Method for improving the production of pyrroloquinoline quinone by methylotrophs

Abstract

The application discloses a method for improving the pyrroloquinoline quinone titer of methylotrophic bacteria, which comprises primary seed culture, secondary seed culture and fed-batch fermentation; the fed-batch fermentation is that the cultured secondary seed liquid is inoculated into a fermentation medium, and under the condition of 28-32 DEG C and pH 6.8-7.0, fermentation is carried out until methanol is consumed, and then uniform feeding is started when the dissolved oxygen rebounds; the fermentation medium at least contains methanol, ammonium sulfate and formaldehyde dehydrogenase; and the feeding contains methanol, yeast extract powder and peptone. The application improves the utilization rate of methanol by the bacteria, reduces the influence of the intermediate product formaldehyde on the growth and metabolism of the bacteria, accelerates the growth rate of the bacteria, fully utilizes the feeding, and improves the pyrroloquinoline quinone yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and specifically relates to a method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria. Background Technology

[0002] Pyrroloquinoline quinone (PQQ) is a cofactor of class III oxidoreductases. It has functions such as protecting against liver damage, promoting organism growth, accelerating the synthesis rate of nerve growth factor, enhancing the tolerance of bacteria to some extreme conditions, and scavenging nitrogen free radicals. It is widely found in animals, plants and microorganisms and has important application value in the fields of food, medicine and cosmetics.

[0003] Literature reports that methyltrophic bacteria possess high and low-cost PQQ synthesis capabilities, making them highly promising PQQ-producing strains. However, when C1 compounds such as methanol and methane are the sole carbon source, the utilization rate of these compounds by methyltrophic bacteria is low, easily leading to resource waste. Furthermore, methyltrophic bacteria produce formaldehyde as an intermediate metabolite when utilizing C1 compounds like methanol. Since formaldehyde can react with proteins and nucleic acids, it has a strong toxic effect on bacterial cells. Therefore, methyltrophic bacteria must rapidly metabolize formaldehyde to prevent adverse reactions from occurring.

[0004] Currently, most research on improving PQQ production in methyltrophic bacteria focuses on the modification of engineered bacteria. For example, CN105624084A discloses a method for targeted domestication and selection of methyltrophic bacteria producing high-yield pyrroloquinoline quinones. By gradually increasing the methanol concentration in the culture medium to induce stress domestication in the methyltrophic bacteria, the positive mutation rate and the increase in pyrroloquinoline quinone production were improved. CN113913448A discloses a method and application for improving pyrroloquinoline quinone production in methyltrophic bacteria. The TCS3 gene in the genome of the methyltrophic bacteria was replaced with a selection marker gene to obtain engineered bacteria for pyrroloquinoline quinone production. However, there are few reports on improving the fermentation process to reduce the toxic side effects of the intermediate metabolite formaldehyde on the bacteria and improve PQQ titer. Improving the utilization rate of C1 compounds such as methanol by methyltrophic bacteria and reducing the toxic effects of the intermediate metabolite formaldehyde on the bacteria can effectively increase PQQ production, achieve energy conservation and emission reduction, and promote green and healthy development. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria. This method involves improving the fermentation medium and fermentation method by adding formaldehyde dehydrogenase to the culture medium, adding organic nitrogen sources to the feed, and so on. The aim is to appropriately accelerate the growth rate of the bacteria and increase methanol utilization, while reducing the toxic side effects of the intermediate product formaldehyde on the bacteria. Ultimately, this achieves the goals of reducing production energy consumption, shortening the fermentation cycle, and increasing the potency of PQQ.

[0006] To achieve its purpose, the technical solution adopted by the present invention is: a method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria, wherein the fermentation process includes primary seed culture, secondary seed culture and fed-batch fermentation;

[0007] The fed-batch fermentation process involves inoculating the cultured secondary seed culture into the fermentation medium at a volume ratio of 5-10%, and fermenting at 28-32℃ and pH 6.8-7.0 until methanol is depleted and dissolved oxygen rebounds, at which point fed-batch fermentation begins at a uniform rate. Fermentation is terminated after 170 hours.

