Method for preparing pyrroloquinoline quinone and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a method for preparing pyrroloquinoline quinone and application. The invention provides a method for preparing pyrroloquinoline quinine, a seed culture medium is designed according to the requirements of microbial growth, microelements in the seed culture medium promote thallus growth, and a good thallus foundation is laid for subsequent synthesis of pyrroloquinoline quinine (PQQ); in a fermentation medium stage, namely a fermentation culture stage, PQQ synthesis enters a key stage, and trace elements beneficial to improving PQQ synthetase activity are pertinently supplemented, so that mass synthesis of PQQ is promoted. According to the method, the specific seed culture medium and the fermentation culture medium are utilized, trace elements are supplemented in the middle stage of fermentation, accurate regulation and control of the PQQ fermentation process are achieved, and the fermentation titer and purity of pyrroloquinoline quinone are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a method for preparing pyrroloquinoline quinone and application thereof. BACKGROUND

[0002] As a kind of material with important biological activity, PQQ (pyrroloquinoline quinone) has a wide application prospect in the industries of medicine, health care products, cosmetics and the like. In recent years, with the continuous growth of market demand for PQQ, the optimization of the fermentation production process of PQQ has become a research hotspot.

[0003] The current PQQ fermentation production technology faces many bottlenecks. From the perspective of fermentation titer, the PQQ yield obtained by the existing fermentation process is at a low level. For example, the PQQ fermentation product concentration in the industry is about 1900 mg / L. The low fermentation product concentration leads to low production efficiency and high production cost, which limits the market supply of PQQ products, and also makes the price of PQQ relatively high, which is not conducive to the popularization and application of PQQ in more fields.

[0004] In terms of impurity control, the existence of impurities in the fermentation broth seriously affects the quality of PQQ products. In particular, the impurities at the RRT 1.2 position have a high content under the existing process. The RRT 1.2 position refers to the impurities corresponding to the position with a relative retention time of 1.2 in the chromatogram with PQQ as the reference.

[0005] The existence of impurities at the RRT 1.2 position not only reduces the purity of PQQ, but also may cause a series of problems in subsequent applications. For example, in the medical application, the impurities may interfere with the pharmacological effect of PQQ and increase the risk of drug side effects; in the field of cosmetics, the impurities may affect the stability and safety of the products and reduce the use experience of consumers.

[0006] In summary, the existing PQQ fermentation process needs to be solved in terms of improving the fermentation titer of PQQ and reducing specific impurities. SUMMARY

[0007] The purpose of the present application is to provide a method for preparing pyrroloquinoline quinone, which has a high fermentation titer and a low content of impurities at the RRT 1.2 position.

[0008] In order to solve the above technical problems, the present application provides the following technical solutions:

[0009] The present application provides a method for preparing pyrroloquinoline quinone, comprising the following steps:

[0010] Inoculating the pyrroloquinoline quinone production strain into a seed culture medium for seed culture to obtain a seed liquid;

[0011] Inoculate the seed liquid into a fermentation medium to obtain a fermentation system, and perform fermentation culture to obtain a fermentation liquor;

[0012] During the fermentation culture, supplement trace element A mother liquor, trace element B mother liquor and trace element C mother liquor at 70-170 h;

[0013] The seed culture medium comprises: methanol 5-20 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, disodium hydrogen phosphate dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.2-5 g / L, trace element B mother liquor 0.2-5 g / L and trace element C mother liquor 0.2-5 g / L;

[0014] The fermentation culture medium comprises: methanol 2-10 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, disodium hydrogen phosphate dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.5-5 g / L, trace element B mother liquor 0.5-5 g / L and trace element C mother liquor 0.5-5 g / L;

[0015] The trace element A mother liquor comprises: zinc sulfate heptahydrate 15-30 g / L, manganese sulfate tetrahydrate 2-10 g / L and copper sulfate pentahydrate 0.2-1.5 g / L;

[0016] The trace element B mother liquor comprises: sodium chloride 0.5-5 g / L, cobalt chloride hexahydrate 0.01-0.2 g / L, calcium chloride dihydrate 10-50 g / L and potassium iodide 0.01-0.2 g / L;

[0017] The trace element C mother liquor comprises: ammonium molybdate tetrahydrate 0.01-0.2 g / L, boric acid 0.01-0.2 g / L and ferrous sulfate 2.5-10 g / L.

[0018] Preferably, the temperature of the seed culture is 23-35℃, and the time is 40-45 h; the initial pH of the seed culture medium is 6.0-7.0, and the dissolved oxygen content is 30%-70%.

[0019] Preferably, the temperature of the fermentation culture is 23-35℃; the initial pH of the fermentation culture medium is 6.0-7.0, and the dissolved oxygen content is 20%-40%.

[0020] Preferably, the time of the fermentation culture is 170-195 h.

[0021] Preferably, during the whole fermentation culture process, when the concentration of methanol in the fermentation system is less than 5 g / L, methanol is added to control the concentration of methanol in the fermentation system to be 2-10 g / L

[0022] Preferably, the trace element A mother liquor, trace element B mother liquor and trace element C mother liquor are supplemented in the following 1) or 2):

[0023] 1) After mixing the trace element A mother liquor, trace element B mother liquor and trace element C mother liquor to obtain a premix, the premix is supplemented at one time; the volume ratio of the trace element A mother liquor, trace element B mother liquor and trace element C mother liquor in the premix is (1-2):(1-2):(1-2);

[0024] 2) After mixing the trace element A mother liquor and trace element B mother liquor to obtain an A+B mixture, the A+B mixture and trace element C mother liquor are supplemented at one time; the volume ratio of the trace element A mother liquor and trace element B mother liquor in the A+B mixture is (1-2):(1-2); the volume ratio of the A+B mixture and trace element C mother liquor is (1-2):(1-2).

