Production method of D-pantothenic acid

Abstract

The invention provides a production method of D-pantothenic acid. The production method of the D-pantothenic acid comprises the following steps: continuously fermenting an escherichia coli strain in a culture medium to produce the D-pantothenic acid, wherein the concentration of the escherichia coli strain is 40-50g / L; the pH value of the continuous fermentation is 6.5-7.0. According to the production method of the D-pantothenic acid, the production efficiency of the D-pantothenic acid is greatly improved, and the application prospect is good.

Description

Technical Field

[0001] The invention relates to a method for producing D-pantothenic acid, and specifically to a method for realizing continuous fermentation production of D-pantothenic acid by intercepting bacterial cells through membrane filtration and repeatedly using the bacterial cells for subsequent batch fermentation of D-pantothenic acid, belonging to the technical field of biological fermentation. Background Art

[0002] D-pantothenic acid, also known as vitamin B5, is an important water-soluble vitamin widely used in pharmaceutical, food, cosmetic, feed and other industries. At present, the preparation methods of D-pantothenic acid are mainly divided into two methods: chemical enzyme synthesis and biological fermentation. The preparation process of chemical enzyme synthesis is relatively complicated and has many by-products. Therefore, the production of D-pantothenic acid by microbial fermentation using green renewable resources as raw materials has received more and more attention. The microbial fermentation method is to sterilize the culture medium containing carbon source, nitrogen source, inorganic salt, etc., and then inoculate the bacteria for culture, so that the bacteria can metabolize autonomously to produce D-pantothenic acid. This method has mild conditions, is environmentally friendly, and has few by-products.

[0003] At present, the fermentation of D-pantothenic acid mainly adopts single tank batch fermentation, that is, each batch fermentation process needs to go through inoculation, growth and reproduction, bacterial aging and then end the fermentation. The batch fermentation process reduces the utilization rate of the bacteria, thereby affecting the production efficiency and conversion rate of the fermentation. Compared with batch fermentation, continuous fermentation can keep the fermentation process in the optimal state for a long time.

[0004] Therefore, it is of great significance to develop a method that can recycle bacteria and realize the continuous fermentation production of D-pantothenic acid, thereby improving the efficiency and conversion rate of the D-pantothenic acid fermentation process. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of low production efficiency or low conversion rate of D-pantothenic acid in the prior art, and provide a method for producing D-pantothenic acid. In the method for producing D-pantothenic acid of the present invention, the production efficiency of D-pantothenic acid is greatly improved, and the C8 homologues in D-pantothenic acid are further reduced.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] The present invention provides a method for producing D-pantothenic acid, which comprises the following steps: continuously fermenting an Escherichia coli strain in a culture medium to produce D-pantothenic acid;

[0008] Wherein, the concentration of the Escherichia coli strain is 40-50g / L;

[0009] The pH of the continuous fermentation is 6.5-7.0.

[0010] In the present invention, preferably, the starting strain of Escherichia coli is K12 strain of Escherichia coli.

[0011] Preferably, the K12 Escherichia coli is Escherichia coli W3110.

[0012] More preferably, the Escherichia coli W3110 strain is a PB17 strain, and the PB17 strain is prepared according to the method in “Preparation Example 1” in the patent document with application number 2024104554005.

[0013] In the present invention, preferably, the culture medium is fed during the continuous fermentation process.

[0014] Preferably, the flow rate of the fed culture medium is 0.4-1.0 L / h.

[0015] Preferably, while the culture medium is fed, the fermentation liquid and the clear liquid containing D-pantothenic acid flow out.

[0016] More preferably, the outflow rate of the D-pantothenic acid-containing clear liquid is 0.2-0.8 L / h.

[0017] More preferably, the outflow rate of the fermentation broth is 0.2-0.4 L / h.

[0018] In the present invention, preferably, the components and contents of the culture medium are, by mass percentage, 0.1-1% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water;

[0019] For example, in terms of mass percentage, the components and contents of the culture medium are:

[0020] Glucose 1%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, the balance is water;

[0021] Or, glucose 0.5%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, and the balance is water;

[0022] Or, glucose 0.8%, peptone 0.5%, yeast extract powder 1%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.007%, anhydrous ferric chloride 0.008%, the balance is water;

[0023] Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, and the balance is water.

[0024] In the present invention, preferably, the culture conditions for the continuous fermentation are selected from one or more of the following:

[0025] a. The continuous fermentation temperature is 35-37 ° C, for example, 35 ° C, 36 ° C or 37 ° C;

[0026] b. The pH of the continuous fermentation is 6.5-7.0, for example 6.8 or 7.0; the pH is generally adjusted by aqueous ammonia;

[0027] c. The continuous fermentation ventilation ratio is 0.8-1.2VVM, for example 1.0VVM;

[0028] d. The DO of the continuous fermentation is 30%-40%, for example 30%.

[0029] In the present invention, preferably, the cycle of the continuous fermentation culture is 7-20 days, such as 167h, 236h, 261h or 270h.

[0030] In the present invention, preferably, the continuous fermentation comprises the following steps:

[0031] The concentrated bacterial liquid of Escherichia coli is inoculated into the culture medium for fermentation; after fermentation for 7-20 days, the fermentation liquid is filtered through a membrane to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid, wherein the outflow rate of the clear liquid containing D-pantothenic acid is 0.2-0.8 L / h, the outflow rate of the fermentation liquid is 0.2-0.4 L / h, and the concentrated bacterial liquid is fed to the next fermentation process; while feeding the concentrated bacteria, the culture medium and ammonia water are fed, and the flow rate of the fed culture medium is 0.4-1.0 L / h.