[0008] The fermentation medium contains at least 0.8-1.2 g / L methanol, 4-6 g / L ammonium sulfate, and 0.5-0.8 mg / L formaldehyde dehydrogenase. The feed additive consists of: 450-550 g / L methanol, 5.0-7.0 g / L yeast extract, 2.0-3.0 g / L peptone, and pH 5.5-6.5. After fermentation, the pH is maintained at 7.0-7.5 by adjusting the fermentation speed.

[0009] The feeding rate is 0.1%-0.2% of the fermentation broth volume per hour, and the total feeding rate is 25%-30% of the total fermentation broth volume.

[0010] Preferably, the primary seed culture is as follows: methyl trophic bacteria glycerol are inoculated into a seed culture medium and cultured with shaking at 28-32℃ for 48-72 hours to obtain a primary seed solution.

[0011] Preferably, the secondary seed culture is as follows: the cultured primary seeds are inoculated into the seed culture medium at an inoculation rate of 1.0-2.0%, and cultured with shaking at 28-32℃ for 48-72 hours to obtain the secondary seed solution.

[0012] Preferably, the methyltrophic bacteria used in this invention are JCM2803 methyltora from JCM Corporation. Compared with ordinary strains, this strain accelerates the cell growth rate in a fermentation medium with methanol as the sole carbon source.

[0013] The seed culture medium of this invention comprises the following components and contents: methanol 8-12 g / L, ammonium sulfate 2.5-3.5 g / L, potassium dihydrogen phosphate 1.2-1.6 g / L, disodium hydrogen phosphate dodecahydrate 7-8 g / L, magnesium sulfate heptahydrate 0.45-0.55 g / L, trace element A 0.6-0.8 ml / L, trace element B 0.6-0.8 ml / L, trace element C 0.6-0.8 ml / L, ferrous sulfate heptahydrate 0.6-0.8 ml / L, and the pH of the culture medium is 6.8-7.0. Preferably, the seed culture medium contains the following components and contents: methanol 10 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 0.5 g / L, trace element A 0.7 ml / L, trace element B 0.7 ml / L, trace element C 0.7 ml / L, ferrous sulfate heptahydrate 0.7 ml / L, and the pH of the culture medium is 6.8-7.0.

[0014] The fermentation medium of this invention comprises the following components and contents: methanol 0.8-1.2 g / L, ammonium sulfate 4.5-5.5 g / L, potassium dihydrogen phosphate 1.2-1.6 g / L, disodium hydrogen phosphate dodecahydrate 7-8 g / L, magnesium sulfate heptahydrate 0.45-0.55 g / L, formaldehyde dehydrogenase 0.5-0.8 mg / L, trace element A 0.6-0.8 ml / L, trace element B 0.6-0.8 ml / L, trace element C 0.6-0.8 ml / L, and ferrous sulfate heptahydrate 0.6-0.8 ml / L. The pH of the medium is 6.8-7.0. Preferably, the fermentation medium contains the following components and concentrations: methanol 1 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 0.5 g / L, formaldehyde dehydrogenase 0.5-0.8 mg / L, trace element A 0.7 ml / L, trace element B 0.7 ml / L, trace element C 0.7 ml / L, ferrous sulfate heptahydrate 0.7 ml / L, and the medium pH is 6.8-7.0.

[0015] The components of trace element A are: zinc sulfate heptahydrate 22.5 g / L, manganese sulfate pentahydrate 4.5 g / L, and copper sulfate heptahydrate 0.75 g / L; the components of trace element B are: sodium chloride 1.5 g / L, potassium iodide 0.03 g / L, cobalt chloride hexahydrate 0.03 g / L, and calcium chloride dihydrate 30 g / L; the components of trace element C are: ammonium heptamolybdate tetrahydrate 0.03 g / L and boric acid 0.03 g / L; and the concentration of ferrous sulfate heptahydrate is 7.5 g / L.