[0025] Preferably, in the 2), the volume of the trace element A+B mixture is 0.08%-0.15% of the fermentation medium; the volume of the trace element C mother liquor is 0.08%-0.15% of the fermentation medium.

[0026] In the 1), the volume of the premix is 0.2%-0.4% of the fermentation medium.

[0027] Preferably, the supplementing in the 1) or 2) is ≥1 time.

[0028] Preferably, the time interval between adjacent two supplementings is 22-26 h.

[0029] The present application provides the use of the method described in the above technical solution in at least one of the following:

[0030] 1) improving the yield of pyrroloquinoline quinone;

[0031] 2) improving the purity of pyrroloquinoline quinone;

[0032] 3) reducing the content of impurities corresponding to the RRT1.2 position of pyrroloquinoline quinone.

[0033] The present application provides a method for preparing pyrroloquinoline quinone, in the PQQ fermentation process, the growth and metabolism of microorganisms are affected by many factors, among which trace elements, although the demand is relatively small, but plays an indispensable role. The present application designs a seed medium according to the needs of microbial growth, and the trace elements in the seed medium promote the growth of the bacterial body, and lay a good bacterial body foundation for the subsequent synthesis of PQQ; in the fermentation culture stage, PQQ synthesis enters the key stage, and the trace elements which are helpful to improve the activity of PQQ synthesis enzyme are added in a targeted manner, so as to maintain the normal metabolic function of microbial cells and reduce the generation of impurities. The present application controls the addition of trace elements accurately, so that the microbial growth environment is in the optimal state. The present application uses specific seed medium and fermentation medium, and supplements trace elements in the middle of fermentation, so as to realize the accurate regulation of PQQ fermentation process, and improve the fermentation titer and purity of pyrroloquinoline quinone. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 and Figure 2 The chromatographic analysis report graph of the fermentation liquid obtained in Example 1 is shown in the following figure: Figures 1-2 The retention time of 10.721 min in corresponds to PQQ;

[0035] Figure 3 and Figure 4 The chromatographic analysis report graph of the fermentation liquid obtained in Example 2 is shown in the following figure: Figures 3-4 The retention time of 11.864 min in corresponds to PQQ;

[0036] Figure 5 and Figure 6 The chromatographic analysis report graph of the fermentation liquid obtained in Example 3 is shown in the following figure: Figures 5-6 The retention time of 11.051 min in corresponds to PQQ;

[0037] Figure 7 and Figure 8 The chromatographic analysis report graph of the fermentation liquid obtained in Example 4 is shown in the following figure: Figures 7-8 The retention time of 10.413 min in corresponds to PQQ.

[0038] Figures 1-8 The peak number in the table of corresponds to the retention time, and the peak number is the number of the peak in the chromatogram in order, Figures 1-8 The retention time in the table of corresponds to the retention time in the chromatogram. DETAILED DESCRIPTION

[0039] The present application provides a method for preparing pyrroloquinoline quinone, comprising the following steps:

[0040] The pyrroloquinoline quinone production strain is inoculated into a seed culture medium for seed culture to obtain a seed liquid;

[0041] The seed liquid is inoculated into a fermentation culture medium to obtain a fermentation system for fermentation culture to obtain a fermentation liquid.

[0042] Trace element A mother liquor, trace element B mother liquor and trace element C mother liquor are supplemented at 70-170 h of the fermentation culture.

[0043] The pyrroloquinoline quinone production strain is inoculated into a seed culture medium for seed culture to obtain a seed liquid. The strain type of the pyrroloquinoline quinone production strain is not specially limited in the present application, and the strain type commonly used in the art for fermentation preparation of pyrroloquinoline quinone can be adopted. In the specific embodiments of the present application, Hyphomicrobium denitrificans is taken as an example for illustration, but it cannot be understood as the whole protection scope of the present application.

[0044] In the present application, the seed culture medium comprises: methanol 5-20 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, disodium hydrogen phosphate dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.2-5 g / L, trace element B mother liquor 0.2-5 g / L and trace element C mother liquor 0.2-5 g / L.