[0032] In the present invention, preferably, the continuous fermentation is to continuously ferment the concentrated bacterial solution containing Escherichia coli in a culture medium; the production method of the concentrated bacterial solution containing Escherichia coli comprises the following steps:

[0033] S1, Escherichia coli strains are cultured to obtain seed tank seeds;

[0034] S2, inoculating the seeds obtained in step S1 into the first fermentation tank for fermentation and culture to obtain a bacterial fermentation liquid;

[0035] S3, filtering the bacterial fermentation liquid obtained in step S2 through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid;

[0036] S4, inoculating the concentrated bacterial liquid obtained in step S3 into a second fermentation tank for fermentation culture to obtain a bacterial fermentation liquid; filtering the bacterial fermentation liquid through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid.

[0037] The conditions and operations of the above steps S1, S2, S3 and S4 are conventional conditions and operations in the art, and the following conditions and operations are particularly preferred in the art:

[0038] In step S1, preferably, the culture includes shake flask culture and seed tank culture in sequence.

[0039] Preferably, the container for shake flask culture is a shake flask.

[0040] Preferably, the culture medium for the shake flask culture is LB culture medium, and the LB culture medium is a conventional LB culture medium in the art.

[0041] Preferably, the culture temperature of the shake flask culture is 30-35°C, such as 30°C.

[0042] Preferably, the rotation speed of the shaker during the shake flask culture is 200-250 rpm, such as 220 rpm.

[0043] Preferably, the shaking flask culture time is 3-5 hours, such as 5 hours.

[0044] Wherein, the shake flask culture obtains shake flask seeds. Preferably, 5-10% of the shake flask seeds are used for the seed pot culture, for example, 10% of the shake flask seeds are used for the seed pot culture.

[0045] Preferably, the container for the seed tank culture is a seed tank.

[0046] Wherein, the medium for seed tank culture is a seed tank medium, preferably, in terms of mass percentage, the components and contents of the seed tank medium are: 1-3% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water;

[0047] For example, in terms of mass percentage, the components and contents of the seed tank culture medium are:

[0048] Glucose 1%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water;

[0049] Or, glucose 2%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water;

[0050] Or, glucose 3%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water;

[0051] Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, and the balance is water.

[0052] The pH of the medium for the seed tank culture before sterilization is, for example, 7.0. The pH is generally adjusted with ammonia water.

[0053] The culture medium for the seed tank culture is generally sterilized at 121° C. for 30 minutes before use.

[0054] Preferably, the temperature of the seed tank culture is 35-37°C, such as 35°C or 37°C.

[0055] Preferably, the pH of the seed tank culture is 6.5-7.0, for example, 6.6, 6.8 or 7.0. The pH is generally adjusted by ammonia water.

[0056] Preferably, the ventilation ratio of the seed tank culture is 0.8-1.2VVM, for example, 1.0VVM.

[0057] Preferably, the DO of the seed tank culture is between 30% and 40%, for example, 30%.

[0058] Preferably, the seed tank culture time is 8-10 h, such as 8.2 h, 8.5 h, 8.8 h or 9.0 h.

[0059] In step S2, preferably, the inoculation ratio of the seeds obtained in the seed tank in step S1 to the first fermentation tank is 5-10%, for example, 10%.

[0060] In step S2, the medium for fermentation culture is a fermentation medium. Preferably, the components and contents of the fermentation medium are as follows, by mass percentage: 0.1-1% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water;

[0061] For example, in terms of mass percentage, the components and contents of the fermentation medium are:

[0062] Glucose 1%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, the balance is water;

[0063] Or, glucose 0.5%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, and the balance is water;

[0064] Or, glucose 0.8%, peptone 0.5%, yeast extract powder 1%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.007%, anhydrous ferric chloride 0.008%, the balance is water;

[0065] Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, and the balance is water.

[0066] Wherein, when the substrate sugar in the fermentation medium is exhausted and the DO rebounds to 70%-90%, preferably, feed medium is added to the first fermentation tank.

[0067] More preferably, the components and contents of the feed medium are, by mass percentage, 50-60% glucose, 0.5-1.0% yeast extract or peptone, and the balance is water; for example, by mass percentage, the components and contents of the feed medium are:

[0068] Glucose 58%, yeast extract powder 0.5%, the rest is water;

[0069] Or, glucose 50%, yeast extract powder 0.5%, the balance is water;

[0070] Or, glucose 50%, peptone 1%, the balance is water;

[0071] Or, glucose 55%, peptone 0.5%, and the balance water.

[0072] More preferably, the feed medium is sterilized at 121° C. for 30 min before use.

[0073] In step S2, preferably, the pH of the fermentation culture medium before sterilization is 6.5-7.0, for example 7.0. The pH is generally adjusted by ammonia water.

[0074] In step S2, the culture medium for fermentation culture is generally sterilized at 121° C. for 30 minutes before use.

[0075] In step S2, preferably, the fermentation culture temperature is 35-37°C, for example, 35°C, 36°C or 37°C.

[0076] In step S2, preferably, the pH of the fermentation culture is 6.5-7.0, for example, 6.8 or 7.0. The pH is generally adjusted by ammonia water.

[0077] In step S2, preferably, the ventilation ratio of the fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM.

[0078] In step S2, preferably, the DO of the fermentation culture is 30%-40%, for example 30%.

[0079] In step S2, preferably, the fermentation culture cycle is 50-70 h, more preferably 60-65 h, such as 61.6 h, 61.9 h or 62 h.

[0080] In step S3, preferably, the filter membrane of the membrane filtration device is a ceramic membrane, and more preferably, the pore size of the ceramic membrane is 50-200 nm, for example, 100 nm, 150 nm or 200 nm.

[0081] In step S3, preferably, the filtration is performed under closed and aseptic conditions.

[0082] In step S3, preferably, the volume of the concentrated bacterial liquid is 15-30% of the volume of the bacterial fermentation liquid obtained in step S2.

[0083] In step S4, preferably, the inoculation ratio is 40%-100% of the volume of the concentrated bacterial solution obtained in step S3.

[0084] In step S4, preferably, the number of the second fermentation tanks is 1 or 2.

