin in the fermentation broth is the highest, and then the content of toxoflavin in the Burkholderia sp.HDXY-02 fermentation broth begins to decrease with the increase of the culture temperature. Therefore, an incubation temperature of 30 ℃ was chosen for subsequent experiments.
Example 4 Effect of carbon sources on Burkholderia sp.HDXY-02 toxoflavin production
Six carbon sources, namely glucose, glycerol, sucrose, mannose, succinic acid, citric acid and lactose, are selected to study the influence of the six carbon sources on the synthesis of Burkholderia sp.HDXY-02 toxoflavin, and the concentration of the carbon source is 1 percent. The initial fermentation medium was KMB medium. The other components remained unchanged. The inoculation amount is 3 percent, the culture temperature is 30 ℃, the rotating speed of a shaking table is 200rpm, the culture is carried out for 48 hours, and the output of the toxoflavin is determined. As can be seen from the graph (FIG. 3), the results show that the yield of toxoflavin is highest when glucose and glycerol are used as carbon sources, and the yield is 485mg/L and 460mg/L respectively, and the two are used as carbon sources to facilitate the accumulation of toxoflavin in Burkholderia sp.HDXY-02 fermentation liquor.
Example 5 Effect of organic Nitrogen Source on Burkholderia sp.HDXY-02 toxoflavin production
The nitrogen source has important influence on the growth of thalli and the synthesis of products, and the organic nitrogen source plays a key role in the accumulation of antibiotic substances. As can be seen in FIG. 4, the yield of Burkholderia sp.HDXY-02 toxoflavin was the highest at 471mg/L, using peptone as nitrogen source.
Example 6 Effect of inorganic Nitrogen sources and amino acids on Burkholderia sp.HDXY-02 toxoflavin production
As shown in fig. 5, the inorganic nitrogen source is significantly detrimental to the synthesis of flavins by Burkholderia sp. Therefore, organic nitrogen sources are the best choice for bacterial growth and accumulation of secondary metabolites. In addition, the organic nitrogen source in the KMB medium is replaced by a single amino acid as a nitrogen source. Tyrosine, phenylalanine and aspartic acid are selected. The results show that the three amino acids have certain promotion effect on the yield of the toxoflavin, and the yields of the Burkholderia sp.HDXY-02 toxoflavin can respectively reach 601mg/L, 582mg/L and 613 mg/L.
Example 7 Effect of inorganic salts on Burkholderia sp.HDXY-02 toxoflavin production
Inorganic salts can affect secondary metabolite accumulation. Several common inorganic salts are selected and added into the fermentation medium. As shown in FIG. 6, the test results showed NaCl, MgSO4、K2HPO4HDXY-02 has relatively high toxoflavin yield, which can reach 504mg/L, 540mg/L and 451mg/L respectively. Hdxy-02 has no significant effect on the accumulation of flavins in burkholderia sp.
Example 8 Effect of Metal ions on Burkholderia sp.HDXY-02 toxoflavin production
Trace elements as bioactive substancesModulators of the composition or physiological activity of the substance. Magnesium, phosphorus, potassium, sulfur, calcium ions, etc. are often added in the form of salts and play an important role in cell growth and metabolism. The results of the experiment (FIG. 7) show Fe by adding various trace elements2+、Ca2+And Mn2+The amount of the additive (b) can be increased to 638mg/L, 520mg/L and 580 mg/L. Hdxy-02 has a negative regulatory effect on flavin accumulation in Burkholderia sp.
Example 9 statistical methods optimization of Burkholderia sp.HDXY-02 toxoflavin-producing Medium
According to the results of the one-way test, a carbon source (glucose glycerol), a nitrogen source (peptone), inorganic salts (NaCl, MgSO)4、K2HPO4) Amino acids (tyrosine, aspartic acid, phenylalanine) and metal ions (Fe)2+、Ca2+And Mn2+) Has promoting effect on accumulation of flavin in Burkholderia sp.HDXY-02 fermentation supernatant. Therefore, the 12 nutrient components are selected as variables for Plackett-Burman design and screening, the design is designed by adopting software Minitab 17.0, 20 combinations are counted, and the high-low level value of each variable is set. The levels of the specific 12 variables are shown in table 1.