[0045] The seed culture medium of the present application comprises methanol 5-20 g / L, which can also be 6-15 g / L; the methanol of the present application can be arbitrarily valued within the above range, for example, in a specific embodiment, the methanol in the seed culture medium is 16 g / L; the role of methanol is to serve as the sole carbon source and energy source. The seed culture medium of the present application comprises ammonium sulfate 1-10 g / L, which can also be 3-7 g / L; the ammonium sulfate of the present application can be arbitrarily valued within the above range, for example, in a specific embodiment, the ammonium sulfate in the seed culture medium is 4, 5, 6 or 7 g / L, and the role of the ammonium sulfate is to provide a nitrogen source. The seed culture medium of the present application comprises potassium dihydrogen phosphate 0.5-5 g / L, which can also be 1.5-3.5 g / L, and the potassium dihydrogen phosphate of the present application can be arbitrarily valued within the above range, for example, in a specific embodiment, the potassium dihydrogen phosphate in the seed culture medium is 2, 3 or 4 g / L, and the role of the potassium dihydrogen phosphate is to maintain the pH stability of the culture medium. The seed culture medium of the present application comprises disodium hydrogen phosphate dodecahydrate 5-15 g / L, which can also be 8-12 g / L, and the disodium hydrogen phosphate dodecahydrate of the present application can be arbitrarily valued within the above range, for example, in a specific embodiment, the disodium hydrogen phosphate dodecahydrate in the seed culture medium is 8, 9, 10 or 11 g / L, and the role of the disodium hydrogen phosphate dodecahydrate is to maintain the pH stability of the culture medium. The seed culture medium of the present application comprises magnesium sulfate heptahydrate 0.2-5 g / L, which can also be 1-4 g / L, and the magnesium sulfate heptahydrate of the present application can be arbitrarily valued within the above range, for example, in a specific embodiment, the magnesium sulfate heptahydrate in the seed culture medium is 2, 3 or 4 g / L, and the role of the magnesium sulfate heptahydrate is to provide magnesium ions (Mg 2+). The seed culture medium of the present application comprises trace element A mother liquor 0.2-5 g / L, which can also be 1.2-3.6 g / L; the trace element A mother liquor of the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the trace element A mother liquor in the seed culture medium is 1, 2, 3 or 4 g / L; the composition of the trace element A mother liquor of the present application comprises zinc sulfate heptahydrate 15-30 g / L, manganese sulfate tetrahydrate 2-10 g / L and copper sulfate pentahydrate 0.2-1.5 g / L; the trace element A mother liquor of the present application comprises zinc sulfate heptahydrate 15-30 g / L, which can also be 20-26 g / L, and the zinc sulfate heptahydrate provides zinc ions; the trace element A mother liquor comprises manganese sulfate tetrahydrate 2-10 g / L, which can also be 4-8 g / L, and the manganese sulfate tetrahydrate provides manganese ions; the trace element A mother liquor comprises copper sulfate pentahydrate 0.2-1.5 g / L, which can also be 0.5-1.3 g / L, and the copper sulfate pentahydrate provides copper ions. The seed culture medium of the present application comprises trace element B mother liquor 0.2-5 g / L, which can also be 1.0-3.5 g / L; the trace element B mother liquor of the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the trace element B mother liquor in the seed culture medium is 1, 2, 3 or 4 g / L. The trace element B mother liquor of the present application comprises sodium chloride 0.5-5 g / L, cobalt chloride hexahydrate 0.01-0.2 g / L, calcium chloride dihydrate 10-50 g / L and potassium iodide 0.01-0.2 g / L. The trace element B mother liquor of the present application comprises sodium chloride 0.5-5 g / L, which can also be 1.0-4.0 g / L, and the sodium chloride maintains the osmotic pressure balance. The trace element B mother liquor of the present application comprises cobalt chloride hexahydrate 0.01-0.2 g / L, which can also be 0.05-0.13 g / L, and the cobalt chloride hexahydrate provides cobalt ions and participates in the methyl transfer reaction. The trace element B mother liquor of the present application comprises calcium chloride dihydrate 10-50 g / L, which can also be 15-40 g / L, and the calcium chloride dihydrate provides calcium ions. The trace element B mother liquor of the present application comprises potassium iodide 0.01-0.2 g / L, which can also be 0.05-0.15 g / L, and the potassium iodide provides iodine ions. 2+, participates in cell wall stability, signal transduction and enzyme activity regulation. The trace element B mother liquor in the application includes potassium iodide 0.01-0.2 g / L, which can also be 0.05-0.16 g / L, and the potassium iodide provides iodine element (I-). The seed culture medium in the application includes trace element C mother liquor 0.2-5 g / L, which can also be 0.8-4.2 g / L; the trace element C mother liquor in the application can be arbitrarily selected within the above range, for example, in a specific embodiment, the trace element C mother liquor in the seed culture medium is 1, 2, 3 or 4 g / L. The trace element C mother liquor in the application includes ammonium molybdate tetrahydrate 0.01-0.2 g / L, boric acid 0.01-0.2 g / L and ferrous sulfate 2.5-10 g / L. The trace element C mother liquor in the application includes ammonium molybdate tetrahydrate 0.01-0.2 g / L, which can also be 0.06-0.13 g / L, and the ammonium molybdate tetrahydrate provides molybdenum element. The trace element C mother liquor in the application includes boric acid 0.01-0.2 g / L, which can also be 0.05-0.16 g / L, and the boric acid may participate in cell wall synthesis and membrane stability. The trace element C mother liquor in the application includes ferrous sulfate 2.5-10 g / L, which can also be 4-8 g / L, and the ferrous sulfate provides Fe 2+ , participates in electron transport chain and energy metabolism.

[0046] As an optional embodiment, the temperature of the seed culture in the application is 23-35℃, which can also be 25-30℃. As an optional embodiment, the time of the seed culture in the application is 35-48 h, which can also be 38-46 h. As an optional embodiment, the initial pH of the seed culture medium in the application is 6.0-7.0. As an optional embodiment, the dissolved oxygen content of the seed culture medium in the application is 30%-70%, which can also be 40%-60%. The inoculation amount and method of the seed culture in the application are not particularly limited, and conventional methods can be used. The setting of the parameters of the seed culture in the application can promote the growth of the strain and increase the viable cell count.

[0047] The seed liquid in the application is inoculated into a fermentation medium to obtain a fermentation system for fermentation culture, and a fermentation liquid is obtained.