[0085] In step S4, preferably, the second fermentation tank is filled with fermentation medium, and the fermentation medium is the same as the fermentation medium described above.

[0086] In step S4, preferably, the fermentation culture temperature is 35-37°C, for example, 35°C, 36°C or 37°C.

[0087] In step S4, preferably, the pH of the fermentation culture is 6.5-7.0, for example, 6.8 or 7.0. The pH is generally adjusted by ammonia water.

[0088] In step S4, preferably, the ventilation ratio of the fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM.

[0089] In step S4, preferably, the DO of the fermentation culture is 30%-40%, for example 30%.

[0090] In step S4, preferably, the filter membrane of the membrane filtration device is a ceramic membrane, and more preferably, the pore size of the ceramic membrane is 50-200 nm, such as 50 nm or 100 nm.

[0091] In step S4, preferably, the fermentation culture time is 25-35 hours, such as 30 hours.

[0092] In the present invention, preferably, the continuous fermentation comprises the following steps:

[0093] S1, using the method of the above step S1;

[0094] S2, using the method of the above step S2;

[0095] S3, adopt the method of the above step S3;

[0096] S4, adopt the method of the above step S4;

[0097] S5, returning the concentrated bacterial liquid obtained in step S4 to the second fermentation tank in step S4 for continuous fermentation culture;

[0098] During the continuous fermentation culture, part of the fermentation liquid containing bacteria in the second fermentation tank is kept flowing out to the membrane filtration device described in step S4, and a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid are obtained after membrane filtration;

[0099] The clear liquid containing D-pantothenic acid flows out of the second fermentation tank, and the concentrated bacterial liquid flows back to the second fermentation tank again for continuous fermentation culture, and the cycle is repeated in sequence; at the same time, fresh culture medium and ammonia water are kept added to the second fermentation tank; at the same time, the second fermentation tank keeps flowing out the bacterial fermentation liquid, so as to maintain the concentration of Escherichia coli in the second fermentation tank at 40-50 g / L.

[0100] In step S5, preferably, the temperature of the continuous fermentation culture is 35-37°C, for example, 35°C, 36°C or 37°C.

[0101] In step S5, preferably, the pH of the continuous fermentation culture is 6.5-7.0, for example, 6.8 or 7.0. The pH is generally adjusted by ammonia water.

[0102] In step S5, preferably, the ventilation ratio of the continuous fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM.

[0103] In step S5, preferably, the DO of the continuous fermentation culture is 30%-40%, for example 30%.

[0104] In step S5, preferably, the outflow rate of the clear liquid containing D-pantothenic acid is 0.2-0.8 L / h, such as 0.3 L / h, 0.5 L / h or 0.7 L / h.

[0105] In step S5, preferably, the components and contents of the fresh culture medium are, by mass percentage, 30-50% glucose, 0.1-0.3% yeast extract, 0.05-0.2% potassium dihydrogen phosphate, 0.2-0.5% ammonium chloride, 0.02-0.05% magnesium sulfate heptahydrate, and the remainder is water; for example, by mass percentage, the components and contents of the fresh culture medium are:

[0106] Glucose 48%, yeast extract powder 0.1%, potassium dihydrogen phosphate 0.05%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.02%, the balance is water;

[0107] Or, glucose 37%, yeast extract powder 0.15%, potassium dihydrogen phosphate 0.1%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.02%, and the balance is water;

[0108] Or, glucose 42%, yeast extract powder 0.2%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.05%, and the balance is water;

[0109] Or, glucose 31%, yeast extract powder 0.2%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.02%, and the balance is water.

[0110] In step S5, preferably, the fresh culture medium is sterilized at 121° C. for 30 min before use.

[0111] In step S5, preferably, the flow rate of the fresh culture medium is 0.4-1.0 L / h, such as 0.5 L / h, 0.7 L / h, 0.9 L / h or 1.0 L / h.

[0112] In step S5, preferably, the outflow rate of the bacteria-containing fermentation liquid is 0.2-0.4 L / h, such as 0.2 L / h or 0.3 L / h.

[0113] In step S5, preferably, the flow rate of the fresh culture medium is equal to the sum of the outflow rate of the clear liquid containing D-pantothenic acid and the outflow rate of the fermentation liquid containing bacteria.

[0114] In the present invention, the clear liquid containing D-pantothenic acid is used for extraction and purification to obtain D-pantothenic acid.

[0115] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0116] The reagents and raw materials used in the present invention are commercially available.

[0117] The positive improvement effect of the present invention is that the advantages of the continuous fermentation of the present invention are one or more of the following:

[0118] (1) The present invention inoculates the bacteria that still have high vitality in the late stage of batch fermentation into fresh fermentation medium to continue fermentation, thereby reducing the bacterial culture process;

[0119] (2) The continuous addition of fresh culture medium in the present invention reduces the accumulation of harmful metabolites, reduces product feedback inhibition, improves process conversion rate, and reduces production costs;

[0120] (3) The present invention can achieve long-term continuous and stable fermentation production of D-pantothenic acid, thereby greatly improving the fermentation production efficiency of D-pantothenic acid;

[0121] (4) The present invention reduces the content of C8 homologues. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 The chromatogram of D-pantothenic acid and C8 homologues in Example 1.

[0123] Figure 2 The chromatogram of D-pantothenic acid and C8 homologues of Comparative Example 1. DETAILED DESCRIPTION

[0124] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0125] The LB medium, seed tank medium, fermentation medium, feed medium and fresh medium used in the following examples and comparative examples were sterilized at 121°C for 30 min before use. The pH of the seed tank medium and fermentation medium was adjusted to 7.0 with ammonia water before sterilization.

[0126] The filtration and concentration involved in the following examples and comparative examples were all carried out under closed and aseptic conditions.

[0127] The Escherichia coli in the following example is K12 strain Escherichia coli W3110, specifically PB17 strain, and the PB17 strain is prepared according to the method in “Preparation Example 1” in the patent document with application number 2024104554005.

