WO2024041228A1 - Feed liquid for production of polysialic acid, and preparation method for polysialic acid - Google Patents

Abstract

The present invention relates to the technical field of microbial fermentation, and in particular to a feed liquid for the production of polysialic acid, and a preparation method for the polysialic acid. By means of investigating the kinetic behavior of Escherichia coli fermentation for producing the polysialic acid under NaCl stress, a production method for increasing the yield of the polysialic acid by means of flow-feed NaCl is provided, wherein the feeding quantity of the flow-feed NaCl is 20 g/L fermentation medium to 40 g/L fermentation medium, and when said feeding quantity is reached 4 h to 6 h before the end of the fermentation, the increase in the yield of the polysialic acid is particularly significant. Furthermore, provided are a NaCl-containing feed liquid and use thereof in the production of the polysialic acid by Escherichia coli fed-batch fermentation. The production method is beneficial for increasing the yield of the polysialic acid, and facilitates the ethanol precipitation and separation of the polysialic acid after the fermentation is completed.

Description

Feeding liquid for producing polysialic acid and preparation method of polysialic acid Technical field

The invention belongs to the technical field of microbial fermentation, and specifically relates to a feeding solution for producing polysialic acid and a polysialic acid preparation method.

Background technique

Polysialic acid is a type of polyanionic linear extracellular polysaccharide composed of a-(2,8) and/or a-(2,9)-linked sialic acid with a degree of polymerization of approximately 8 to 400. Polysialic acid not only has important physiological and pharmacological effects, but also has potential application value in drug delivery systems and modification of drug macromolecules. For example, polysialic acid can promote nerve development and regeneration, and can also be used in place of polyethylene glycol to modify macromolecular drugs such as proteins to increase the half-life of drugs. It can also be used as a framework material for gel drug delivery systems to slow down the release rate of drugs in the body. In addition, polysialic acid can produce sialic acid through acid hydrolysis, which is an important intermediate for the preparation of sialic acid through fermentation. As a new food raw material, sialic acid has been approved for marketing in the European Union, Japan, Malaysia, Singapore, China and other countries/regions.

Polysialic acid is mainly produced through fermentation of Escherichia coli, including Escherichia coli K1, Escherichia coli K12, Escherichia coli K92, Escherichia coli K235, etc. The optimal carbon and nitrogen sources required for the synthesis of polysialic acid by different Escherichia coli are slightly different. Various carbon sources can enter E. coli cells through the phosphoenolpyruvyl transferase system or NanT transporter, and synthesize polysialic acid through complex biochemical reactions and polysaccharide assembly pathways. The generated polysialic acid is finally processed by KpsS, KpsD, etc. Transported outside the cell. During the de novo synthesis of polysialic acid by E. coli, the genes involved in polysialic acid synthesis include kpsF , kpsM , kpsT , etc. However, the initiation mechanism of polysialic acid synthesis in E. coli is still unclear. A variety of natural E. coli strains or E. coli mutated strains have been used for the fermentation production of polysialic acid, among which E. coli K235 has been studied more intensively in recent years (CN101195661A, CN112553120A, etc.).

There are many reports on the process of preparing polysialic acid by fermentation of Escherichia coli. In published literature, carbon sources used for the fermentation production of polysialic acid include glucose, glycerol, sorbitol, xylose, etc.; organic nitrogen sources and inorganic nitrogen sources include: corn steep starch, yeast extract, tryptone, and L-proline. Acid, aspartic acid, asparagine, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , etc. The combination of different carbon sources and nitrogen sources will affect the synthesis of polysialic acid. Among them, sorbitol is the most commonly used carbon source; corn steep starch, yeast extract, tryptone, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ is the most commonly used nitrogen source. E. coli grows well when glucose is used as a carbon source, but the acetate produced by metabolism will lower the pH of the culture solution, thereby promoting the degradation of polysialic acid or inhibiting bacterial growth. When xylose was used as the carbon source, the pyruvate content in the culture was higher.

On the basis of extensive screening and optimization of carbon sources and nitrogen sources of the culture medium, the field also compared batch fermentation of polysialic acid and fed-batch fermentation, and found that fed-batch fermentation is beneficial to improving Polysialic acid fermentation yield. Fed-batch fermentation can overcome many shortcomings of batch fermentation, such as delaying bacterial aging; the ingredients of fed-batch fermentation include carbon sources, nitrogen sources, water and other substances (Cao Junwei. Microbial Engineering [M]. Beijing: Science and Technology Publishing House, 2nd Edition 2007.).

On this basis, in order to further improve the fermentation yield of polysialic acid, the field has also conducted extensive research on the dynamic regulation of polysialic acid fermentation culture conditions in recent years, including the dynamic regulation of temperature, pH, and stirring speed during the fermentation culture process. . It has been found that the staged regulation of the above indicators is also beneficial to improving the fermentation yield of polysialic acid (CN109182423A). In addition, sodium pyruvate and hydrogen peroxide stress are also beneficial to improving the fermentation yield of polysialic acid (CN104046671A). However, the current fermentation yield of polysialic acid is still low and needs to be further improved.

technical problem

In order to solve the above technical problems, the present invention studied the kinetic characteristics of Escherichia coli fermentation to produce polysialic acid under NaCl stress conditions, improved the process of Escherichia coli fermentation to produce polysialic acid based on the existing polysialic acid fermentation process, and further improved The fermentation yield of polysialic acid.

