Method for producing pyrroloquinoline quinone by utilizing tanshinone to induce hyphomicrobium fermentation

Abstract

The invention belongs to the technical field of microbial fermentation, and discloses a method for producing pyrroloquinoline quinine by utilizing tanshinone to induce hyphomicrobe fermentation, which comprises the following steps of: screening hyphomicrobe by utilizing a selective culture medium containing 0.01 mg / L of tanshinone to obtain a tolerant strain, and respectively adding 0.1 mg / L of tanshinone mother liquor in 12, 24, 36, 48 and 60 hours of fermentation during fermentation culture, the fermentation conditions are regulated and controlled until the final concentration is 0.01 mg / L, so that the yield of the pyrroloquinoline quinine is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a method for inducing Hyphomicrobium to ferment and produce pyrroloquinoline quinone by tanshinone. Background Art

[0002] Pyrroloquinoline quinone (PQQ) is the third cofactor of oxidoreductase discovered after NAD / NADP and FMN / FAD; PQQ naturally exists in nature and is the only active small molecule found in all animal and plant tissues so far and participates in growth and development, with physiological functions such as promoting mitochondrial production, preventing myocardial damage, and scavenging free radicals. At present, PQQ is mainly produced by microbial fermentation relying on strains such as Hyphomicrobium, but the traditional process has problems such as low metabolic activity of the bacteria and insufficient PQQ synthesis efficiency. In the prior art, although the yield can be partially increased by optimizing the medium components or adjusting the fermentation parameters, the problems of energy loss caused by oxidative stress of the bacteria and coenzyme reaction inhibition have not been effectively solved; some studies have tried to improve the fermentation level of the production strain by adding special media or improving the fermentation method, but no good results have been obtained. Summary of the Invention

[0003] The first technical problem to be solved by the present invention is: to provide a method for inducing Hyphomicrobium to ferment and produce pyrroloquinoline quinone by tanshinone. This method screens for tolerant strains by tanshinone and regulates the fermentation conditions, significantly increasing the yield of pyrroloquinoline quinone.

[0004] To solve the above technical problem, the technical solution of the present invention is:

[0005] A method for inducing Hyphomicrobium to ferment and produce pyrroloquinoline quinone by tanshinone, comprising the following steps:

[0006] a. Gradiently dilute the Hyphomicrobium for producing pyrroloquinoline quinone with sterilized water to obtain a dilution;

[0007] b. Inoculate the dilution of different gradients on a selective medium respectively, and incubate at a constant temperature of 30°C for 44 - 52 hours; select colonies with uniform and single color, convex colonies, and a colony diameter of 1 - 2 mm, coat and inoculate them on the selective medium, and subculture continuously for 5 times;

[0008] The formula of the selective medium is 5 g / L of methanol, 10 g / L of ammonium sulfate, 5 g / L of disodium hydrogen phosphate, 20 g / L of agar powder, 2 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 2 g / L of calcium chloride, 0.01 mg / L of tanshinone, 0.1 g / L of zinc sulfate, and the rest is deionized water, with the pH value being natural;

[0009] c. Pick colonies with uniform and single color, convex colonies, and a colony diameter of 1 - 2 mm from the selection medium plates after 5 passages in step b, coat and inoculate them on the plate medium, and incubate at a constant temperature of 30 °C for 44 - 52 hours;

[0010] The formula of this plate medium is 5 g / L of methanol, 10 g / L of ammonium sulfate, 5 g / L of disodium hydrogen phosphate, 20 g / L of agar powder, 2 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 2 g / L of calcium chloride, 0.1 g / L of zinc sulfate, and the rest is deionized water, with the pH value being natural;

[0011] d. Use a part of the colonies after plate culture in step c for preservation, and inoculate another part into the shake flask seed medium, and incubate at a constant temperature of 30 °C and a rotation speed of 200 - 240 rpm until the OD 600 value is 1.6 - 2.0 to obtain the seed liquid;

[0012] The formula of this seed medium is 5 g / L of methanol, 10 g / L of ammonium sulfate, 5 g / L of disodium hydrogen phosphate, 20 g / L of agar powder, 2 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 2 g / L of calcium chloride, 0.1 g / L of zinc sulfate, and the rest is deionized water, with the pH value being 6.80;