[0048] The fermentation culture in the application is preferably completed by using a fermentation tank. Before the fermentation culture in the application is carried out, fermentation tank preparation work needs to be carried out, and the accurate work of the fermentation tank includes: selecting a fermentation tank with appropriate specifications, and comprehensively cleaning and disinfecting the fermentation tank to ensure that the fermentation environment is sterile. Check whether the stirring device, aeration device, temperature and pH control system and other parts of the fermentation tank are normally running to ensure that the fermentation process can be stably carried out.

[0049] The fermentation medium comprises: methanol 2-10 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, disodium hydrogen phosphate dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.5-5 g / L, trace element B mother liquor 0.5-5 g / L and trace element C mother liquor 0.5-5 g / L.

[0050] The fermentation medium comprises methanol 2-10 g / L, which can also be 4-7 g / L, and the methanol acts as a sole carbon source and energy source. The methanol in the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the methanol in the seed medium is 8, 9 or 10 g / L. The fermentation medium comprises ammonium sulfate 1-10 g / L, which can also be 2-8 g / L, and the ammonium sulfate provides a nitrogen source. The ammonium sulfate in the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the ammonium sulfate in the seed medium is 5, 6 or 7 g / L. The fermentation medium comprises potassium dihydrogen phosphate 0.5-5 g / L, which can also be 1.3-4 g / L, and the potassium dihydrogen phosphate maintains the pH of the medium stable. The potassium dihydrogen phosphate in the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the potassium dihydrogen phosphate in the seed medium is 2, 3 or 4 g / L. The fermentation medium comprises disodium hydrogen phosphate dodecahydrate 5-15 g / L, which can also be 8-12 g / L, and the disodium hydrogen phosphate dodecahydrate maintains the pH of the medium stable (phosphate buffer system). The disodium hydrogen phosphate dodecahydrate in the present application can be arbitrarily selected within the above range, for example, in specific embodiments, the disodium hydrogen phosphate dodecahydrate in the seed medium is 8, 9, 10 or 11 g / L. The fermentation medium comprises magnesium sulfate heptahydrate 0.2-5 g / L, which can also be 1.0-4.6 g / L, and the magnesium sulfate heptahydrate provides magnesium ions. The compositions of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor have been discussed above, and will not be repeated here.

[0051] The potassium dihydrogen phosphate and the disodium hydrogen phosphate dodecahydrate in the seed medium and the fermentation medium of the present application form a phosphate buffer system, which stabilizes the pH of the seed medium and the fermentation medium.

[0052] The present application does not have special restrictions on the sources of the components in the seed medium, the fermentation medium, the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor, and any sources known to those skilled in the art can be used.

[0053] As an optional implementation, the inoculation amount of the seed liquid into the fermentation medium is 10% to 20% of the volume of the fermentation medium, or it can be 20% to 30%. The present invention does not have any particular limitation on the inoculation method; conventional methods are acceptable.

[0054] As an optional embodiment, the fermentation culture temperature of the present invention is 23–35°C, or 25–32°C. As an optional embodiment, the initial fermentation medium of the present invention has a pH of 6.0–7.0, and the dissolved oxygen content of the fermentation culture is 20%–40%, or 30%. The OD of the fermentation broth of the present invention… 600 When the concentration of pyrroloquinoline quinone (PQU) is <35, it is considered the initial fermentation stage. During this stage, the fermentation temperature is controlled within the range of 23–35°C, and a suitable dissolved oxygen level (20%–40%) is maintained through a ventilation device. Simultaneously, the stirring device is activated, with the rotation speed set to 100–700 r / min to ensure sufficient contact between the bacteria and the culture medium. The primary purpose of the initial fermentation stage is to allow the pyrroloquinoline quinone-producing bacteria to adapt to the fermentation environment and begin rapid growth and reproduction.

[0055] The fermentation broth OD of the fermentation culture of the present invention 600 When the value is ≥35, the fermentation process regulation stage begins. During this stage, the fermentation temperature is controlled within the range of 25–30℃, and a suitable dissolved oxygen level (20%–40%) is maintained through an aeration device. Simultaneously, the stirring device is activated, with a rotation speed set to 100–700 r / min to ensure sufficient contact between the bacteria and the culture medium. The main purpose of the fermentation process regulation stage is to encourage the pyrroloquinoline quinone-producing bacteria to produce more pyrroloquinoline quinone.

[0056] The fermentation time described in this invention is the sum of the initial fermentation stage and the fermentation process control stage. As an optional implementation, the fermentation time is 170–195 hours, or 172–193 hours. In specific embodiments of this invention, the fermentation time is 172 hours, 192 hours, or 193 hours. The fermentation continues until the PQQ content increase slows down or stops increasing. In specific embodiments of this invention, fermentation is considered complete when the PQQ content increase is less than 10%.

[0057] The trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor are supplemented in the fermentation culture described in the application at 70-170 h. As an alternative embodiment, the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor are supplemented in the following 1) or 2): 1) the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor are mixed to obtain a premix, and the premix is supplemented at one time; the volume ratio of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor in the premix is (1-2):(1-2):(1-2); 2) the trace element A mother liquor and the trace element B mother liquor are mixed to obtain an A+B mixture, and the A+B mixture and the trace element C mother liquor are supplemented at one time; the volume ratio of the trace element A mother liquor and the trace element B mother liquor in the A+B mixture is (1-2):(1-2). As an alternative embodiment, the volume ratio of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor in the premix in 1) is 1:1:1. As an alternative embodiment, the volume ratio of the trace element A mother liquor and the trace element B mother liquor in the A+B mixture in 2) is 1:1.