Technical solutions

One of the technical solutions of the present invention provides a fed-batch fermentation production method for producing polysialic acid using Escherichia coli. Compared with the publicly reported fed-batch fermentation production method of polysialic acid, the ingredients of the fed-batch fermentation production method during the fermentation culture process are mainly carbon sources or a combination of carbon sources and nitrogen sources. The mixture; the typical feature of this fed-batch fermentation production method is that the ingredients fed during the fermentation culture process also contain NaCl, thereby further improving the fermentation yield of polysialic acid.​

Furthermore, NaCl can be fed by intermittent feeding or continuous feeding. In a preferred solution, the NaCl is fed in an intermittent feeding manner. In another preferred solution, the NaCl is fed in a continuous fed manner.​

Further, when feeding feed, the feeding amount of NaCl is: 20g/L fermentation medium to 40g/L fermentation medium. In a preferred solution, the feeding amount of NaCl is: 20g/L fermentation medium to 35g/L fermentation medium. The term "fermentation medium" is also called "fed-batch fermentation basic medium" in the art, which refers to the medium added to the fermentation tank before the start of fermentation culture. Based on published reports, those skilled in the art should know that during fed-batch fermentation, the total volume of the fermentation broth usually does not exceed 70% of the fermentor volume, and the volume of the fermentation medium is usually 60% of the fermentor volume. That is, the feed volume usually does not exceed 10% of the fermentation tank volume. In other words, based on the volume of the fermentation medium and the final volume of the fermentation broth at the end of the fermentation, the feeding amount of NaCl can also be expressed as the concentration of NaCl in the fermentation broth. In fed-batch fermentation, the fermentation liquid in the fermentor is composed of fermentation medium, inoculated seed liquid, feeding liquid, etc. Taking the volume of the fermentation medium as V1 (unit: L), the volume of the fermentation liquid in the fermentor as V2 (unit: L), and the NaCl feed amount calculated based on the fermentation medium as C1 (unit: g/L), then The amount of NaCl fed in the fermentation broth is C2 (unit: g/L) = (V1xC1)/V2.​

Based on published reports, those skilled in the art should know that the feeding speed depends on factors such as the feeding volume and the concentration of the fed aqueous solution. The greater the concentration of the fed aqueous solution, the smaller the required feeding volume and the slower the required feeding speed. vice versa. In the fed-batch fermentation process in this field, the ingredients to be fed are usually added to water to prepare an aqueous solution, and fed in the form of an aqueous solution during the fermentation culture process. The term "fed-feed aqueous solution" refers to an aqueous solution for fed-feeding prepared from the ingredients that require fed-feeding and water. When the "fed-batch aqueous solution" contains a carbon source, it is customarily called a fed-batch solution or a fed-batch solution. When different fed-batch ingredients are fed separately, the "fed-batch aqueous solution" is usually divided into a fed-batch aqueous solution and other fed-batch aqueous solutions, such as pH adjuster aqueous solution, H ₂ O ₂ aqueous solution, and NaCl aqueous solution. Based on differences in fed-batch methods, differences in flow acceleration, or differences in the effects of fed-batch ingredients, in the same fermentation culture process, a variety of "fed-batch aqueous solutions" can be prepared for fed-bending separately, such as the preparation of ingredients for adjusting pH. The carbon source is prepared into a separate aqueous solution and the carbon source is prepared into a separate aqueous solution, and the two are separately subjected to fed-batch operations. This is routine practice in the field. It can be seen from this that when the term "feeding solution" is used in this context, it specifically refers to the "fed-feed aqueous solution" containing the carbon source. When the term "fed-batch aqueous solution" is used in this context, it refers to feed solutions and other fed-batch aqueous solutions.

NaCl is not an energy substance for E. coli and can be absorbed but not metabolized. That is, NaCl in the fermentation broth is cumulative. Based on this, in a preferred solution, when fed feeding, the NaCl feeding rate is: the cumulative feeding amount of NaCl 4h ~ 8h before the end of fermentation culture is 20g/L fermentation medium ~ 40g/L fermentation culture base. Further preferably, the NaCl feeding rate is: the cumulative feeding amount of NaCl 4h to 8h before the end of the fermentation culture is 20g/L fermentation medium to 35g/L fermentation medium. Still further preferably, the NaCl feeding rate is: the cumulative feeding amount of NaCl 4h to 6h before the end of the fermentation culture is 20g/L fermentation medium to 35g/L fermentation medium. Still further preferably, the NaCl feeding rate is: the cumulative feeding amount of NaCl between 9h and 10h before the end of fermentation culture is 5g/L fermentation medium - 15g/L fermentation medium; the cumulative feeding amount of NaCl between 4h and 6h before the end of fermentation culture. The cumulative feeding amount is 20g/L fermentation medium to 35g/L fermentation medium.

In the publicly reported fed-batch fermentation production method of polysialic acid, the ingredients of fed-batch include carbon source, nitrogen source, pH regulator (such as NaOH, ammonia, liquid ammonia), hydrogen peroxide, etc. In most of the publicly reported methods, the carbon source and the nitrogen source are added to water to form an aqueous solution as the feeding solution, that is, the carbon source and the nitrogen source are added simultaneously. Some published reports do not include a nitrogen source in the ingredients of the fed feed. In the publicly reported fed-batch fermentation production method of polysialic acid, the carbon source and nitrogen source are fed by intermittent feeding or continuous feeding. pH regulators (such as NaOH, ammonia) are used to regulate the pH of the fermentation broth at different stages. Feeding is done in an intermittent feeding manner, and is usually not fed synchronously with carbon sources. In published reports, hydrogen peroxide is fed in an intermittent feeding manner. NaCl can be fed in an intermittent or continuous manner. Therefore, NaCl can be used in conjunction with the above-mentioned feeding ingredients and feeding methods. For example, NaCl and carbon source or NaCl, carbon source and nitrogen source can be added to the water to facilitate simultaneous feeding (intermittent feeding or continuous feeding can be used); NaCl can also be mixed with hydrogen peroxide. Synchronous feeding and feeding into an aqueous solution (intermittent feeding can be used); NaCl can also be mixed with a carbon source and hydrogen peroxide to form an aqueous solution and fed into the aqueous solution simultaneously, or NaCl can be mixed with a carbon source, a nitrogen source and hydrogen peroxide. The aqueous solution is fed and fed synchronously (intermittent feeding can be used).