[0013] e. Prepare a 0.1 mg / L tanshinone stock solution; then inoculate the well - cultured seed liquid in step d into a 5 L fermenter with 3 L of fermentation broth, and the inoculation amount is 5 - 10% of the volume of the medium; the initial pH of the fermentation broth is 6.50, the initial air flow rate is 2.5 L / min, the maintenance pressure is 0.02 - 0.05 Mpa, the fermentation temperature is 28 - 32 °C, and the rotation speed is 200 - 300 rpm for fermentation. During the fermentation process, add a 50% methanol solution (slowly add dropwise, as long as there is no methanol accumulation during the fermentation process), the temperature is automatically controlled by the fermenter, the pH value is maintained at 6.50 ± 0.05 by adding 50% ammonia water by volume, keep the DO value (dissolved oxygen) at 20 - 30%, add the 0.1 mg / L tanshinone stock solution at 12, 24, 36, 48, and 60 hours of fermentation respectively, inject it at a flow rate of 5 mL / min through a peristaltic pump until the final concentration reaches 0.01 mg / L. Take samples every 6 hours to measure the PQQ content (HPLC method, C18 column, mobile phase methanol: 0.1% formic acid water = 15:85); add 2 g / L of methanol at 72 hours of fermentation, terminate the fermentation at 96 hours, and centrifuge to collect the supernatant.

[0014] Preferably, the serial dilution in step a is from 10 -1 to 10 -6 .

[0015] Preferably, the color of the colonies selected in both step b and step c is white, and both are incubated at a constant temperature of 30 °C for 48 hours.

[0016] Preferably, in step d, the seed liquid culture conditions are culturing at 30 °C and 220 rpm until the OD600 value of the bacterial liquid reaches 1.8.

[0017] Preferably, in step e, the inoculation amount is 10%; the fermentation medium is 5 g / L of methanol, 10 g / L of ammonium sulfate, 5 g / L of disodium hydrogen phosphate, 2 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 2 g / L of calcium chloride, 0.1 g / L of zinc sulfate, 0.2 mg / L of folic acid, 0.3 mg / L of calcium pantothenate, 0.05 mg / L of folic acid, 0.02 mg / L of cobalt chloride, 0.03 mg / L of potassium iodide, and the rest is purified water, with a pH value of 6.50.

[0018] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0019] The present invention utilizes the characteristics that tanshinone can scavenge ROS (reactive oxygen species), reduce MDA (malondialdehyde) in the bacterial cells, and activate the key enzymes in the pyrroloquinoline quinone synthesis pathway; first, tanshinone (0.01 mg / L) is used to naturally screen for tolerant strains, and then tanshinone is added five times during the logarithmic fermentation phase (12 - 60 hours), combined with dissolved oxygen feedback regulation (maintaining 20% - 30% saturation) and dynamic pH balance (6.50 ± 0.05). The yield of pyrroloquinoline quinone can be increased by 25% compared with the prior art. Specific Embodiments

[0020] The technical solution of the present invention will be further described below in conjunction with embodiments:

[0021] Example 1:

[0022] a. Dilute the Hyphomicrobium sp. for producing pyrroloquinoline quinone with sterilized water by a factor of 10 -1 to 10 -6 times to obtain a 10 -6 diluted solution;

[0023] b. Inoculate the 10 -6 diluted solution onto the selective medium and incubate at 30 °C for 48 hours; select colonies with uniform and single color, convex colonies, and a colony diameter of 1 - 2 mm, and spread and inoculate them onto the selective medium for 5 consecutive passages;

[0024] The formula of the selective medium is 5 g / L of methanol, 10 g / L of ammonium sulfate, 5 g / L of disodium hydrogen phosphate, 20 g / L of agar powder, 2 g / L of potassium dihydrogen phosphate, 1 g / L of magnesium sulfate, 2 g / L of calcium chloride, 0.01 mg / L of tanshinone, 0.1 g / L of zinc sulfate, and the rest is deionized water, with a natural pH value;

[0025] c. Select colonies with uniform and single color, convex colonies, and a colony diameter of 1 - 2 mm from the selective medium plates after 5 passages in step b, coat and inoculate them on the plate medium, and incubate at a constant temperature of 30 °C for 48 hours;

[0026] The formula of this plate medium is 5 g / L methanol, 10 g / L ammonium sulfate, 5 g / L disodium hydrogen phosphate, 20 g / L agar powder, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.1 g / L zinc sulfate, and the rest is deionized water, with the pH value being natural;

[0027] d. Use a part of the colonies after plate culture in step c for preservation, and inoculate another part into the shake flask seed medium, and incubate at a constant temperature of 30 °C and a rotation speed of 220 rpm until the OD 600 value reaches 1.8 to obtain the seed liquid;

[0028] The formula of this seed medium is 5 g / L methanol, 10 g / L ammonium sulfate, 5 g / L disodium hydrogen phosphate, 20 g / L agar powder, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.1 g / L zinc sulfate, and the rest is deionized water, with the pH value being 6.80;