[0058] As an alternative embodiment, the supplementing in 1) is performed for ≥1 time, which can be 1-2 times. As an alternative embodiment, the supplementing in 2) is performed for ≥1 time, which can be 5-6 times. If the first mode of supplementing is adopted, the first mode of supplementing is adopted all the time.

[0059] As an alternative embodiment, when the supplementing in 1) is performed, the time interval between two adjacent supplementings is 46-50 h, which can be 48 h. The time interval for supplementing can provide the necessary nutrients for the growth of the strain, and avoid the negative impact of the instantaneous high concentration of nutrients in the culture medium on the growth of the strain. As an alternative embodiment, the volume of the premix supplemented each time in 1) is 0.2%-0.4% of the volume of the fermentation liquid, which can also be 0.3%. The volume of the fermentation liquid described in the application is the volume of the fermentation liquid before the mixed liquor is supplemented.

[0060] As an optional embodiment, when the supplement is carried out in the manner of 2), the time interval between two adjacent supplements is 22-26h, and can be 24h, and the setting of the time interval of the supplement can ensure the nutritional requirement of the strain in the growth process. As an optional embodiment, the volume ratio of the trace element A+B mixture and the trace element C mother liquor in each supplement in 2) is (1-2):(1-2), and can be 1:1. As an optional embodiment, the volume of the trace element A+B mixture in the present application is 0.08%-0.15% of the fermentation medium, and can also be 0.10%. As an optional embodiment, the volume of the trace element C mother liquor in the present application is 0.08%-0.15% of the fermentation medium, and can also be 0.10%. In the specific embodiments of the present application, 400mL of the trace element A+B mixture and 400mL of the trace element C mother liquor are supplemented for 5 times at 72h, 96h, 120h, 144h and 168h of the fermentation culture, respectively. If the supplement in the second manner is adopted, the supplement in the second manner is adopted all the time.

[0061] The trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor in the present application can promote the synthesis of PQQ in a large amount. For example, the supplement of zinc in the trace element A mother liquor can help to maintain the integrity of the microbial cell membrane, improve the tolerance of the microorganism to the environmental stress, and further ensure the stability of the fermentation process. The supplement of iron ions in the trace element C mother liquor can significantly improve the activity of the key enzyme related to the synthesis of PQQ, and promote the synthesis of PQQ. The present application supplements the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor to improve the activity of the PQQ synthesis enzyme, and promote the synthesis of PQQ in a large amount. At the same time, the dissolved oxygen and the stirring speed are stabilized to ensure the smooth progress of the fermentation process.

[0062] The compositions of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor in the present application have been discussed above, and will not be repeated here.

[0063] As an optional embodiment, when the concentration of methanol is less than 5g / L, the present application starts to add methanol in the whole fermentation culture stage, and the flow rate is adjusted according to the concentration of methanol in the fermentation broth, so that the concentration of methanol in the fermentation tank is controlled to be 2-10g / L. The present application does not have special limitations on the method and flow rate of the flow addition of methanol, and the conventional method can be adopted.

[0064] The present application provides the application of the above technical solution in improving the yield of pyrroloquinoline quinone.

[0065] The present application provides the application of the above technical solution in improving the purity of pyrroloquinoline quinone.

[0066] The present application provides the application of the above technical solution in reducing the content of the impurities corresponding to the RRT1.2 position of pyrroloquinoline quinone.

[0067] In existing technologies for preparing pyrroloquinoline quinone, the focus is mainly on the supply of major nutrients such as carbon and nitrogen sources, neglecting the crucial role of trace elements in the fermentation process. Due to the lack of a scientific strategy for supplementing trace elements, microorganisms cannot obtain an optimal growth and metabolic environment during fermentation, thus affecting the synthesis efficiency of PQQ and the amount of impurities generated, resulting in an impurity content of 2.5%–3.0% at the RRT1.2 position. The method of this invention produces pyrroloquinoline quinone with high yield, high purity, and low content at the RRT1.2 position.

[0068] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0069] The culture medium in the seed tank is referred to as seed culture medium, and the culture medium in the fermenter is referred to as fermentation culture medium.

[0070] Example 1

[0071] 1. Culture medium formulation

[0072] (1) The seed culture medium consists of: 16 g / L methanol, 6 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 10 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L magnesium sulfate heptahydrate, 2 g / L trace element A stock solution, 2 g / L trace element B stock solution and 2 g / L trace element C stock solution.

[0073] (2) The fermentation medium consists of: 10 g / L methanol, 6 g / L ammonium sulfate, 3 g / L potassium dihydrogen phosphate, 10 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L magnesium sulfate heptahydrate, 2 g / L trace element A mother liquor, 2 g / L trace element B mother liquor and 2 g / L trace element C mother liquor.

[0074] Feeding: Mother liquor of trace element A, mother liquor of trace element B, and mother liquor of trace element C.

[0075] The mother liquor for trace element A in the seed culture medium, fermentation culture medium, and feed consisted of: 15 g / L zinc sulfate heptahydrate, 2 g / L manganese sulfate tetrahydrate, and 0.2 g / L copper sulfate pentahydrate; the mother liquor for trace element B consisted of: 0.5 g / L sodium chloride, 0.1 g / L cobalt chloride hexahydrate, 40 g / L calcium chloride dihydrate, and 0.2 g / L potassium iodide; and the mother liquor for trace element C consisted of: 0.2 g / L ammonium molybdate tetrahydrate, 0.2 g / L boric acid, and 3 g / L ferrous sulfate.