Regarding the aforementioned fed-batch fermentation production method using Escherichia coli to produce polysialic acid, the present invention further provides a fed-batch fermentation method for producing polysialic acid. The fed-batch fermentation production method includes the following steps:

F1: Inoculate the E. coli seed liquid into the fermentation tank containing fermentation medium;

F2: Fermentation culture is carried out under the fermentation conditions of 32℃~42℃, pH 6.4~8.0, stirring speed 75rpm~700rpm, and ventilation volume 0.5vvm~2vvm;

F3: During the fermentation culture process of step F2, feed material is added to the fermentation tank; the components of the fed material include: NaCl and carbon source. The feeding amount and feeding speed of NaCl are as described above. The carbon source of the fed feed is selected from at least one of glucose, xylose, glycerol, and sorbitol.​

In a preferred solution, the fermentation culture temperature in step F2 is 34°C to 42°C, the pH is 6.4 to 7.1, and the stirring speed is 150rpm to 700rpm. In another preferred embodiment, the components of the fed feed in step F3 also include a nitrogen source. The nitrogen source of the fed feed is selected from at least one of corn steep starch, yeast extract, tryptone, L-proline, aspartic acid, asparagine, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ kind. In a further preferred embodiment, the carbon source of the fed feed in step F3 is selected from one of glucose, xylose, and sorbitol; the nitrogen source of the fed feed is selected from NH ₄ Cl, L-proline, (NH ₄ ) ₂ SO ₄ , one of asparagine. Based on published reports, those skilled in the art should know that among the ingredients of the fed-batch feed, the selection of carbon sources and nitrogen sources is related to the metabolic characteristics of E. coli itself. For example, xylose and L-proline are used as E. coli bacteria respectively. The carbon source and nitrogen source of K1 are beneficial to improving the polysialic acid yield. When glucose and (NH ₄ ) ₂ SO ₄ are used as the carbon source and nitrogen source of E. coli K1 respectively, the polysialic acid yield is slightly lower than xylose and L- The combination of proline; glucose and L-proline as carbon source and nitrogen source respectively for E. coli K92 are beneficial to improve the yield of polysialic acid. Escherichia coli K235, Escherichia coli C8, Escherichia coli SA8, Escherichia coli CASOV-8, etc. use glucose or sorbitol as the carbon source and NH ₄ Cl or (NH ₄ ) ₂ SO ₄ as the nitrogen source, which are beneficial to increasing the production of polysialic acid. Rate.

Further, in step F3, the concentration of the carbon source in the feeding liquid used during fed feeding is 20g/L ~ 800g/L; the concentration of the nitrogen source in the feeding liquid used during fed feeding is 12g/L ~ 300g /L. Further preferably, in step F3, the concentration of the carbon source in the feeding liquid used during fed feeding is 60g/L to 800g/L carbon source; the concentration of the nitrogen source in the feeding liquid used during fed feeding is 50g/L. L～300g/L.

Further, the ingredients of the fed feed in step F3 also include pH regulator and/or hydrogen peroxide. The pH adjuster is selected from one of NaOH, ammonia water, and liquid ammonia.

In the aforementioned step F3, the concentration range of the carbon source and nitrogen source in the feeding liquid used during fed feeding is the conventional range in this field for the production of polysialic acid by Escherichia coli fermentation. As mentioned before, the feeding speed depends on the feeding volume, the concentration of the feeding solution and other factors. The greater the concentration of the feeding solution, the smaller the required feeding volume and the slower the required feeding speed. Skilled technicians have the ability to make reasonable selections of fed feeding time, feeding speed, feeding concentration, feeding amount, etc. of carbon and nitrogen sources according to the consumption rate of carbon and nitrogen sources during fermentation and culture.

The ranges of parameters such as temperature, pH, stirring speed, ventilation volume and other parameters of the fermentation culture in the aforementioned step F2 are the conventional ranges in this field for the production of polysialic acid by Escherichia coli fermentation.

Further, the fermentation medium in step F1 contains carbon source, nitrogen source, K ₂ HPO4 or K ₂ HPO4 hydrate 0.5g/L to 27g/L, MgSO ₄ or MgSO ₄ hydrate 0.15g/L to 1.5g/ L.

Further, the carbon source of the fermentation medium in step F1 is selected from at least one of sorbitol, glycerol, glucose, and xylose; the nitrogen source of the fermentation medium in step F1 is selected from NH ₄ Cl, (NH ₄ ) ₂ SO _4. At least one of corn steep starch, yeast extract, tryptone, L-proline, aspartic acid, and asparagine. In a preferred embodiment, the carbon source of the fermentation medium is selected from at least one of the following components: sorbitol 10g/L to 60g/L, glycerol 20g/L to 40g/L, and glucose 6g/L to 40g/L. L, xylose 8g/L～15g/L; the nitrogen source of the fermentation medium is selected from at least one of the following components: NH ₄ Cl 2g/L～8g/L, (NH ₄ ) ₂ SO ₄ 2.5g/ L～5g/L, corn steep starch 8g/L～20g/L, yeast extract 1.2g/L～10g/L, tryptone 0.4g/L～16g/L, L-proline 5g/L～19g /L, aspartic acid or asparagine 9g/L ~ 15g/L. In a further preferred embodiment, the carbon source of the fermentation medium is selected from at least one of the following components: sorbitol 10g/L~60g/L, glucose 7g/L, xylose 8g/L~15g/L; The nitrogen source of the fermentation medium is selected from at least one of the following components: (NH ₄ ) ₂ SO ₄ 5g/L, yeast extract 10g/L, tryptone 1.5g/L ~ 10g/L, L-proline Acid 17g/L ~ 19g/L, asparagine 9g/L ~ 12g/L.

The fermentation medium can also further add trace elements, including but not limited to FeSO ₄ or its hydrate, CuSO ₄ or its hydrate, CaCl ₂ or its hydrate, K ₂ SO ₄ or its hydrate, KH ₂ PO ₄ Or NaH ₂ PO ₄ .

Some typical and preferred fermentation medium formulas can be found in, but are not limited to: CN1916010A, CN104046671A, etc. Based on published reports, those skilled in the art should know that the selection of carbon sources and nitrogen sources in the fermentation medium is related to the metabolic characteristics of E. coli itself. For example, xylose and L-proline are used as carbon sources for E. coli K1 respectively. and nitrogen sources are beneficial to improving the yield of polysialic acid; glucose and L-proline are used as carbon sources and nitrogen sources respectively for E. coli K92, which are beneficial to improving the yield of polysialic acid. Escherichia coli K235, Escherichia coli C8, Escherichia coli SA8, Escherichia coli CASOV-8, etc. use glucose or sorbitol as the carbon source and NH ₄ Cl or (NH ₄ ) ₂ SO ₄ as the nitrogen source, which is beneficial to improving the yield of polysialic acid. .