[0128] Embodiment 1:

[0129] a. Inoculate D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, culture it in a shaker at 30℃ and 220rpm for 5h, and then inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 1% glucose, 1% peptone, 0.5% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 35℃, pH controlled at 6.6 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0130] b After 9 hours of cultivation in the seed tank, the fermenter containing 21L fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 1% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions are temperature 35℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 58% glucose, 0.5% yeast extract powder, and the balance is water. The fermentation cycle is 62h, and 37L of fermentation liquid containing D-pantothenic acid is obtained.

[0131] c The fermentation broth was filtered and concentrated using a ceramic membrane with a pore size of 100 nm. The clear liquid containing D-pantothenic acid obtained by filtration entered the extraction process. When the volume of the concentrated bacterial liquid reached 6 L, the filtration was stopped. The concentrated bacterial liquid was pumped into another fermentation tank containing 19 L of fermentation medium to continue repeated batch fermentation under unchanged fermentation conditions.

[0132] d. The second fermentation tank is coupled with a membrane filtration device. When the batch fermentation is repeated for 30 hours, the membrane device is turned on to start continuous fermentation. The fermentation broth is filtered and concentrated using a ceramic membrane with a pore size of 100 nm. The outflow rate of the filtered clear liquid containing D-pantothenic acid is 0.3 L / h. The obtained clear liquid containing D-pantothenic acid enters the extraction process, and the concentrated bacterial liquid is returned to the fermentation tank for continued fermentation. At the same time, fresh culture medium and ammonia water (control pH to 6.8) are added to the fermentation tank. In terms of mass percentage, the components and contents of the fresh culture medium are: 48% glucose, 0.1% yeast extract powder, 0.05% potassium dihydrogen phosphate, 0.2% ammonium chloride, 0.02% magnesium sulfate heptahydrate, and the balance is water. The flow rate of the fresh culture medium is 0.5 L / h. In addition, the discharge rate of the bacterial fermentation broth is 0.2 L / h to maintain the concentration of Escherichia coli in the tank at 40-50 g / L. The continuous fermentation cycle is 167 hours, and the total mass of D-pantothenic acid obtained is 11.82 kg.

[0133] Embodiment 2:

[0134] a. Inoculate D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, culture it in a shaker at 30℃ and 220rpm for 4.9h, and then inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 2% glucose, 1% peptone, 0.5% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 35℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0135] b After 8.8h of cultivation in the seed tank, the fermenter containing 21L fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 0.5% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions in the fermenter are temperature 37℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 50% glucose, 0.5% yeast extract powder, and the balance is water. The fermentation cycle is 61.9h, and 38.5L of fermentation broth containing D-pantothenic acid is obtained.

[0136] c The fermentation liquid was filtered and concentrated using a ceramic membrane with a pore size of 100 nm. The clear liquid containing D-pantothenic acid obtained by filtration entered the extraction process. When the volume of the concentrated bacterial liquid reached 8 L, the filtration was stopped. The concentrated bacterial liquid was pumped into another fermentation tank containing 17 L of fermentation medium to continue repeated batch fermentation under unchanged fermentation conditions.

[0137] d. The second fermentation tank is coupled with a membrane filtration device. When the batch fermentation is repeated for 30 hours, the membrane device is turned on to start continuous fermentation. The fermentation broth is filtered and concentrated using a ceramic membrane with a pore size of 50 nm. The outflow rate of the filtered clear liquid containing D-pantothenic acid is 0.7 L / h. The obtained clear liquid containing D-pantothenic acid enters the extraction process, and the concentrated bacterial liquid is returned to the fermentation tank for continued fermentation. At the same time, fresh culture medium and ammonia water (control pH to 6.6) are added to the fermentation tank. In terms of mass percentage, the components and contents of the fresh culture medium are: 37% glucose, 0.15% yeast extract powder, 0.1% potassium dihydrogen phosphate, 0.2% ammonium chloride, 0.02% magnesium sulfate heptahydrate, and the balance is water. The flow rate of the fresh culture medium is 0.9 L / h. In addition, the discharge rate of the bacterial fermentation broth is 0.2 L / h to maintain the concentration of Escherichia coli in the tank at 40-50 g / L. The continuous fermentation cycle is 236 hours, and the total mass of D-pantothenic acid obtained is 19.445 kg.

[0138] Embodiment 3:

[0139] a. Inoculate the D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, and culture it in a shaker at 30°C and 220rpm for 4.5h. Then, inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 3% glucose, 1% peptone, 0.5% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 37°C, pH controlled at 7.0 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0140] b After 8.2h of cultivation in the seed tank, the fermenter containing 22.5L of fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 0.8% glucose, 0.5% peptone, 1% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.007% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions in the fermenter are temperature 37℃, pH controlled at 7.0 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 50% glucose, 1% peptone, and the balance is water. The fermentation cycle is 61.6h, and 40L of fermentation liquid containing D-pantothenic acid is obtained.

[0141] c The fermentation broth was filtered and concentrated using a ceramic membrane with a pore size of 200 nm. The clear liquid containing D-pantothenic acid obtained by filtration entered the extraction process. When the volume of the concentrated bacterial liquid reached 12 L, the filtration was stopped. The concentrated bacterial liquid was pumped into two other fermentation tanks containing 19 L of fermentation medium to continue repeated batch fermentation, 6 L / tank, and the fermentation conditions remained unchanged.