[0076] 2. Preparation method

[0077] (1) Seed culture method: inoculate the PQQ-producing strain Hyphomicrobium denitrificans into a seed tank containing seed culture medium, and then cultivate at 30°C and 150 rpm until OD 600 ≥ 2.0, and the dissolved oxygen content of the seed culture is 30% to 70%. At the 42nd hour of seed tank cultivation, seed liquid is obtained.

[0078] (2) Fermenter cultivation

[0079] Transfer 40 L ± 5 L of seed liquid into a 500 L fermenter for fermentation cultivation. The volume of the fermentation medium is 280 to 300 L. The initial fermentation cultivation conditions are: temperature 30°C, initial pH of the fermentation medium 7.0, dissolved oxygen content 30%, aeration rate 1 vvm, and the stirring device is started with a rotation speed of 100 to 700 r / min to ensure that the bacterial cells and the medium are in full contact.

[0080] At the 30th to 50th hour of fermenter cultivation: when the biomass reaches a certain range (OD 600 value is 35 to 50), enter the fermentation process control stage. The fermentation temperature of this stage is 25 to 30°C, and the aeration rate and stirring speed are fine-tuned to keep the dissolved oxygen saturation at 20% to 40%, and the aeration flow rate is adjusted to 0.5 to 3 m 3 / h.

[0081] At the 72nd hour of fermentation cultivation, add 1 L of trace element mixture at one time, which accounts for 0.2% to 0.3% of the volume of the fermentation liquid. The mixture is composed of trace element A stock solution, trace element B stock solution, and trace element C stock solution, and the volume ratio of the three stock solutions in the mixture is 1:1:1.

[0082] Methanol feeding: when the methanol concentration is less than 5 g / L, start feeding methanol in the entire fermentation cultivation stage, and adjust the feeding rate according to the methanol concentration in the fermentation liquid to control the methanol concentration in the fermenter to be 2 to 10 g / L.

[0083] (3) Fermentation termination: when the fermentation is carried out to the 193rd hour, detect the PQQ content and impurities in the fermentation liquid. When the PQQ content increases by less than 10%, terminate the fermentation.

[0084] Example 2

[0085] 1. Medium formula

[0086] The seed culture medium comprises: methanol 16 g / L, ammonium sulfate 6 g / L, potassium dihydrogen phosphate 3 g / L, sodium phosphate dibasic dodecahydrate 10 g / L, magnesium sulfate heptahydrate 3 g / L, trace element A mother liquor 2 g / L, trace element B mother liquor 2 g / L and trace element C mother liquor 2 g / L;

[0087] The fermentation culture medium comprises: methanol 10 g / L, ammonium sulfate 6 g / L, potassium dihydrogen phosphate 3 g / L, sodium phosphate dibasic dodecahydrate 10 g / L, magnesium sulfate heptahydrate 3 g / L, trace element A mother liquor 2 g / L, trace element B mother liquor 2 g / L and trace element C mother liquor 2 g / L;

[0088] The trace element mother liquor comprises: zinc sulfate heptahydrate 15 g / L, manganese sulfate tetrahydrate 2 g / L and copper sulfate pentahydrate 0.2 g / L; sodium chloride 0.5 g / L, cobalt chloride hexahydrate 0.1 g / L, calcium chloride dihydrate 40 g / L and potassium iodide 0.2 g / L; ammonium molybdate tetrahydrate 0.2 g / L, boric acid 0.2 g / L and ferrous sulfate 3 g / L.

[0089] 2. Preparation method

[0090] (1) Seed culture method: the strain is inoculated into a seed tank containing the seed culture medium, and then cultured at 30°C and 150 rpm until OD 600 ≥2.0 is reached, and the seed tank is cultured for 43 h to obtain a seed liquid;

[0091] (2) Fermentor culture:

[0092] 1) 40L±5L seed liquid is transferred into a 500L fermentor for fermentation culture; the volume of the fermentation culture medium is 280-300L; the initial fermentation culture conditions are: temperature 30°C, initial pH of the fermentation culture medium 7.0, dissolved oxygen content 30%, and aeration rate 1vvm.

[0093] 2) When the biomass reaches a certain range (OD 600 value is 35-50), the fermentation process is controlled, the fermentation temperature is 30°C, the aeration rate and stirring speed are adjusted, the dissolved oxygen saturation is maintained at 20%-40%, and the aeration flow rate is adjusted to 0.5-3 m 3 / h.

[0094] At 72h and 120h of the fermentation culture, 1L of trace element mixture was added at one time, respectively, and the mixture was composed of trace element A mother liquor, trace element B mother liquor and trace element C mother liquor, and the volume ratio of trace element A mother liquor, trace element B mother liquor and trace element C mother liquor in the mixture was 1:1:1. The mixture accounted for about 0.2% to 0.3% of the volume of the fermentation liquid.

[0095] Methanol feeding: during the whole fermentation culture stage, when the methanol concentration was less than 5g / L, the methanol feeding was started. The feeding speed was adjusted according to the methanol concentration in the fermentation liquid, and the methanol concentration in the fermentation tank was controlled to be 2-10g / L,

[0096] (3) Fermentation termination: when the fermentation was carried out to 192h, the fermentation was terminated when the PQQ content increased by less than 10%.