[0029] e. Prepare a 0.1 mg / L tanshinone stock solution; then inoculate the well - cultured seed liquid in step d into a 5 L fermenter with 3 L of fermentation broth, and the inoculation amount is 10% of the medium volume; the fermentation medium formula is 5 g / L methanol, 10 g / L ammonium sulfate, 5 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.1 g / L zinc sulfate, 0.2 mg / L folic acid, 0.3 mg / L calcium pantothenate, 0.05 mg / L folic acid, 0.02 mg / L cobalt chloride, 0.03 mg / L potassium iodide, and the rest is purified water; the initial pH value of the fermentation broth is 6.50, the initial air flow rate is 2.5 L / min, the maintenance pressure is 0.02 - 0.05 Mpa, the fermentation temperature is 30 °C, and the rotation speed is 300 rpm for fermentation. During the fermentation process, add a 50% methanol solution (slowly flow - add, as long as there is no methanol accumulation during the fermentation process), the temperature is automatically controlled by the fermenter, the pH value is maintained at 6.50 ± 0.05 by adding 50% ammonia water by volume fraction, keep the DO value at 20 - 30%, add the 0.1 mg / L tanshinone stock solution at 12, 24, 36, 48, and 60 hours of fermentation respectively, inject it through a peristaltic pump at a flow rate of 5 mL / min until the final concentration reaches 0.01 mg / L. Take samples every 6 hours to measure the PQQ content (HPLC method, C18 column, mobile phase methanol: 0.1% formic acid water = 15:85); add 2 g / L methanol at 72 hours of fermentation and terminate the fermentation at 96 hours, and centrifuge to collect the supernatant; detect its OD 600Values were measured, and the content of pyrroloquinoline quinone in the supernatant was detected by HPLC. The results are shown in Table 1.

[0030] Example 2:

[0031] In step b of Example 1, 0.01 mg / L of tanshinone was not added to the solid medium, and other conditions were the same as those in Example 1.

[0032] Example 3:

[0033] In step e of Example 1, the interval time of the tanshinone solution was extended 5 times, and 0.1 mg / L of the tanshinone stock solution was added at 12, 30, 48, 66, and 84 hours respectively. Other conditions were the same as those in Example 1.

[0034] Example 4:

[0035] In step e of Example 1, the addition times of tanshinone were reduced, and 0.1 mg / L of the tanshinone stock solution was added at 24, 48, and 72 hours respectively. Other conditions were the same as those in Example 1.

[0036] Example 5:

[0037] In step e of Example 1, the addition times of tanshinone were increased, and 0.1 mg / L of the tanshinone stock solution was added at 12, 24, 36, 48, 60, 72, and 84 hours respectively. Other conditions were the same as those in Example 1.

[0038] Example 6:

[0039] In step e of Example 1, the final concentration of tanshinone added each time was increased from 0.01 mg / L to 0.02 mg / L, and other conditions were the same as those in Example 1.

[0040] Example 7:

[0041] In step e of Example 1, the final concentration of tanshinone added each time was decreased from 0.01 mg / L to 0.005 mg / L, and other conditions were the same as those in Example 1.

[0042] Comparative Example 1:

[0043] Tanshinone was not added during the screening and fermentation processes of Example 1, and the remaining conditions were the same as those in Example 1.

[0044] Results and Analysis

[0045] The OD 600 values and yields of the pyrroloquinoline quinone fermentation broths in Examples 1 to 7 and Comparative Example 1 were summarized in Table 1:

[0046] Table 1 OD values and pyrroloquinoline quinone yields of the fermentation broths in Examples 1 - 7 and Comparative Example 1

[0047] OD value Pyrroloquinoline quinone (PQQ) g / L Example 1 115 0.835 Example 2 80 0.698 Example 3 103 0.702 Example 4 110 0.796 Example 5 105 0.799 Example 6 92 0.704 Example 7 123 0.689 Comparative Example 1 85 0.668

[0048] From the above data, the following conclusions can be drawn: The tanshinone provided by the present invention can effectively promote the production of pyrroloquinoline quinone by Hyphomicrobium, and the yield of pyrroloquinoline quinone can reach 0.835 g / L, which is 25% higher than that of the prior art; adding tanshinone at different times has a great impact on the synthesis of pyrroloquinoline quinone; reducing and increasing the number of times of adding tanshinone will both reduce the synthesis amount of pyrroloquinoline quinone, and increasing the addition amount will decrease the biological OD value; reducing the concentration of tanshinone added each time will increase the biomass, but the increase in the synthesis amount of pyrroloquinoline quinone is not obvious, and increasing the concentration of tanshinone added each time will decrease the biological OD value; therefore, it is optimal to add tanshinone with a final concentration of 0.01 mg / L in 5 times at 12, 24, 36, 48, and 60 hours.