[0142] d. The second fermentation tank is coupled with a membrane filtration device. When the batch fermentation is repeated for 30 hours, the membrane device is turned on to start continuous fermentation. The fermentation broth is filtered and concentrated using a ceramic membrane with a pore size of 100 nm. The outflow rate of the filtered clear liquid containing D-pantothenic acid is 0.5 L / h. The obtained clear liquid containing D-pantothenic acid enters the extraction process, and the concentrated bacterial liquid is returned to the fermentation tank for continued fermentation. At the same time, fresh culture medium and ammonia water (control pH to 7) are added to the fermentation tank. In terms of mass percentage, the components and contents of the fresh culture medium are: 42% glucose, 0.2% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.2% ammonium chloride, 0.05% magnesium sulfate heptahydrate, and the balance is water. The flow rate of the fresh culture medium is 0.7 L / h. In addition, the discharge rate of the bacterial fermentation broth is 0.2 L / h to maintain the concentration of Escherichia coli in the tank at 40-50 g / L. The continuous fermentation cycles of the two tanks were 270 h and 362 h, respectively, and the total mass of D-pantothenic acid obtained was 37.17 kg and 55.31 kg, respectively.

[0143] Embodiment 4:

[0144] a. Inoculate D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, culture it in a shaker at 30℃ and 220rpm for 5h, and then inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 2.5% glucose, 0.8% peptone, 0.8% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 37℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0145] b After 8.5h of cultivation in the seed tank, the fermenter containing 22.5L of fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 0.8% glucose, 0.8% peptone, 1% yeast extract, 0.4% potassium dihydrogen phosphate, 0.7% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.008% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions in the fermenter are temperature 36℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 55% glucose, 0.5% peptone, and the balance is water. The fermentation cycle is 62h, and 38.6L of fermentation liquid containing D-pantothenic acid is obtained.

[0146] c The fermentation liquid was filtered and concentrated using a ceramic membrane with a pore size of 150 nm. The clear liquid containing D-pantothenic acid obtained by filtration entered the extraction process. When the volume of the concentrated bacterial liquid reached 7 L, the filtration was stopped. The concentrated bacterial liquid was pumped into another fermentation tank containing 18 L of fermentation medium to continue repeated batch fermentation under unchanged fermentation conditions.

[0147] d. The second fermentation tank is coupled with a membrane filtration device. When the batch fermentation is repeated for 30 hours, the membrane device is turned on to start continuous fermentation. The fermentation broth is filtered and concentrated using a ceramic membrane with a pore size of 50 nm. The outflow rate of the filtered clear liquid containing D-pantothenic acid is 0.7 L / h. The obtained clear liquid containing D-pantothenic acid enters the extraction process, and the concentrated bacterial liquid is returned to the fermentation tank for continued fermentation. At the same time, fresh culture medium and ammonia water (control pH to 6.8) are added to the fermentation tank. In terms of mass percentage, the components and contents of the fresh culture medium are: 31% glucose, 0.2% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.02% magnesium sulfate heptahydrate, and the balance is water. The flow rate of the fresh culture medium is 1.0 L / h. In addition, the discharge rate of the bacterial fermentation broth is 0.3 L / h to maintain the concentration of Escherichia coli in the tank at 40-50 g / L. The continuous fermentation cycle is 261 hours, and the total mass of D-pantothenic acid obtained is 22.989 kg.

[0148] Comparative Example 1:

[0149] The difference from Example 1 is that this comparative example does not carry out continuous fermentation but single batch fermentation.

[0150] a. Inoculate D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, culture it in a shaker at 30℃ and 220rpm for 4.6h, and then inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 1% glucose, 1% peptone, 0.5% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 35℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0151] b After 8.5h of cultivation in the seed tank, the fermenter containing 20L fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 0.5% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions in the fermenter are temperature 36℃, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 55% glucose, 0.5% peptone, and the balance is water. The fermentation was completed after 61.6 h, and a total of 15.3 L of feed medium was added to obtain 36.8 kg of fermentation liquid containing D-pantothenic acid, and all the fermentation liquid entered the extraction process.

[0152] Comparative Example 2:

[0153] The difference from Example 1 is that this comparative example does not carry out continuous fermentation.

[0154] a. Inoculate D-pantothenic acid-producing Escherichia coli into a shake flask containing 1L LB medium, culture it in a shaker at 30°C and 220rpm for 4.9h, and then inoculate it into a seed tank containing 9L seed tank medium at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the seed tank medium are: 0.5% glucose, 1% peptone, 0.5% yeast extract powder, 0.5% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.05% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.005% anhydrous ferric chloride, and the balance is water. The seed tank culture conditions are temperature 37°C, pH controlled at 6.8 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed.

[0155] b After 8.6h of cultivation in the seed tank, the fermenter containing 20L fermentation medium was inoculated at a 10% inoculation ratio. In terms of mass percentage, the components and contents of the fermentation medium are: 1% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.2% potassium dihydrogen phosphate, 0.5% ammonium chloride, 0.07% magnesium sulfate heptahydrate, 0.005% manganese chloride tetrahydrate, 0.008% anhydrous ferric chloride, and the balance is water; the pH of the fermentation medium before sterilization is 7.0. The fermentation culture conditions in the fermenter are temperature 35℃, pH controlled at 6.9 with ammonia water, ventilation ratio 1.0VVM, and DO controlled at 30% by speed. When the substrate sugar in the fermentation medium is exhausted and DO rebounds to 70%-90%, feeding begins. In terms of mass percentage, the components and contents of the feed medium are: 50% glucose, 1% yeast extract powder, and the balance is water. The fermentation was completed after 61.4 h, and a total of 16.6 L of feed medium was added to obtain 36.4 kg of fermentation liquid containing D-pantothenic acid, and all the fermentation liquid entered the extraction process.

[0156] Effect Example 1

[0157] The number of grams (g / kg) of D-pantothenic acid contained in each kilogram of bacterial fermentation broth, the conversion rate of D-pantothenic acid to glucose, the content of C8 homologues in D-pantothenic acid, and the total productivity of D-pantothenic acid in Examples 1-4 and Comparative Examples 1-2.

[0158] 1. Number of grams of D-pantothenic acid in each kilogram of bacterial fermentation liquid (g / kg)

[0159] (1) Methods:

[0160] The concentration of D-pantothenic acid in the bacterial fermentation broth is detected by liquid chromatography, that is, the number of grams (g / kg) of D-pantothenic acid contained in each kilogram of bacterial fermentation broth.