[0097] Example 3

[0098] 1. Medium formula

[0099] (1) The composition of the seed culture medium was: methanol 16g / L, ammonium sulfate 6g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate dodecahydrate 10g / L, magnesium sulfate heptahydrate 3g / L, trace element A mother liquor 2g / L, trace element B mother liquor 2g / L, and trace element C mother liquor 2g / L.

[0100] (2) The composition of the fermentation medium was: methanol 10g / L, ammonium sulfate 6g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate dodecahydrate 10g / L, magnesium sulfate heptahydrate 3g / L, trace element A mother liquor 2g / L, trace element B mother liquor 2g / L, and trace element C mother liquor 2g / L.

[0101] Trace element mother liquor: the composition of trace element A mother liquor was: zinc sulfate heptahydrate 15g / L, manganese sulfate tetrahydrate 2g / L, and copper sulfate pentahydrate 0.2g / L; the composition of trace element B mother liquor was: sodium chloride 0.5g / L, cobalt chloride hexahydrate 0.1g / L, calcium chloride dihydrate 40g / L, and potassium iodide 0.2g / L; the composition of trace element C mother liquor was: ammonium molybdate tetrahydrate 0.2g / L, boric acid 0.2g / L, and ferrous sulfate 3g / L.

[0102] 2. Preparation method

[0103] (1) Seed culture method: the strain was inoculated into a seed tank containing seed culture medium, and then cultured at 30°C and 150rpm until OD 600 ≥2.0 at 43h, to obtain a seed liquid;

[0104] (2) Fermentor culture:

[0105] The 40L±5L seed liquid is transferred into a 500L fermenter for fermentation culture; the volume of the fermentation medium is 280-300L; the initial fermentation culture conditions are: temperature 30°C, initial pH of the fermentation medium 7.0, dissolved oxygen content 30%, and aeration rate 1vvm.

[0106] The fermentation culture is carried out for 30-50h: when the biomass reaches a certain range (OD 600 value is 35-50), the fermentation process is regulated, the fermentation temperature is 30°C, the aeration rate and stirring speed are finely adjusted, the dissolved oxygen saturation is kept at 20%-40%, and the aeration flow rate is adjusted to 0.5-3m 3 / h.

[0107] When the culture is carried out for 72h, 96h, 120h, 144h, and 168h, 400mL of trace element (A+B) mixed solution and 400mL of trace element C mother liquor are added for 5 times; the trace element (A+B) mixed solution is composed of trace element A mother liquor and trace element B mother liquor, and the volume ratio of the trace element A mother liquor to the trace element B mother liquor is 1:1.

[0108] Methanol feeding: during the whole fermentation culture stage, when the methanol concentration is less than 5g / L, the methanol feeding is started, and the feeding speed is adjusted according to the methanol concentration in the fermentation broth, so as to control the methanol concentration in the fermenter to be 2-10g / L.

[0109] (3) Fermentation termination: when the fermentation is carried out for 192h, the PQQ content and impurities in the fermentation broth are detected, and when the PQQ content increase is less than 10%, the fermentation is terminated.

[0110] Example 4

[0111] 1. Medium formula

[0112] (1) The composition of the seed culture medium is: methanol 16g / L, ammonium sulfate 6g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate dodecahydrate 10g / L, magnesium sulfate heptahydrate 3g / L, trace element A mother liquor 2g / L, trace element B mother liquor 2g / L, and trace element C mother liquor 2g / L.

[0113] (2) The composition of the fermentation medium is: methanol 10g / L, ammonium sulfate 6g / L, potassium dihydrogen phosphate 3g / L, disodium hydrogen phosphate dodecahydrate 10g / L, magnesium sulfate heptahydrate 3g / L, trace element A mother liquor 2g / L, trace element B mother liquor 2g / L, and trace element C mother liquor 2g / L;

[0114] Trace element mother liquor: the composition of trace element A mother liquor is: zinc sulfate heptahydrate 15 g / L, manganese sulfate tetrahydrate 2 g / L and copper sulfate pentahydrate 0.2 g / L; the composition of trace element B mother liquor is: sodium chloride 0.5 g / L, cobalt chloride hexahydrate 0.1 g / L, calcium chloride dihydrate 40 g / L and potassium iodide 0.2 g / L; the composition of trace element C mother liquor is: ammonium molybdate tetrahydrate 0.2 g / L, boric acid 0.2 g / L and ferrous sulfate 3 g / L.

[0115] 2. Preparation method

[0116] (1) Seed culture method: the strain is inoculated into a seed tank containing seed culture medium, and then cultured at 30°C and 150 rpm until OD 600 ≥ 2.0 is reached, and the seed tank is cultured for 39 h to obtain a seed liquid.

[0117] (2) Fermenter culture

[0118] 40 L ± 5 L seed liquid is transferred into a fermenter with a volume of 500 L; the volume of the fermentation medium is 280-300 L; the initial conditions for transfer are: temperature 30°C, initial pH of the fermentation medium 7.0, dissolved oxygen content 30%, aeration rate 1 vvm,

[0119] Fermentation culture for 30-50 h: when the biomass reaches a certain range (OD 600 value 35-50), the fermentation process is controlled, the fermentation temperature is 30°C, the aeration rate and stirring speed are finely adjusted, the saturation of dissolved oxygen is maintained at 20%-40%, and the aeration flow rate is adjusted to 0.5-3 m 3 / h.

[0120] At 72 h, 96 h, 120 h, 144 h and 168 h of fermentation culture, 400 mL of trace element A+B mixed solution and 400 mL of trace element C mother liquor are supplemented in five times; the trace element A+B mixed solution is composed of trace element A mother liquor and trace element B mother liquor, and the volume ratio of trace element A mother liquor to trace element B mother liquor is 1:1.