Further, in step F1, the inoculum amount of the seed liquid is calculated as 0.05% to 8% in volume percentage.

Further, in step F1, 4.09g/L to 4.14g/L sodium pyruvate is further added to the fermentation tank. The fed ingredients in step F3 also include hydrogen peroxide. For the feeding method of hydrogen peroxide, please refer to CN104046671A, that is, at the 5th, 10th, 15th, and 20th hours of fermentation culture, 2mmol/L fermentation medium, 4mmol/L fermentation medium, 8mmol/L fermentation medium and the feeding amount of 8mmol/L fermentation medium were used for fed feeding. Sodium pyruvate and hydrogen peroxide have been proven to be beneficial to improving the yield of polysialic acid produced by E. coli K235 fermentation.

 Further, the preparation method of the seed liquid described in step F1 includes the following steps:

Z1: Inoculate Escherichia coli into the first-level seed culture medium, culture it at 34°C to 42°C, pH 6.4-7.8, and shaker speed 150rpm-300rpm for 6h-12h to obtain the first-level seed culture medium;

Z2: Inoculate the primary seed culture liquid into the secondary seed culture medium, and cultivate it for 6 to 12 hours at 34°C to 42°C, pH 6.4 to 7.8, and 150rpm to 300rpm to obtain the secondary seed culture liquid, that is, the seed liquid.

Preferably, the primary seed culture medium and the secondary seed culture medium are any one selected from the following culture media: M1: tryptone 8g/L~12g/L, yeast extract 4g/L~10g/L, NaCl 1g/L～12g/L, the balance is water; M2: tryptone 10g/L, beef extract 2g/L～5g/L, NaC1 5g/L, the balance is water; M3: tryptone 10g/L, Beef extract 2g/L～5g/L, NaC1 5g/L, yeast extract 2g/L, the balance is water; M4: glucose 25g/L, (NH ₄ ) ₂ SO ₄ 5g/L, tryptone 52g/L , K ₂ HPO ₄ 20g/L, MgSO ₄ 0.4g/L, and the balance is water. Among them, M1 is the commonly used LB medium and its improved medium; M2 and M3 are the commonly used meat extract peptone medium and its improved medium; LB medium and its improved medium, meat extract peptone medium and its improved medium It is a common basic medium widely used in bacterial culture. M4 can be used for E. coli SA-8 (deposit number: CGMCC No. 5585).

The culture temperature, pH, and shaking table rotation speed in the aforementioned steps Z1 and Z2 are the conventional ranges in this field for the production of polysialic acid by Escherichia coli fermentation. Some typical and preferred primary seed culture medium, secondary seed culture medium and culture conditions can be found in but are not limited to CN108588152A and CN109136308A.

The aforementioned yeast extract is also called yeast extract powder, and tryptone is also called tryptic casein peptone.

The above technical solution provides a fed-batch fermentation production method for producing polysialic acid using Escherichia coli. The method may further include step F4 of extracting and separating polysialic acid from the fermentation broth. Because the fed-batch fermentation production method using Escherichia coli to produce polysialic acid provided by the first technical solution and the second technical solution of the present invention does not introduce new impurities compared to the polysialic acid fermentation production method that has been publicly reported. Therefore, step F4 can adopt the method commonly used in the art to extract and separate polysialic acid from fermentation broth. Specifically, you can centrifuge at 3000 to 8000 rpm for 10 to 30 minutes, and then filter to remove the bacterial cells in the fermentation broth (see CN1896263A); or use a 50nm to 300nm ceramic membrane to filter to remove the bacterial cells in the fermentation broth (see CN109369730A ); Saturated CaCl ₂ can be used to remove phosphate in the fermentation broth (see Li Wenqiang. Research on control of sialic acid fermentation and its separation and purification [D]. Jiangnan University, 2005); ethanol precipitation or ultrafiltration concentration combined with ethanol can be used Precipitation method (see Wu Jianrong, Zhan Xiaobei, Zhu Zhu. Extraction of sialic acid from Escherichia coli fermentation broth [J]. Chinese Journal of Pharmaceutical Industry, 2003, 34(001):8-10.) or ethanol precipitation method combined with chlorine Polysialic acid in fermentation broth can be separated by cetylpyridinium complex method (see: Zhou Yajuan. Purification process and amplification study of polysialic acid in fermentation broth [D]. Jiangnan University, 2013.); Sevag method can be used Or trifluorotrichloroethane method or trichloroacetic acid method or centrifugation method (15000rpm~20000rpm centrifugation for 10min~30min) to remove protein; dialysis, electrodialysis or ultrafiltration can be used to remove salt (see: CN111386350A; Zhan Xiaobei, Yu Junhua, Wu Jianrong, et al. Effect of NTG on polysialic acid production of E.coli mutant strain [J]. Journal of Food and Biotechnology, 2002, 21(5):456-459.). When the ethanol precipitation method is used to separate polysialic acid in the fermentation broth, the fed-in NaCl in the fed-batch fermentation production method for producing polysialic acid using Escherichia coli provided by the first technical solution and the second technical solution of the present invention is beneficial to Promote the precipitation of polysialic acid in ethanol solution (please refer to CN101195661A; CN113005161A; Yu Danfeng. Research on polysialic acid and sialic acid extraction process [D]. Jiangnan University, 2008; Liu Jinlong. Research on the preparation of polysialic acid by microbial fermentation [D]. D]. Jiangnan University, 2011; Zhou Yajuan. Purification process and amplification research of polysialic acid in fermentation broth [D]. Jiangnan University, 2013); Activated carbon can be used for decolorization when the fermentation medium contains sulfates such as MgSO ₄ Barium chloride or Ca(OH) ₂ precipitation can be used to remove sulfate.

Based on the aforementioned fed-batch fermentation production method using Escherichia coli to produce polysialic acid, the second technical solution of the present invention provides a feed liquid for producing polysialic acid through fed-batch fermentation using Escherichia coli. The feeding liquid contains a carbon source, or a carbon source and a nitrogen source, and the fed liquid also contains NaCl; the concentration of NaCl in the fed liquid is 150g/L to 200g/L.

Further, the feeding solution contains 20g/L to 800g/L carbon source, or contains 20g/L to 800g/L carbon source and 12g/L to 300g/L nitrogen source.