[0049] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for producing pyrroloquinoline quinone by inducing sclerotium microorganism fermentation using tanshinone, characterized in that: The following steps are involved: a. The raw silk microorganisms for the production of pyrroloquinoline quinone were gradiently diluted with sterile water to obtain a dilution; b. Inoculate the dilutions of different gradients on the selection medium, and culture at 30°C for 44 to 52 hours; select the colonies with uniform color, raised colonies, and a colony diameter of 1 to 2 mm, spread them on the selection medium, and subculture for 5 times; The formula of the selection medium is 5g / L methanol, 10g / L ammonium sulfate, 5g / L disodium hydrogen phosphate, 20g / L agar powder, 2g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, 2g / L calcium chloride, 0.01mg / L tanshinone, 0.1g / L zinc sulfate, and the rest is deionized water, with a natural pH value; c. Select colonies with uniform color, raised colonies, and a colony diameter of 1 to 2 mm from the selective medium plate after five passages in step b, spread them on the plate medium, and culture them at a constant temperature of 30°C for 44 to 52 hours; The formula of the plate culture medium is 5g / L methanol, 10g / L ammonium sulfate, 5g / L disodium hydrogen phosphate, 20g / L agar powder, 2g / L potassium dihydrogen phosphate, 1g / L magnesium sulfate, 2g / L calcium chloride, 0.1g / L zinc sulfate, and the rest is deionized water, with a natural pH value; d. A portion of the colonies cultured on the plate in step c was used for preservation, and a portion was inoculated into a shake flask seed culture medium and cultured at 30°C and 200-240 rpm until the bacterial solution OD 600 The value is 1.6 to 2.0, and the seed solution is obtained; The seed culture medium is formulated as follows: 5 g / L methanol, 10 g / L ammonium sulfate, 5 g / L disodium hydrogen phosphate, 20 g / L agar powder, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.1 g / L zinc sulfate, and the rest is deionized water, with a pH value of 6.80; e. Prepare 0.1mg / L tanshinone mother liquor; then inoculate the seed solution cultured in step d into a 5L fermentation tank with 3L fermentation broth, and the inoculation amount is 5-10% of the volume of the culture medium; the initial pH of the fermentation broth is 6.50, the initial air flow rate is 2.5L / min, the pressure is maintained at 0.02-0.05Mpa, the fermentation temperature is 28-32°C, the speed is 200-300rpm, and the fermentation is performed. 50% methanol solution is added during the fermentation process, and the temperature is automatically controlled by the fermentation tank. The pH value was maintained at 6.50±0.05 by adding 50% ammonia water by volume, and the DO value was maintained at 20-30%. 0.1 mg / L tanshinone mother solution was added at 12, 24, 36, 48, and 60 hours of fermentation, respectively, and injected at a flow rate of 5 mL / min through a peristaltic pump until the final concentration reached 0.01 mg / L. Samples were taken every 6 hours to determine the PQQ content. 2 g / L methanol was added at 72 hours of fermentation, and the fermentation was terminated at 96 hours, and the supernatant was collected by centrifugation.

2. The method for producing pyrroloquinoline quinone by fermentation of tanshinone-induced mycelial microorganisms as claimed in claim 1, characterized in that: The gradient dilution in step a is from 10 -1 to 10 -6 .

3. The method for producing pyrroloquinoline quinone by fermentation of tanshinone-induced mycelial microorganisms as claimed in claim 1, characterized in that: The colonies selected in both step b and step c are white in color and are cultured at a constant temperature of 30° C. for 48 hours.

4. The method for producing pyrroloquinoline quinone by fermentation of tanshinone-induced mycelial microorganisms as claimed in claim 1, characterized in that: In the step d, the seed solution culture conditions are 30° C. and 220 rpm until the bacterial solution obtains an OD600 value of 1.

8.

5. The method for producing pyrroloquinoline quinone by fermentation of tanshinone-induced mycelial microorganisms as claimed in claim 1, characterized in that: The inoculation amount in step e is 10%; the fermentation medium comprises 5 g / L methanol, 10 g / L ammonium sulfate, 5 g / L disodium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate, 2 g / L calcium chloride, 0.1 g / L zinc sulfate, 0.2 mg / L folic acid, 0.3 mg / L calcium pantothenate, 0.05 mg / L folic acid, 0.02 mg / L cobalt chloride, 0.03 mg / L potassium iodide, and the rest is purified water, with a pH value of 6.50.