[0161] Among them, the liquid chromatography instrument and detection conditions are as follows:

[0162] Equipment: Shimadzu LC-20AD,

[0163] Chromatographic column: ODS-35um 4.6*250mm(UP),

[0164] Mobile phase A: 0.006 M potassium dihydrogen phosphate,

[0165] Mobile phase B: methanol (chromatographic grade),

[0166] Detection time: 35min,

[0167] Detection wavelength: 210nm,

[0168] Column oven temperature: 35°C,

[0169] Pump mode: low pressure isocratic gradient, total flow rate 1.2 ml / min, mobile phase A: mobile phase B 9:1,

[0170] Injection volume: 20 μL.

[0171] (2) Results:

[0172] The amount of D-pantothenic acid in grams per kilogram of the bacterial fermentation liquid in Examples 1-4 and Comparative Examples 1-2 is shown in Table 2.

[0173] 2. Conversion rate of D-pantothenic acid to glucose

[0174] (1) Methods:

[0175] Conversion rate of D-pantothenic acid to glucose = total D-pantothenic acid production (g) / glucose dosage (g) × 100%.

[0176] (2) Results:

[0177] The conversion rates of D-pantothenic acid to glucose in Examples 1-4 and Comparative Examples 1-2 are shown in Table 2.

[0178] 3. Content of C8 homologues in D-pantothenic acid

[0179] C8 homologues:

[0180] (1) Methods:

[0181] C8 homologues in D-pantothenic acid were detected by liquid chromatography. The peak area ratio of C8 homologues to D-pantothenic acid was calculated using the following formula: C8 homologue ratio = C8 homologue peak area / D-pantothenic acid peak area * 100%.

[0182] Among them, the liquid chromatography instrument and detection conditions are as follows:

[0183] Equipment: Shimadzu LC-20AD,

[0184] Chromatographic column: ODS-35um 4.6*250mm(UP),

[0185] Mobile phase A: 0.006 M potassium dihydrogen phosphate,

[0186] Mobile phase B: methanol (chromatographic grade),

[0187] Detection time: 35min,

[0188] Detection wavelength: 210nm,

[0189] Column oven temperature: 35°C,

[0190] Pump mode: low pressure isocratic gradient, total flow rate 1.2 ml / min, mobile phase A: mobile phase B 9:1,

[0191] Injection volume: 20 μL.

[0192] (2) Results:

[0193] The chromatograms of D-pantothenic acid and C8 homologues in Example 1 are as follows: Figure 1 The chromatograms of D-pantothenic acid and C8 homologues in Comparative Example 1 are shown in Figure 2 shown.

[0194] The values ​​of "peak area of ​​C8 homologues" and "peak area of ​​D-pantothenic acid" of Examples 1-4 and Comparative Examples 1-2, and the calculated results of "peak area ratio of C8 homologues to D-pantothenic acid" are shown in Table 1. As can be seen from Table 1, the content of C8 homologues in the continuous fermentation production method of D-pantothenic acid of the present invention is significantly lower than that in single batch fermentation.

[0195] Table 1

[0196]

[0197] 4. Total productivity (g / h)

[0198] (1) Methods:

[0199] Total productivity (g / h) = total D-pantothenic acid production (g) / total cycle, that is, the total production of D-pantothenic acid obtained in each stage of the entire fermentation cycle is calculated. The total production of D-pantothenic acid is as described in the above examples and comparative examples.

[0200] The total productivity in Examples 1-4 and Comparative Examples 1-2 is shown in Table 2.

[0201] Table 2

[0202]

[0203] As shown in Table 2, the number of grams of D-pantothenic acid per kilogram of bacterial fermentation liquid (g / kg), the conversion rate of D-pantothenic acid to glucose, the content of C8 homologues in D-pantothenic acid, and the total productivity of D-pantothenic acid in the continuous fermentation production method of D-pantothenic acid of the present invention are significantly better than those of single batch fermentation.

Claims

1. A method for producing D-pantothenic acid, characterized in that: The method comprises the following steps: continuously fermenting the Escherichia coli strain in a culture medium to produce D-pantothenic acid; Wherein, the concentration of the Escherichia coli strain is 40-50g / L; The pH of the continuous fermentation is 6.5-7.

0.

2. The method for producing D-pantothenic acid according to claim 1, characterized in that: It meets one or more of the following conditions: (1) The starting strain of Escherichia coli is K12 strain Escherichia coli; preferably, the K12 strain Escherichia coli is Escherichia coli W3110; more preferably, the Escherichia coli W3110 strain is PB17 strain; (2) During the continuous fermentation process, feeding the culture medium; Preferably, the flow rate of the fed culture medium is 0.4-1.0 L / h; Preferably, while the culture medium is fed, the fermentation liquid and the clear liquid containing D-pantothenic acid flow out; More preferably, the outflow rate of the clear solution containing D-pantothenic acid is 0.2-0.8 L / h; More preferably, the outflow rate of the fermentation broth is 0.2-0.4 L / h; (3) The components and contents of the culture medium are, by mass percentage, 0.1-1% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water; For example, in terms of mass percentage, the components and contents of the culture medium are: Glucose 1%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 0.5%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, and the balance is water; Or, glucose 0.8%, peptone 0.5%, yeast extract powder 1%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.007%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; (4) The culture conditions for the continuous fermentation are selected from one or more of the following: a. The continuous fermentation temperature is 35-37 ° C, for example, 35 ° C, 36 ° C or 37 ° C; b. The continuous fermentation pH is 6.5-7.0, for example 6.8 or 7.0; c. The continuous fermentation ventilation ratio is 0.8-1.2VVM, for example 1.0VVM; d. The continuous fermentation DO is 30%-40%, for example 30%; (5) The continuous fermentation culture cycle is 7-20 days, for example, 167h, 236h, 261h or 270h.