[0016] This invention utilizes formaldehyde dehydrogenase to convert formaldehyde into formic acid, ultimately producing CO2 and water, thus reducing the toxic effects of formaldehyde on microorganisms. This invention leverages formaldehyde dehydrogenase to rapidly metabolize formaldehyde, achieving the goal of reducing the formaldehyde content of intermediate metabolites in the fermentation broth to a certain extent without affecting bacterial growth and the synthesis of target metabolites.

[0017] The beneficial effects of this invention are:

[0018] 1. This invention adds a certain amount of formaldehyde dehydrogenase to the culture medium to accelerate the metabolism of formaldehyde into formic acid, and prevents excessive toxicity of formaldehyde accumulation from affecting the growth and metabolism of bacteria.

[0019] 2. The addition of an organic nitrogen source to the feed in this invention promotes the early growth rate of the cells and the secretion of the target product PQQ, thereby increasing efficacy and shortening the fermentation cycle.

[0020] 3. This invention improves the utilization rate of methanol by the strain, reduces the impact of intermediate product formaldehyde on the growth and metabolism of the strain, accelerates the growth rate of the strain, and makes full use of feed. While increasing the yield of pyrroloquinoline quinone, it shortens the fermentation cycle and saves energy costs for subsequent industrial production. Attached Figure Description

[0021] Figure 1 The PQQ titer was compared between two cultures: one with no formaldehyde dehydrogenase in the culture medium and fed only methanol (Comparative Example 1), and the other with fed methanol and organic nitrogen (Comparative Example 2).

[0022] Figure 2 Comparison of formaldehyde content in fermentation broth when only methanol is fed and formaldehyde dehydrogenase is present in the culture medium (Comparative Example 1 and Comparative Example 3).

[0023] Figure 3 Comparison of PQQ titers when the supplemental feed is only methanol and the culture medium contains or does not contain formaldehyde dehydrogenase (Comparative Example 1 and Comparative Example 3). Detailed Implementation

[0024] The effects are illustrated below with reference to the embodiments and accompanying drawings.

[0025] The methyltrophic bacteria used in this embodiment is JCM2803, a type of methyltrophic bacterium from JCM Corporation.

[0026] The seed culture medium consisted of the following components and concentrations: methanol 10 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 0.5 g / L, trace element A 0.7 ml / L, trace element B 0.7 ml / L, trace element C 0.7 ml / L, and ferrous sulfate heptahydrate 0.7 ml / L. The pH of the culture medium was 6.8.

[0027] The composition and content of the fermentation medium are as follows: methanol 1g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 1.4g / L, disodium hydrogen phosphate dodecahydrate 7.56g / L, magnesium sulfate heptahydrate 0.5g / L, formaldehyde dehydrogenase 0.6-0.7mg / L (excluding comparative examples 1-2), trace element A 0.7ml / L, trace element B 0.7ml / L, trace element C 0.7ml / L, ferrous sulfate heptahydrate 0.7ml / L, the pH of the medium is 6.8, and the volume of the fermentation medium is 3L, which is 60% of the fermenter volume.

[0028] The components of trace element A are: zinc sulfate heptahydrate 22.5 g / L, manganese sulfate pentahydrate 4.5 g / L, and copper sulfate heptahydrate 0.75 g / L; the components of trace element B are: sodium chloride 1.5 g / L, potassium iodide 0.03 g / L, cobalt chloride hexahydrate 0.03 g / L, and calcium chloride dihydrate 30 g / L; the components of trace element C are: ammonium heptamolybdate tetrahydrate 0.03 g / L and boric acid 0.03 g / L; and the concentration of ferrous sulfate heptahydrate is 7.5 g / L.

[0029] Comparative Example 1: The feed consisted only of methanol, and the culture medium did not contain formaldehyde dehydrogenase.