Further, the feeding solution contains 60g/L to 800g/L carbon source, or contains 60g/L to 800g/L carbon source and 50g/L to 300g/L nitrogen source.

Further, the carbon source in the feeding liquid is selected from at least one of glucose, glycerol, sorbitol, and xylose; the nitrogen source in the feeding liquid is selected from corn steep starch, yeast extract, and tryptone. , L-proline, asparagine, at least one of NH ₄ Cl, (NH ₄ ) ₂ SO ₄ . In a preferred embodiment, the carbon source in the feeding liquid is selected from one of glucose, sorbitol, and xylose; the nitrogen source in the feeding liquid is selected from L-proline, asparagine, One of NH ₄ Cl and (NH ₄ ) ₂ SO ₄ .

beneficial effects

(1) The fed-batch fermentation production method for producing polysialic acid using Escherichia coli provided by the present invention improves the yield of polysialic acid fermentation by feeding NaCl during the fermentation culture stage.

(2) In the fed-batch fermentation production method using Escherichia coli to produce polysialic acid provided by the present invention, the NaCl fed during the fermentation culture stage can promote the dispersion of polysialic acid in the ethanol solution when the ethanol precipitation method is used to separate the polysialic acid. The precipitation speed in the fermentation broth is conducive to the separation of polysialic acid in the fermentation broth using ethanol precipitation.

(3) On the premise of meeting the carbon source demand of E. coli, the feeding solution provided by the invention adds NaCl to facilitate the simultaneous feeding of NaCl, thereby exerting the beneficial effect of NaCl on improving the yield of polysialic acid.

Description of drawings

The description drawings that constitute a part of the present invention are used to assist in further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Below, the embodiments of the present invention are described in detail with reference to the accompanying drawings, wherein:

Figure 1 is a kinetic curve diagram of cell concentration and polysialic acid yield of Escherichia coli K1 fermentation culture in Example 1; where the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, PS420 are the NaCl feeding amounts of 0, 15, Polysialic acid yield (g/L) corresponding to 20, 25, 30, 35g/L fermentation medium; where the solid lines OD0, OD180, OD240, OD300, OD360, OD420 are the NaCl feeding amounts of 0, 15, The bacterial cell concentrations corresponding to 20, 25, 30, and 35g/L fermentation medium are the OD values at a wavelength of 600nm.

Figure 2 is a kinetic curve diagram of cell concentration and polysialic acid yield of Escherichia coli K92 fermentation culture in Example 1; the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, and PS420 are the NaCl feeding amounts of 0, 15, Polysialic acid yield (g/L) corresponding to 20, 25, 30, 35g/L fermentation medium; where the solid lines OD0, OD180, OD240, OD300, OD360, OD420 are the NaCl feeding amounts of 0, 15, The bacterial cell concentrations corresponding to 20, 25, 30, and 35g/L fermentation medium are the OD values at a wavelength of 600nm.

Figure 3 is a kinetic curve diagram of cell concentration and polysialic acid production rate of Escherichia coli K235 fermentation culture in Example 1; where the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, PS420 are the NaCl feeding amounts of 0, 15, Polysialic acid yield (g/L) corresponding to 20, 25, 30, 35g/L fermentation medium; where the solid lines OD0, OD180, OD240, OD300, OD360, OD420 are the NaCl feeding amounts of 0, 15, The bacterial cell concentrations corresponding to 20, 25, 30, and 35g/L fermentation medium are the OD values at a wavelength of 600nm.

Figure 4 is a histogram of the polysialic acid yield of E. coli K1 under different NaCl feeding strategies in Example 2; S11 is not fed NaCl; S12: NaCl is a single-point fed feeding, and NaCl is fed at 27h, NaCl The feeding amount is 20g/L fermentation medium; S13: NaCl is fed in a continuous flow, and NaCl is added from 8h to 27h. The NaCl concentration is 300g/L, and the NaCl feeding rate is 200mL/h; S14: NaCl is fed intermittently. Fed feeding, the cumulative feeding amounts of NaCl at 22h and 27h were 5 and 20 g/L fermentation medium; S15: NaCl was intermittent fed feeding, and the cumulative feeding amounts of NaCl at 22h and 27h were 10 and 20 g/L respectively. g/L fermentation medium; S16: NaCl is fed intermittently, and the cumulative feeding amounts of NaCl at 22h and 27h are 15 and 20 g/L fermentation medium respectively.​

Figure 5 is a histogram of the polysialic acid yield of E. coli K92 under different NaCl feeding strategies in Example 2; S21 does not feed NaCl; S22: NaCl is fed at a single point, and NaCl is fed at the 27th hour. The feeding amount is 20g/L fermentation medium; S23: NaCl is fed continuously, and NaCl is added from 8h to 27h. The NaCl concentration is 300g/L, and the NaCl feeding speed is 200mL/h; S24: NaCl is fed intermittently. Fed feeding, the cumulative feeding amounts of NaCl at 22h and 27h are 5 and 20 g/L fermentation medium respectively; S25: NaCl is intermittent fed feeding, and the cumulative feeding amounts of NaCl at 22h and 27h are 10 and 20 g/L respectively. g/L fermentation medium; S26: NaCl is fed intermittently, and the cumulative feeding amounts of NaCl at 22h and 27h are 15 and 20 g/L fermentation medium respectively.

Figure 6 is a histogram of the polysialic acid yield of E. coli K235 under different NaCl feeding strategies in Example 2; S31 does not feed NaCl; S32: NaCl is fed at a single point, and NaCl is fed at 27h. NaCl The feeding amount is 20g/L fermentation medium; S33: NaCl is fed in a continuous flow, and NaCl is added from 8h to 27h. The NaCl concentration is 300g/L, and the NaCl feeding speed is 200mL/h; S34: NaCl is fed intermittently. Fed feeding, the cumulative feeding amounts of NaCl at 22h and 27h are 5 and 20 g/L fermentation medium respectively; S35: NaCl is intermittent fed feeding, and the cumulative feeding amounts of NaCl at 22h and 27h are 10 and 20 g/L respectively. g/L fermentation medium; S36: NaCl is fed intermittently, and the cumulative feeding amounts of NaCl at 22h and 27h are 15 and 20 g/L fermentation medium respectively.