[0121] Methanol feeding: during the entire fermentation culture stage, when the methanol concentration is less than 5 g / L, methanol feeding is started, and the methanol concentration in the fermenter is controlled to be 2-10 g / L.

[0122] (3) Fermentation termination: when the fermentation is carried out to 172 h, the PQQ content and impurities in the fermentation broth are detected, and when the increase amplitude of the PQQ content is less than 10%, the fermentation is terminated.

[0123] Test Example 1

[0124] After the fermentation of Examples 1-4, the PQQ content and the impurity content at RRT 1.2 in the fermentation broth obtained in Examples 1-4 were accurately determined by using high performance liquid chromatography (HPLC) (see 2025 edition of the Pharmacopoeia of the People's Republic of China, General Rules 0512) and other analytical means, and the results are shown in Table 1 and Table 2. Figures 1-8 and Table 1.

[0125] Table 1: Test results of each example

[0126]

[0127]

[0128] In summary, the method provided by the present application has high PQQ yield, high purity and low impurity content of RRT 1.2.

[0129] Although the above examples have made a detailed description of the present application, it is only a part of the examples of the present application, not all the examples, and other examples can be obtained according to the present examples without creativity, which all belong to the protection scope of the present application.

Claims

1. A method of preparing pyrroloquinoline quinone, characterized by, The method comprises the following steps: pyrroloquinoline quinone production strain is inoculated into seed culture medium for seed culture to obtain seed liquid; the seed liquid is inoculated into fermentation culture medium to obtain fermentation system, and fermentation culture is carried out to obtain fermentation liquid; trace element A mother liquor, trace element B mother liquor and trace element C mother liquor are supplemented in the 70th to 170th hour of the fermentation culture; the seed culture medium comprises: methanol 5-20 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, sodium phosphate dibasic dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.2-5 g / L, trace element B mother liquor 0.2-5 g / L and trace element C mother liquor 0.2-5 g / L; the fermentation culture medium comprises: methanol 2-10 g / L, ammonium sulfate 1-10 g / L, potassium dihydrogen phosphate 0.5-5 g / L, sodium phosphate dibasic dodecahydrate 5-15 g / L, magnesium sulfate heptahydrate 0.2-5 g / L, trace element A mother liquor 0.5-5 g / L, trace element B mother liquor 0.5-5 g / L and trace element C mother liquor 0.5-5 g / L; the trace element A mother liquor comprises: zinc sulfate heptahydrate 15-30 g / L, manganese sulfate tetrahydrate 2-10 g / L and copper sulfate pentahydrate 0.2-1.5 g / L; the trace element B mother liquor comprises: sodium chloride 0.5-5 g / L, cobalt chloride hexahydrate 0.01-0.2 g / L, calcium chloride dihydrate 10-50 g / L and potassium iodide 0.01-0.2 g / L; the trace element C mother liquor comprises: ammonium molybdate tetrahydrate 0.01-0.2 g / L, boric acid 0.01-0.2 g / L and ferrous sulfate 2.5-10 g / L.

2. The method of claim 1, wherein, The temperature of the seed culture is 23-35 DEG C, and the time is 40-45 h; the initial pH of the seed culture medium is 6.0-7.0, and the dissolved oxygen content is 30%-70%.

3. The method of claim 1, wherein, The temperature of the fermentation culture is 23-35 DEG C; the initial pH of the fermentation culture medium is 6.0-7.0, and the dissolved oxygen content is 20%-40%.

4. The method according to claim 1 or 3, characterized in that, The time of the fermentation culture is 170-195 h.

5. The method of claim 1, wherein, During the whole fermentation culture process, when the concentration of methanol in the fermentation system is less than 5 g / L, methanol is added to control the concentration of methanol in the fermentation system to be 2-10 g / L.

6. The method of claim 1, wherein, The supplementing mode of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor is as follows: 1) the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor are mixed to obtain a premix, and the premix is supplemented at one time; the volume ratio of the trace element A mother liquor, the trace element B mother liquor and the trace element C mother liquor in the premix is (1-2):(1-2):(1-2); 2) the trace element A mother liquor and the trace element B mother liquor are mixed to obtain A+B mixed liquid, and the A+B mixed liquid and the trace element C mother liquor are supplemented at one time; the volume ratio of the trace element A mother liquor and the trace element B mother liquor in the A+B mixed liquid is (1-2):(1-2); the volume ratio of the A+B mixed liquid and the trace element C mother liquor is (1-2):(1-2).

7. The method of claim 1, wherein, In the 2), the volume of the trace element A+B mixture is 0.08% to 0.15% of the fermentation medium; the volume of the trace element C mother liquor is 0.08% to 0.15% of the fermentation medium. In the 1), the volume of the premix solution is 0.2% to 0.4% of the fermentation medium.

8. The method according to claim 6 or 7, characterized in that, The number of replenishments of the 1) or 2) is ≥1 time, respectively.

9. The method of claim 8, wherein, The time interval between adjacent two replenishments is 22 to 26 hours.

10. Use of the method of any one of claims 1 to 9 in at least one of the following: 1) increasing the yield of pyrroloquinoline quinone; 2) increasing the purity of pyrroloquinoline quinone; 3) reducing the content of impurities corresponding to the RRT 1.2 position of pyrroloquinoline quinone.

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