[0030] A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria includes the following steps:

[0031] a. Primary seed culture: The methyl trophic bacteria glycerol bacteria stored in the -80℃ freezer were taken out and thawed. The bacterial suspension was inoculated into a 500mL primary shake flask containing 100mL seed culture medium at a volume ratio of 0.6%, and cultured at 30℃ and 200rpm for 72h.

[0032] b. Secondary seed culture: The primary seeds cultured in step a were inoculated at a volume ratio of 1.5% into a 1L secondary shake flask containing 240mL of seed culture medium and cultured at 30℃ and 200rpm for 48h.

[0033] c. Fed-batch fermentation: The secondary seed cultured in step b is inoculated at a volume ratio of 8% into a 5L fermenter containing 3L of fermentation medium (without formaldehyde dehydrogenase). The fermentation is carried out at 30℃, 150-450 rpm, pH 6.8-7.0, and an aeration rate of 2.5 vvm until methanol depletion and dissolved oxygen rebound (approximately 16 hours). Then, a feedstock containing only 500 g / L of methanol is added at a uniform rate of 3-5 mL / h. During the feeding process, the methanol concentration is controlled at 0.3-0.5 g / L by adjusting the feeding rate, for a total of 720 mL. Fermentation is terminated after 170 hours. After fermentation, the pH is controlled within the range of 7.0-7.5 by adjusting the fermentation speed.

[0034] d. Potency test: The potency of PQQ was 638.19 mg / L after fermentation.

[0035] The method for detecting PQQ valence is as follows:

[0036] 1) Preparation of standard solution: Weigh 40 mg (accurate to 0.01 mg) of pyrroloquinoline quinone disodium salt standard and place it in a 100 mL volumetric flask. Add acetonitrile aqueous solution (acetonitrile:water = 1:3, volume ratio) to dissolve and dilute to the mark. Shake well to obtain a stock solution of pyrroloquinoline quinone disodium salt standard with a concentration of 0.4 mg / mL. Prepare a series of pyrroloquinoline quinone disodium salt standard solutions with concentrations of 0.04 mg / mL, 0.08 mg / mL, 0.12 mg / mL, 0.16 mg / mL, 0.20 mg / mL, and 0.24 mg / mL from this solution.

[0037] 2) Reagent treatment: Weigh 20 mL of the fermentation broth and place it in a 50 mL volumetric flask. Add acetonitrile aqueous solution to dissolve and dilute to the mark, then shake well. Then accurately transfer 5.0 mL of the above solution into a 10 mL volumetric flask, dilute to the mark with acetonitrile aqueous solution, shake well, and use the filtrate after vacuum filtration through a 0.22 μm aqueous microporous membrane for determination.

[0038] 3) Reference conditions for liquid chromatography:

[0039] Chromatographic column: TCI Kaseisorb LC ODS2000 (4.6 mm × 150 mm, 5 μm) or equivalent column; column temperature: 30℃; flow rate: 1.0 mL / min; wavelength: 250 nm; injection volume: 20 μL. Mobile phase: Mobile phase A: 10 mmol / L dipotassium hydrogen phosphate-15 mmol / L tetrabutylammonium bromide mixture (pH 7.4); Mobile phase B: acetonitrile. Gradient elution was used, as shown in Table 1.

[0040] Table 1 Gradient elution program

[0041]

[0042]

[0043] Test: Under specified chromatographic conditions, 20 μL of each standard solution and sample solution were injected into the liquid chromatograph for chromatographic analysis. The peak area of ​​disodium pyrroloquinoline quinone in the chromatograms of the standard and sample solutions was recorded. A standard curve was plotted with the peak area of ​​disodium pyrroloquinoline quinone in the standard solution as the Y-axis and the corresponding concentration of the standard solution as the X-axis, thus obtaining the standard curve for disodium pyrroloquinoline quinone. Based on the peak area of ​​disodium pyrroloquinoline quinone in the chromatogram of the sample solution, the concentration of disodium pyrroloquinoline quinone in the sample solution was determined from the standard curve.