Best Mode of Carrying Out the Invention

The technical solutions of the present invention are illustrated below through specific examples, but the examples are not considered to limit the scope of the present invention. Unless otherwise stated, the bacterial strains, reagents, instruments and equipment used in the following examples were all obtained from commercial sources, or prepared using reagents purchased on the market using conventional operating procedures in this field. In addition, the bacterial strains used in the following examples can also be obtained from microorganism depositories. The solutions used for feeding and the medium used for culture in the following examples should be sterilized in advance.

Through the parameters shown in the following examples, those skilled in the art have the ability to base on the given feeding amount, inoculation amount, and fermentation tanks of different specifications (such as 5L fermentation tank, 20L fermentation tank, 50L fermentation tank, 100L fermentation tank). , 200L fermentation tank, 500L fermentation tank, etc.) The acceptable feed volume is converted into the concentration of the required fed water solution. Therefore, some simple deductions or substitutions can be made without departing from the basic principles of the present invention. Those skilled in the art are also capable of adjusting and changing the inoculum amount, temperature, rotation speed, pH, aeration, etc. used in fermentation culture according to the growth characteristics of the strain without departing from the scope of the present invention. The trace elements used in the fermentation medium of the following examples, such as CaCl ₂ , FeSO ₄ , CuSO _4, etc., are beneficial to the growth of strains, but they are usually already present in the medium raw materials and are not necessary to be added. This is for those skilled in the art. Publicly known (please refer to "Microbiological Engineering" edited by Cao Junwei, Beijing: Science and Technology Press, 2007, 2nd edition, page 84).

In the following examples, the polysialic acid yield (g/L, that is, the concentration of polysialic acid in the fermentation broth at the end of the fermentation) and bacterial concentration were measured using the resorcinol method and the absorbance method (measurement of the polysialic acid concentration at a wavelength of 600 nm) respectively. OD value). Detailed methods and operating steps can be found in "Research on the Control of Sialic Acid Fermentation and Its Isolation and Purification" (Li Wenqiang; Master's thesis of Jiangnan University in 2005. For details, see sections 2.2.4.1 and 2.2.4.6 of this thesis). When measuring the yield of polysialic acid, the fermentation broth was sampled, centrifuged at 7000r/min for 15min to remove bacteria, and an ultrafiltration membrane (molecular weight cutoff 2kD) was used to remove small molecules. Then the resorcinol method was used for determination.

In the following examples, the calculation formula for the increase in sialic acid yield (%) is (Px-P0)/P0x100, where P0 is the sialic acid yield (g/L or mg/L) of the reference point, and Px is the inspection point. Sialic acid yield (g/L or mg/L).

Embodiments of the invention

Example 1 Kinetic characteristics of polysialic acid production by Escherichia coli fermentation

1. Purpose: To investigate the effect of NaCl feeding amount on the growth of E. coli cells and the yield of sialic acid fermentation.

2. Method

(1) Strains

Escherichia coli K1 (Accession number: DSM 107164), Escherichia coli K92 (Accession number: ATCC 35860), Escherichia coli K235 (Accession number: ACTT13027).​

(2) Culture medium and culture methods

Culture medium and culture method of Escherichia coli K1

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 10, yeast extract 10, NaCl 1.2, the balance is water.

Fermentation medium (g/L): tryptone 10, yeast extract 10, xylose 14, L-proline 17.1, NaCl 1.2, K ₂ SO ₄ 1.1, CaCl ₂ 0.013, MgSO ₄ ·7H ₂ O 0.15, FeSO ₄ ·7H ₂ O 0.001, CuSO ₄ ·5H ₂ O 0.001, K ₂ HPO ₄ 6.67, KH ₂ PO ₄ 0.25, and the balance is water.

Feeding solution K11 (g/L): xylose 500, L-proline 300, the balance is water.

Culture steps and culture conditions:

(1) Inoculate E. coli into the first-level seed culture medium, and culture it for 6 hours at 37°C, pH 7.5, and shaker speed 250 rpm to obtain the first-level seed culture medium.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation amount of 2% (V/V), and culture it for 12 hours at 37°C, pH 7.5, and 250 rpm to obtain the secondary seed culture liquid, which is the seed liquid.

(3) Add 12L fermentation medium to the 20L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 2% (V/V); 37°C, pH 6.8, stirring speed 600rpm, ventilation volume Fermentation and culture for 24 hours under fermentation conditions of 1vvm; feed solution K11 was added continuously from 9h to 20h after the start of fermentation, and the feeding speed was 25mL/h; NaCl was added to water to form a flow-feed NaCl aqueous solution, once every 9h after the start of fermentation. Add NaCl 0 (that is, no NaCl is added to the water), 180g, 240g, 300g, 360g, 420g into the fermentation tank. The NaCl feeding volume is 1.36L (the feeding amount of NaCl is 0g/L fermentation medium ~ 35g/L fermentation Culture medium; that is, 0g/L fermentation broth ~ 30.22g/L fermentation broth), that is, the concentrations of NaCl aqueous solution for fed-batch are 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L, and 308.82 respectively. g/L.

Calculate the polysialic acid yield (g/L) and bacterial concentration (OD value at 600nm) at different time points when feeding different amounts of NaCl.

Culture medium and culture method of Escherichia coli K92

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 10, yeast extract 5, NaCl 10, the balance is water.

Fermentation medium (g/L): Glucose 7, Asparagine 11.3, NaCl 1.0, K ₂ SO ₄ 1.0, CaCl ₂ ·6H ₂ O 0.02, MgSO ₄ ·7H ₂ O 0.2, FeSO ₄ ·7H ₂ O 0.001, CuSO ₄ ·5H ₂ O 0.001, K ₂ HPO ₄ 0.5, NaH ₂ PO ₄ 10.8, and the balance is water.

Feeding solution K921 (g/L): glucose 270, asparagine 200.

Culture steps and culture conditions:

(1) Inoculate E. coli into the first-level seed culture medium, and culture it for 12 hours at 37°C, pH 7.2, and shaker speed 180 rpm to obtain the first-level seed culture liquid.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 2% (V/V), and culture it for 8 hours at 37°C, pH 7.2, and 180 rpm to obtain the secondary seed culture liquid, that is, the seed liquid.