3. The method for producing D-pantothenic acid according to claim 1, characterized in that: The continuous fermentation comprises the following steps: The concentrated bacterial liquid of Escherichia coli is inoculated into the culture medium for fermentation; after fermentation for 7-20 days, the fermentation liquid is filtered through a membrane to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid, wherein the outflow rate of the clear liquid containing D-pantothenic acid is 0.2-0.8 L / h, the outflow rate of the fermentation liquid is 0.2-0.4 L / h, and the concentrated bacterial liquid is fed to the next fermentation process; while feeding the concentrated bacteria, the culture medium and ammonia water are fed, and the flow rate of the fed culture medium is 0.4-1.0 L / h.

4. The method for producing D-pantothenic acid according to any one of claims 1 to 3, characterized in that The continuous fermentation is to continuously ferment the concentrated bacterial solution containing Escherichia coli in a culture medium; the production method of the concentrated bacterial solution containing Escherichia coli comprises the following steps: S1, Escherichia coli strains are cultured to obtain seed tank seeds; S2, inoculating the seeds obtained in step S1 into the first fermentation tank for fermentation and culture to obtain a bacterial fermentation liquid; S3, filtering the bacterial fermentation liquid obtained in step S2 through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid; S4, inoculating the concentrated bacterial liquid obtained in step S3 into a second fermentation tank for fermentation culture to obtain a bacterial fermentation liquid; filtering the bacterial fermentation liquid through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid.

5. The method for producing D-pantothenic acid according to claim 4, characterized in that: It meets one or more of the following conditions: (1) In step S1, the culture includes shake flask culture and seed tank culture in sequence; (2) In step S2, the inoculation ratio of the seeds in the seed tank obtained in step S1 to the first fermentation tank is 5-10%, for example, 10%; (3) In step S2, the medium for fermentation culture is a fermentation medium, and the components and contents of the fermentation medium are, by mass percentage, 0.1-1% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water; For example, in terms of mass percentage, the components and contents of the fermentation medium are: Glucose 1%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 0.5%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, and the balance is water; Or, glucose 0.8%, peptone 0.5%, yeast extract powder 1%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.007%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; (4) In step S2, the pH of the fermentation culture medium before sterilization is 6.5-7.0, for example, 7.0; (5) In step S2, the fermentation culture medium is sterilized at 121° C. for 30 min before use; (6) In step S2, the fermentation culture temperature is 35-37°C, for example, 35°C, 36°C or 37°C; (7) In step S2, the pH of the fermentation culture is 6.5-7.0, for example, 6.8 or 7.0; (8) In step S2, the ventilation ratio of the fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM; (9) In step S2, the DO of the fermentation culture is 30%-40%, for example 30%; (10) In step S2, the fermentation culture period is 50-70 h, preferably 60-65 h, for example 61.6 h, 61.9 h or 62 h.

6. The method for producing D-pantothenic acid according to claim 5, characterized in that: Step S1 satisfies one or more of the following conditions: (1) The container for shake flask culture is a shake flask; (2) The medium for the shake flask culture is LB medium; (3) The culture temperature of the shake flask culture is 30-35°C, for example 30°C; (4) The rotation speed of the shaker during the shake flask culture is 200-250 rpm, for example, 220 rpm; (5) The shaking flask culture time is 3-5 hours, for example 5 hours; (6) The shake flask culture produces shake flask seeds, and 5-10% of the shake flask seeds are used for the seed tank culture, for example, 10% of the shake flask seeds are used for the seed tank culture; (7) The container for the seed tank culture is a seed tank; (8) The medium for the seed tank culture is a seed tank medium, and the components and contents of the seed tank medium are, by mass percentage, 1-3% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water; For example, in terms of mass percentage, the components and contents of the seed tank culture medium are: Glucose 1%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; Or, glucose 2%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; Or, glucose 3%, peptone 1%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; (9) The pH of the culture medium for the seed tank culture before sterilization is 7.0; (10) The culture medium for the seed tank culture is sterilized at 121°C for 30 minutes before use; (11) The temperature of the seed tank culture is 35-37°C, for example 35°C or 37°C; (12) The pH of the seed tank culture is 6.5-7.0, for example, 6.6, 6.8 or 7.0; (13) The ventilation ratio of the seed tank culture is 0.8-1.2 VVM, for example, 1.0 VVM; (14) The DO of the seed tank culture is 30%-40%, for example 30%; (15) The time of the seed tank culture is 8-10 hours, for example, 8.2 hours, 8.5 hours, 8.8 hours or 9.0 hours.

7. The method for producing D-pantothenic acid according to claim 5, characterized in that: In step S2, when the substrate sugar in the fermentation medium is exhausted and the DO rebounds to 70%-90%, feed medium is added to the first fermentation tank; Preferably, the components and contents of the feed medium are, by mass percentage, 50-60% glucose, 0.5-1.0% yeast extract or peptone, and the remainder water; for example, by mass percentage, the components and contents of the feed medium are: Glucose 58%, yeast extract powder 0.5%, the balance is water; Or, glucose 50%, yeast extract powder 0.5%, the balance is water; Or, glucose 50%, peptone 1%, the balance is water; Or, glucose 55%, peptone 0.5%, the balance is water; Preferably, the feed medium is sterilized at 121° C. for 30 min before use.