[0044] Calculation results: The content of disodium pyrroloquinoline quinone in the sample is expressed as its mass fraction W1, and the value is expressed as a percentage (%), calculated according to formula (1):

[0045]

[0046] In the formula:

[0047] C – The concentration of pyrroloquinoline quinone disodium salt in the sample solution, determined from the standard curve, in milligrams per milliliter (mg / mL);

[0048] V—Sample dilution volume, in milliliters (mL);

[0049] f—Purity of the disodium pyrroloquinoline quinone standard;

[0050] m — The mass of the sample is measured in milligrams (mg);

[0051] W2 — The mass fraction of water in the sample, expressed as a percentage (%);

[0052] The arithmetic mean of the parallel measurement results is taken as the measurement result, and the result is expressed to two decimal places.

[0053] Precision: The relative standard deviation of two independent determinations obtained under repeatability conditions is no greater than 2.0%.

[0054] Comparative Example 2: Feeding was normal, and the culture medium did not contain formaldehyde dehydrogenase.

[0055] A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria includes the following steps:

[0056] a. Primary seed culture: The methyl trophic bacteria glycerol bacteria stored in the -80℃ freezer were taken out and thawed. The bacterial suspension was inoculated into a 500mL primary shake flask containing 100mL seed culture medium at an inoculation rate of 0.6%, and cultured at 30℃ and 200rpm for 72h.

[0057] b. Secondary seed culture: The primary seeds cultured in step a were inoculated at a rate of 1.5% into a 1L secondary shake flask containing 240mL of seed culture medium and cultured at 30℃ and 200rpm for 48h.

[0058] c. Fed-batch fermentation: The secondary seed cultured in step b was inoculated at a volume ratio of 8% into a 5L fermenter containing 3L of fermentation medium (free of formaldehyde dehydrogenase). The fermentation was carried out at 30℃, 150-450 rpm, pH 6.8-7.0, and an aeration rate of 2.5 vvm until methanol depletion and dissolved oxygen rebound (approximately 16 hours). Then, a fed-batch culture was initiated at a uniform rate of 4-6 mL / h (feed mixture composition and content: methanol 500 g / L, yeast extract 5 g / L, peptone 2.0 g / L, pH 5.5-6.5). During the feeding process, the methanol concentration was controlled at 0.3-0.5 g / L by adjusting the feeding rate. A total of 880 mL was fed, and fermentation was terminated after 170 hours. After fermentation, the pH was maintained within the range of 7.0-7.5.

[0059] d. Potency test: The potency of PQQ was 1050 mg / L after fermentation.

[0060] Comparative Example 3: The feed consisted only of methanol, and the culture medium contained formaldehyde dehydrogenase.

[0061] A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria includes the following steps:

[0062] a. Primary seed culture: The methyl trophic bacteria glycerol bacteria stored in the -80℃ freezer were taken out and thawed. The bacterial suspension was inoculated into a 500mL primary shake flask containing 100mL seed culture medium at an inoculation rate of 0.6%, and cultured at 30℃ and 200rpm for 72h.

[0063] b. Secondary seed culture: The primary seeds cultured in step a were inoculated at a rate of 1.5% into a 1L secondary shake flask containing 240mL of seed culture medium and cultured at 30℃ and 200rpm for 48h.

[0064] c. Fed-batch fermentation: The secondary seed cultured in step b is inoculated at a volume ratio of 8% into a 5L fermenter containing 3L of fermentation medium, which contains 0.6mg / L formaldehyde dehydrogenase. The fermentation is carried out at 30℃, 150-450rpm, pH 6.8-7.0, and an aeration rate of 2.5 vvm until methanol depletion and dissolved oxygen rebound (approximately 16 hours). Then, a feedstock containing only 500g / L methanol is added at a uniform rate of 4-5mL / h. During the feeding process, the methanol concentration is controlled at 0.3-0.5g / L by adjusting the feeding rate, for a total of 710mL. Fermentation is terminated after 170 hours. After fermentation, the pH is maintained within the range of 7.0-7.5.