(3) Add 12L fermentation medium to the 20L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 2% (V/V); 37°C, pH 7.1, stirring speed 150rpm, ventilation volume Fermentation and culture for 24 hours under the fermentation conditions of 2vvm; the feeding solution K921 was continuously added from 9h to 20h after the fermentation started, and the feeding speed was 25mL/h; NaCl was added to the water to form a fed NaCl aqueous solution, and the feeding solution was added once every 9h after the fermentation started. Add NaCl 0 (that is, no NaCl is added to the water), 180g, 240g, 300g, 360g, 420g into the fermentation tank. The NaCl feeding volume is 1.36L (the feeding amount of NaCl is 0g/L fermentation medium ~ 35g/L fermentation Culture medium; that is, 0g/L fermentation broth ~ 30.22g/L fermentation broth), that is, the concentrations of NaCl aqueous solution for fed-batch are 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L, and 308.82 respectively. g/L.

Calculate the polysialic acid yield (g/L) and bacterial concentration (OD value at 600nm) at different time points when feeding different amounts of NaCl.

Culture medium and culture method of Escherichia coli K235

First-level seed culture medium, second-level seed culture medium (g/L): tryptone 10, beef extract 3, NaCl 5, the balance is water.

Fermentation medium (g/L): sorbitol 10, (NH ₄ ) ₂ SO ₄ 5, K ₂ HPO ₄ 2.5, MgSO ₄ 0.9, tryptone 1.5, the balance is water.

Feeding solution K2351 (g/L): sorbitol 800, (NH ₄ ) ₂ SO ₄ 100.

Culture steps and culture conditions:

(1) Inoculate Escherichia coli into the first-level seed culture medium, and culture it for 12 hours at 37°C, pH 7.0, and shaker speed 250 rpm to obtain the first-level seed culture medium.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 2% (V/V), and culture it for 6 hours at 37°C, pH 7.0, and 250 rpm to obtain the secondary seed culture liquid, which is the seed liquid.

(3) Add 12L fermentation medium to the 20L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 2% (V/V); 37°C, pH 6.8, stirring speed 400rpm, ventilation volume Fermentation and culture were carried out for 24 hours under fermentation conditions of 2vvm; feeding solution K2351 was continuously added from 9h to 20h after the fermentation started, and the feeding speed was 25mL/h; NaCl was added to the water, and NaCl was added to the fermentation tank at one time at the 9th hour after the fermentation started 0 (i.e. no NaCl is added to the water), 180g, 240g, 300g, 360g, 420g, the NaCl feeding volume is 1.36L (the feeding amount of NaCl is 0g/L fermentation medium ~ 35g/L fermentation medium; that is, 0g/L Fermentation broth ~ 30.22g/L fermentation broth), that is, the concentrations of fed-batch NaCl aqueous solution are 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L, and 308.82g/L respectively.

Calculate the polysialic acid yield (g/L) and bacterial concentration (OD value at 600nm) at different time points when feeding different amounts of NaCl.

3. Results

(1) E. coli K1 experimental results

The kinetic characteristics of polysialic acid production by E. coli K1 fed-batch fermentation are shown in Figure 1. From 9h to 24h, the bacterial concentration and polysialic acid production rate in the fermentation broth showed an increasing trend with time. Among them, NaCl feeds 0g (OD 0 in the figure) and 180g (OD 180 in the figure), the bacterial concentration curves are basically similar; NaCl feeds 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD in the figure) When OD 360) and 420g (OD 420 in the figure), the bacterial concentration curve is lower than the bacterial concentration curve when the NaCl feed is 0g. The order of bacterial growth rate is: NaCl feed 0g≈NaCl feed 180g>NaCl feed 240g>NaCl feed 300g>NaCl feed 360g>NaCl feed 420g.

When NaCl was fed with 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), the yield of polysialic acid was significantly higher than that of NaCl from 12h to 16h. When feeding 0g. Compared with 0g NaCl feeding, when NaCl feeding was 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), 420g (PSA 420 in the figure), the polysialic acid production at the 16th hour The yield of polysialic acid increased by 4.78% to 7.35%; the yield of polysialic acid increased by about 8.19% to 19.30% in the 14th hour; the yield of polysialic acid in the 12th hour increased by about 9.20% to 21.84%; and under the same NaCl feeding amount, the yield of polysialic acid in the 12th hour increased by about 8.19% to 19.30%. The percentage increase in acid yield was higher than that at 14h. At this stage (12h to 16h), the order of polysialic acid yield from high to low is: NaCl feed 420g ≥ NaCl feed 360g > NaCl feed 300g > NaCl feed 240g > NaCl feed 180g ≈ NaCl feed 0g. Among them, at the 12th hour, when NaCl was fed 240g (PSA 240 in the picture), 300g (PSA 300 in the picture), 360g (PSA 360 in the picture), and 420g (PSA 420 in the picture), compared with NaCl feeding 0g, sialic acid The yield increase percentages were 9.20%, 12.64%, 21.84%, and 21.84% respectively. From 18h to 20h, the gap in polysialic acid yields with different NaCl feeding amounts narrowed. From 22h to 24h, the polysialic acid yields of 360g and 420g NaCl feeds were lower than those of 0g NaCl feeds.

(2) E. coli K92 experimental results

The kinetic characteristics of polysialic acid production by E. coli K92 fed-batch fermentation are shown in Figure 2. The concentration of bacteria in the fermentation broth first increased and then decreased from 9h to 24h, and the OD value at 600nm wavelength decreased slightly from 22h to 24h; the polysialic acid yield increased with time. Among them, when NaCl was fed 0g (OD 0 in the figure) and 180g (OD 180 in the figure), the bacterial concentration curves were basically similar; when NaCl was fed 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD When OD 360) and 420g (OD 420 in the figure), the bacterial concentration curve is lower than the bacterial concentration curve when NaCl is fed 0g. The order of bacterial growth rate is: NaCl feed 0g≈NaCl feed 180g>NaCl feed 240g>NaCl feed 300g>NaCl feed 360g>NaCl feed 420g.