TABLE 1 levels of 12 variables in Plackett-Burman design
The results of the assay of the production of toxoflavin by Burkholderia sp.HDXY-02 using 20 different combinations of fermentation media are shown in Table 2. The results show that: table 3 statistically analyzes the significance level of each trophic factor effect, with 12 nutrient formulations of 20 different fermentation media having a significant effect on toxoflavin yield. The confidence interval is set as 95% (p <0.05), and the results of the Minitab statistical analysis show that 3 factors have significant promotion effects on the yield of toxoflavin, and the factors are respectively: glucose (p ═ 0.016), peptone (p ═ 0.003) and phenylalanine (p ═ 0.021). Therefore, these 3 trophic factors were selected as key components for further optimization and their optimal concentrations were determined.
TABLE 2 Plackett-Burman design array charts and actual toxoflavin production for each combination
TABLE 3 Plackett-Burman design statistical analysis
R2＝87.06％；R2(adjust)＝64.88％
Three factors, glucose, peptone and phenylalanine, were selected with a confidence level greater than 95% for the Plackett-Burman design screen, and were further optimized by the central combinatorial design as the core response surface. The Box-Behnken design's three-factor level table (table 4) shows the encoded and actual values for each selected variable. The central combination trial at 3-factor 3 level had a total of 15 trial combinations, all trial batches having fermentation toxoflavin production, as shown in table 4, and each trial was repeated three times and averaged.
TABLE 4 Box-Behnken design and results
TABLE 5 statistical analysis of Box-Behnken design results
R2＝95.48％；R2(adjust)＝87.33％
Applying multiple regression analysis to the 15 combined test results to establish a regression analysis equation:
Y＝-7960+496.4X1+373X2+3785X3–13.29X1 2–8.79X2 2–4330X3 2–0.22X1X2+38.6X1X3–21.0X2X3
and each group of test results in the Y value have a predicted value obtained by utilizing a regression equation and correspond to the true value of the test. As can be seen in Table 5, the two sets of values are similar and have good consistency, and the correlation coefficient R of the regression equation2And the correlation between the fitting value and the actual value of each variable of the regression equation is 95.48 percent, the shape of the fitting response surface is considered according to the regression equation, and the influence of 3 factors of glucose, peptone and phenylalanine on the yield of toxoflavin is analyzed, and the result is shown in the attached figure 11.
In conclusion, correlation coefficients are calculated on the basis of establishing a regression equation and variance analysis is carried out, and the result shows that the model of the regression equation can sufficiently predict the variation trend of the variable in a given range. And the model prediction value is close to the test value, so that the regression effect of the regression equation is obvious. Regression coefficients and significance analysis showed that glucose and peptone had a significant effect on toxoflavin production (p < 0.05). The squares of glucose and phenylalanine also had a significant effect (p < 0.05).
Calculating the model to obtain the optimal culture medium of glucose (19.24g/L), peptone (20.45g/L), phenylalanine (0.472g/L) and K2HPO4(1.5g/L)MgSO4(0.75 g/L). The shaking flask test is carried out again by utilizing the optimal culture medium formula, and the toxoflavin yield can reach 1533 mg/L. The fermentation strategy greatly improves Burkholderia sp.HDXY-02 toxoflavin yield by about 3 times compared with that before optimization. To check the accuracy of model prediction, under the optimization conditions: a total of 3 replicates were tested. Under the condition, the average toxoflavin production is 1533mg/L, and the method has no obvious difference compared with a model predicted value, so that the reliability of an equation and the effectiveness of a response surface analysis method are proved. Under the same conditions, the yield of toxoflavin is 387mg/L by using KMB culture medium before optimization.
Example 10 fermenter validation
In order to verify the applicability of model simulation, the scientificity of the result is verified through an amplification test of a 10L fermentation tank, the result shows that the fermentation time reaches 1.87g/L after 60 hours of fermentation, the data of the fermentation tank shows that the optimal culture medium obtained by a response surface method under the condition of a shake flask is still suitable for a scale-up fermentation test, and the fermentation tank test also verifies the result of response surface optimization of Burkholderia sp.HDXY-02 toxoflavin production culture medium again.
Claims (1)
1.Burkholderia bacterium (B) increasingBurkholderiasp.) The use method of the culture medium with HDXY-02 toxoflavin synthesis amount is characterized in that:
the Burkholderia HDXY-02 strain is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 14054;
the formula of the culture medium is as follows: 19.24g/L glucose, 20.45g/L peptone and 0.472g/L, K phenylalanine2HPO4 1.5 g/L、MgSO4 0.75g/L, initial pH7.0;
fermentation culture conditions: the seed age is 14h, the inoculum size is 3%, the rotating speed of a shaking table is 200rpm, the liquid loading capacity of a triangular flask is 250mL, 75 mL of liquid culture medium is filled in the triangular flask, and the shake culture is carried out for 48h at the temperature of 30 ℃.
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