8. The method for producing D-pantothenic acid according to claim 4, characterized in that: It meets one or more of the following conditions: (1) In step S3, the filter membrane of the membrane filtration device is a ceramic membrane. Preferably, the pore size of the ceramic membrane is 50-200 nm, for example, 100 nm, 150 nm or 200 nm; (2) In step S3, the filtration is performed under closed and sterile conditions; (3) In step S3, the volume of the concentrated bacterial liquid is 15-30% of the volume of the bacterial fermentation liquid obtained in step S2; (4) In step S4, the inoculation ratio is 40%-100% of the volume of the concentrated bacterial solution obtained in step S3; (5) In step S4, the number of the second fermentation tanks is 1 or 2; (6) In step S4, the second fermentation tank is filled with a fermentation medium, and the components and contents of the fermentation medium are, by mass percentage, 0.1-1% glucose, 0.5-1% peptone, 0.5-1% yeast extract powder, 0.2-0.5% potassium dihydrogen phosphate, 0.5-1% ammonium chloride, 0.05-0.1% magnesium sulfate heptahydrate, 0.005-0.01% manganese chloride tetrahydrate, 0.005-0.01% anhydrous ferric chloride, and the balance is water; For example, in terms of mass percentage, the components and contents of the fermentation medium are: Glucose 1%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 0.5%, peptone 0.5%, yeast extract powder 0.5%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.008%, and the balance is water; Or, glucose 0.8%, peptone 0.5%, yeast extract powder 1%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.07%, manganese chloride tetrahydrate 0.007%, anhydrous ferric chloride 0.008%, the balance is water; Or, glucose 2.5%, peptone 0.8%, yeast extract powder 0.8%, potassium dihydrogen phosphate 0.5%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.05%, manganese chloride tetrahydrate 0.005%, anhydrous ferric chloride 0.005%, the balance is water; (7) In step S4, the fermentation culture temperature is 35-37°C, for example, 35°C, 36°C or 37°C; (8) In step S4, the pH of the fermentation culture is 6.5-7.0, for example, 6.8 or 7.0; (9) In step S4, the ventilation ratio of the fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM; (10) In step S4, the DO of the fermentation culture is 30%-40%, for example, 30%; (11) In step S4, the filter membrane of the membrane filtration device is a ceramic membrane. Preferably, the pore size of the ceramic membrane is 50-200 nm, for example, 50 nm or 100 nm; (12) In step S4, the fermentation culture time is 25-35 hours, for example, 30 hours.

9. The method for producing D-pantothenic acid according to claim 1, wherein The continuous fermentation comprises the following steps: S1, Escherichia coli strains are cultured to obtain seed tank seeds; S2, inoculating the seeds obtained in step S1 into the first fermentation tank for fermentation and culture to obtain a bacterial fermentation liquid; S3, filtering the bacterial fermentation liquid obtained in step S2 through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid; S4, inoculating the concentrated bacterial liquid obtained in step S3 into a second fermentation tank for fermentation culture to obtain a bacterial fermentation liquid; filtering the bacterial fermentation liquid through a membrane filtration device to obtain a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid; S5, returning the concentrated bacterial liquid obtained in step S4 to the second fermentation tank in step S4 for continuous fermentation culture; During the continuous fermentation culture, part of the fermentation liquid containing bacteria in the second fermentation tank is kept flowing out to the membrane filtration device described in step S4, and a clear liquid containing D-pantothenic acid and a concentrated bacterial liquid are obtained after membrane filtration; The clear liquid containing D-pantothenic acid flows out of the second fermentation tank, and the concentrated bacterial liquid flows back to the second fermentation tank again for continuous fermentation culture, and the cycle is repeated in sequence; at the same time, fresh culture medium and ammonia water are kept added to the second fermentation tank; at the same time, the second fermentation tank keeps flowing out the bacterial fermentation liquid, so as to maintain the concentration of Escherichia coli in the second fermentation tank at 40-50 g / L.

10. The method for producing D-pantothenic acid according to claim 9, characterized in that: It meets one or more of the following conditions: (1) The conditions and operations in steps S1 and S2 are the same as those in any one of claims 4 to 7; (2) The conditions and operations in the method of steps S3 and S4 may be the same as the conditions and operations described in claim 4 or 8; (3) In step S5, the temperature of the continuous fermentation culture is 35-37°C, for example, 35°C, 36°C or 37°C; (4) In step S5, the pH of the continuous fermentation culture is 6.5-7.0, for example, 6.8 or 7.0; (5) In step S5, the ventilation ratio of the continuous fermentation culture is 0.8-1.2 VVM, for example, 1.0 VVM; (6) In step S5, the DO of the continuous fermentation culture is 30%-40%, for example, 30%; (7) In step S5, the outflow rate of the clear liquid containing D-pantothenic acid is 0.2-0.8 L / h, for example, 0.3 L / h, 0.5 L / h or 0.7 L / h; (8) In step S5, the components and contents of the fresh culture medium are, by mass percentage, 30-50% glucose, 0.1-0.3% yeast extract, 0.05-0.2% potassium dihydrogen phosphate, 0.2-0.5% ammonium chloride, 0.02-0.05% magnesium sulfate heptahydrate, and the remainder is water; for example, by mass percentage, the components and contents of the fresh culture medium are: Glucose 48%, yeast extract powder 0.1%, potassium dihydrogen phosphate 0.05%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.02%, the balance is water; Or, glucose 37%, yeast extract powder 0.15%, potassium dihydrogen phosphate 0.1%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.02%, and the balance is water; Or, glucose 42%, yeast extract powder 0.2%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.2%, magnesium sulfate heptahydrate 0.05%, and the balance is water; Or, glucose 31%, yeast extract powder 0.2%, potassium dihydrogen phosphate 0.2%, ammonium chloride 0.5%, magnesium sulfate heptahydrate 0.02%, and the balance is water; (9) In step S5, the fresh culture medium is sterilized at 121°C for 30 minutes before use; (10) In step S5, the flow rate of the fresh culture medium is 0.4-1.0 L / h, for example, 0.5 L / h, 0.7 L / h, 0.9 L / h or 1.0 L / h; (11) In step S5, the outflow rate of the bacterial fermentation liquid is 0.2-0.4 L / h, for example, 0.2 L / h or 0.3 L / h; (12) In step S5, the flow rate of the fresh culture medium is equal to the sum of the outflow rate of the clear liquid containing D-pantothenic acid and the outflow rate of the fermentation liquid containing bacteria.

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