[0065] d. Potency test: The potency of PQQ was 882.6 mg / L after fermentation.

[0066] Example 1:

[0067] A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria includes the following steps:

[0068] a. Primary seed culture: The glycerol bacteria stored in the -80℃ freezer were taken out and thawed. The bacterial suspension was inoculated into a 500mL primary shake flask containing 100mL seed culture medium at an inoculation rate of 0.6%. The culture was carried out at 30℃ and 200rpm for 72h.

[0069] b. Secondary seed culture: The primary seeds cultured in step a were inoculated at a rate of 1.5% into a 1L secondary shake flask containing 240mL of seed culture medium and cultured at 30℃ and 200rpm for 48h.

[0070] c. Fed-batch fermentation: The secondary seed cultured in step b was inoculated at a volume ratio of 8% into a 5L fermenter containing 3L of fermentation medium, which contained 0.7mg / L formaldehyde dehydrogenase. The fermentation was carried out at 30℃, 150-450rpm, pH 6.8-7.0, and an aeration rate of 2.5vvm until methanol depletion and dissolved oxygen rebound (approximately 16 hours). Then, a fed-batch culture was started at a uniform rate of 4-6mL / h (composed of: 500g / L methanol, 5g / L yeast extract, 2.0g / L peptone, pH 5.5-6.5). During the feeding process, the methanol concentration was controlled at 0.3-0.5g / L by adjusting the feeding rate. A total of 890mL was fed, and fermentation was terminated after 170 hours. After fermentation, the pH was maintained within the range of 7.0-7.5.

[0071] d. Potency test: The potency of PQQ was 1359 mg / L after fermentation.

[0072] The experimental results show that the addition of formaldehyde dehydrogenase to the culture medium and the addition of an organic nitrogen source to the feed significantly increased the PQQ titer (comparison of PQQ titer in culture medium without formaldehyde dehydrogenase, feed consisting only of methanol, and feed consisting of methanol + organic nitrogen). Figure 1 As shown; when the feed consisted only of methanol, and the culture medium contained or lacked formaldehyde dehydrogenase (Comparative Example 1 and Comparative Example 3), the PQQ titers were compared as follows: Figure 3 As shown in the figure, when the two are used together, the PQQ potency increase rate is as high as 113% (Example 1 compared with Comparative Example 1). It can be concluded that the use of formaldehyde dehydrogenase and organic nitrogen source in this invention can significantly increase the yield of PQQ.

[0073] The method provided by this invention improves the utilization rate of methanol by the microbial strain, shortens the fermentation cycle, and reduces the toxic side effects of the intermediate metabolite formaldehyde on the microbial cells (see [link]). Figure 2This improved the efficiency of enzyme production by the microorganisms; the feed was fully utilized, and the problem of slow growth rate during the exponential growth phase of the microorganisms was solved, which is conducive to increasing biomass and reducing energy consumption. Compared with the original fermentation conditions, the yield of PQQ was significantly increased.

Claims

1. A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria, wherein the fermentation process includes primary seed culture, secondary seed culture and fed-batch fermentation; The fed-batch fermentation process involves inoculating the cultured secondary seed culture into the fermentation medium at a volume ratio of 5-10%, fermenting at 28-32℃ and pH 6.8-7.0 until methanol is depleted and dissolved oxygen rebounds, then starting fed-batch fermentation, and terminating fermentation after 170 hours. The fermentation medium contains at least 0.8-1.2 g / L methanol, 4-6 g / L ammonium sulfate, and 0.5-0.8 mg / L formaldehyde dehydrogenase; the feed consists of 450-550 g / L methanol, 5.0-7.0 g / L yeast extract, 2.0-3.0 g / L peptone, and pH 5.5-6.

5. in, The feed flow rate is 0.1%-0.2% of the fermentation broth volume per hour, and the total feed flow rate is 25%-30% of the total fermentation broth volume.

2. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 1, characterized in that, The primary seed culture is as follows: methyl trophic bacteria glycerol are inoculated into seed culture medium and cultured with shaking at 28-32℃ for 48-72 hours to obtain primary seed liquid.

3. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 2, characterized in that, The secondary seed culture is as follows: the cultured primary seeds are inoculated into the seed culture medium at an inoculation rate of 1.0-2.0%, and cultured with shaking at 28-32℃ for 48-72 hours to obtain the secondary seed solution.

4. A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 2 or 3, characterized in that, The seed culture medium comprises the following components and contents: methanol 8-12 g / L, ammonium sulfate 2.5-3.5 g / L, potassium dihydrogen phosphate 1.2-1.6 g / L, disodium hydrogen phosphate dodecahydrate 7-8 g / L, magnesium sulfate heptahydrate 0.45-0.55 g / L, trace element A 0.6-0.8 ml / L, trace element B 0.6-0.8 ml / L, trace element C 0.6-0.8 ml / L, ferrous sulfate heptahydrate 0.6-0.8 ml / L, pH=6.8-7.0; The composition and content of trace element A are as follows: zinc sulfate heptahydrate 22.5 g / L, manganese sulfate pentahydrate 4.5 g / L, copper sulfate heptahydrate 0.75 g / L; the composition and content of trace element B are as follows: sodium chloride 1.5 g / L, potassium iodide 0.03 g / L, cobalt chloride hexahydrate 0.03 g / L, calcium chloride dihydrate 30 g / L; the composition and content of trace element C are as follows: ammonium heptamolybdate tetrahydrate 0.03 g / L, boric acid 0.03 g / L; the concentration of ferrous sulfate heptahydrate is 7.5 g / L.

5. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 4, characterized in that, The seed culture medium contains the following components and concentrations: methanol 10 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 0.5 g / L, trace element A 0.7 ml / L, trace element B 0.7 ml / L, trace element C 0.7 ml / L, ferrous sulfate heptahydrate 0.7 ml / L, and pH = 6.8-7.

0.

6. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 1, characterized in that, The fermentation medium comprises the following components and contents: methanol 0.8-1.2 g / L, ammonium sulfate 4.5-5.5 g / L, potassium dihydrogen phosphate 1.2-1.6 g / L, disodium hydrogen phosphate dodecahydrate 7-8 g / L, magnesium sulfate heptahydrate 0.45-0.55 g / L, formaldehyde dehydrogenase 0.5-0.8 mg / L, trace element A 0.6-0.8 ml / L, trace element B 0.6-0.8 ml / L, trace element C 0.6-0.8 ml / L, ferrous sulfate heptahydrate 0.6-0.8 ml / L, pH=6.8-7.0; The composition and content of trace element A are as follows: zinc sulfate heptahydrate 22.5 g / L, manganese sulfate pentahydrate 4.5 g / L, copper sulfate heptahydrate 0.75 g / L; the composition and content of trace element B are as follows: sodium chloride 1.5 g / L, potassium iodide 0.03 g / L, cobalt chloride hexahydrate 0.03 g / L, calcium chloride dihydrate 30 g / L; the composition and content of trace element C are as follows: ammonium heptamolybdate tetrahydrate 0.03 g / L, boric acid 0.03 g / L; the concentration of ferrous sulfate heptahydrate is 7.5 g / L.

7. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 6, characterized in that, The fermentation medium contains the following components and concentrations: methanol 1 g / L, ammonium sulfate 5 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 0.5 g / L, formaldehyde dehydrogenase 0.5-0.8 mg / L, trace element A 0.7 ml / L, trace element B 0.7 ml / L, trace element C 0.7 ml / L, ferrous sulfate heptahydrate 0.7 ml / L, and pH = 6.8-7.

0.

8. The method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in claim 1, characterized in that, The methyltrophic bacteria is *Thiazinomethylobacterium* JCM2803.

9. A method for improving the potency of pyrroloquinoline quinone in methyltrophic bacteria as described in any one of claims 1-3, 5-8, characterized in that, After fermentation, maintain the pH at 7.0-7.5.

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