When NaCl was fed with 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), the yield of polysialic acid was significantly higher than that of NaCl from 12h to 16h. When feeding 0g. Compared with 0g NaCl feeding, when NaCl feeding was 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), 420g (PSA 420 in the figure), the polysialic acid production at the 16th hour The yield of polysialic acid increased by 1.37% to 4.79%; the yield of polysialic acid increased by about 8.25% to 17.53% at the 14th hour; the yield of polysialic acid at the 12th hour increased by about 8.16% to 16.33%; and when the NaCl feeding amount is the same, the yield of polysialic acid at the 14th hour increased by about 8.25% to 17.53%. The percentage increase in acid yield was higher than that at 12h. At this stage (12h to 16h), the order of polysialic acid yield from high to low is: NaCl feed 420g>NaCl feed 360g>NaCl feed 300g>NaCl feed 240g>NaCl feed 180g≈NaCl feed 0g. Among them, at the 14th hour, when NaCl was fed 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), compared with NaCl feeding 0g, sialic acid The yield increase percentages were 8.25%, 12.37%, 15.46%, and 17.53% respectively. From 18h to 24h, the polysialic acid production of NaCl fed 420g and 360g was successively lower than that of NaCl fed 0g, and the difference expanded with time.

(3) E. coli K235 experimental results

The kinetic characteristics of polysialic acid production by E. coli K235 fed-batch fermentation are shown in Figure 3. From 9h to 24h, the bacterial concentration and polysialic acid production rate in the fermentation broth showed an increasing trend with time. Among them, NaCl feeds 0g (OD 0 in the figure) and 180g (OD 180 in the figure), the bacterial concentration curves are basically similar; NaCl feeds 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD in the figure) When OD 360) and 420g (OD 420 in the figure), the bacterial concentration curve is lower than the bacterial concentration curve when NaCl is fed 0g. The order of bacterial growth rate is: NaCl feed 0g≈NaCl feed 180g>NaCl feed 240g>NaCl feed 300g>NaCl feed 360g>NaCl feed 420g.

When NaCl was fed with 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), the yield of polysialic acid was significantly higher than that of NaCl from 12h to 18h. When feeding 0g. Compared with 0g NaCl feeding, when NaCl feeding was 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), the polysialic acid production at the 18th hour The yield of polysialic acid increased by 2.61% to 5.44%; the yield of polysialic acid at the 16th hour increased by 5.15% to 12.06%; the yield of polysialic acid at the 14th hour increased by about 10.70% to 21.85%; the yield of polysialic acid at the 12th hour increased by about 7.38% to 18.20 %; and when the NaCl feeding amount is the same, the percentage increase in polysialic acid yield at the 14th hour is higher than that at the 12th hour. The order of polysialic acid yield at this stage from high to low is: NaCl feed 420g>NaCl feed 360g>NaCl feed 300g>NaCl feed 240g>NaCl feed 180g≈NaCl feed 0g. Among them, when NaCl was fed 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), and 420g (PSA 420 in the figure), compared with NaCl feeding 0g, the sialic acid production at the 14th hour The rate improvement percentages are 10.70%, 14.96%, 17.60%, and 21.85% respectively. From 20h to 24h, the polysialic acid yields of 240g, 300g, 360g and 420g of NaCl feed were all significantly lower than 0g of NaCl feed.

4. Conclusion

In the process of preparing polysialic acid by fed-batch fermentation of Escherichia coli K1, Escherichia coli K92 and Escherichia coli K235, the feeding amount of fed-batch NaCl is 0 g/L fermentation medium to 15g/L fermentation medium. The effect on bacterial growth and sialic acid fermentation yield is not obvious; the feeding amount of fed NaCl is 20g/L fermentation medium ~ 35g/L fermentation medium, that is, 17.27g/L fermentation broth ~ 30.22g/L fermentation The yield of polysialic acid can be increased when the solution is dissolved. Among them, the yield of polysialic acid increased significantly 4h to 8h after the NaCl feeding was completed. The yield of polysialic acid increased the most 4h to 6h after the NaCl fed feeding was completed. Among them, the polysialic acid production rate of E. coli K1 increased the most 4 hours after NaCl fed feeding; the polysialic acid production rate of E. coli K92 and E. coli K235 increased the most 6 hours after NaCl fed feeding.

Example 2 Investigation into the feeding strategy of NaCl fed feeding

1. Purpose To investigate the effect of NaCl intermittent feeding and continuous feeding on the sialic acid fermentation yield of E. coli cells when the feeding volume is the same and the carbon source and nitrogen source are feeding at the same speed. Based on the relevant research in Example 1, the NaCl feeding amount was selected to be 20g/L fermentation medium, and the fermentation culture was continued for 4 hours after the NaCl fed feeding was completed.

2. Method

(1) Strains

Escherichia coli K1 (Accession number: DSM 107164), Escherichia coli K92 (Accession number: ATCC 35860), Escherichia coli K235 (Accession number: ACTT13027).​

(2) Culture medium and culture methods

Culture medium and culture method of Escherichia coli K1

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 12, yeast extract 8, NaCl 1.2, the balance is water.

Fermentation medium (g/L): tryptone 10, yeast extract 10, xylose 15, L-proline 19, NaCl 1.2, K ₂ SO ₄ 1.1, CaCl ₂ 0.013, MgSO ₄ ·7H ₂ O 0.15, FeSO ₄ ·7H ₂ O 0.001, CuSO ₄ ·5H ₂ O 0.001, K ₂ HPO ₄ 6.67, KH ₂ PO ₄ 0.25, and the balance is water.

Feeding solution K1A1 (g/L): xylose 100, L-proline 60;

Feeding solution K1B1 (g/L): xylose 200, L-proline 120;

Feeding solution K1C1 (g/L): xylose 100, L-proline 60, NaCl 150;

NaCl aqueous solution D1 (g/L): 300.

Culture steps and culture conditions:

(1) Inoculate Escherichia coli into the first-level seed culture medium, and culture it for 8 hours at 37°C, pH 6.8, and shaker speed 200 rpm to obtain the first-level seed culture liquid.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 3% (V/V), and culture it for 12 hours at 37°C, pH 6.8, and 200 rpm to obtain the secondary seed culture liquid, which is the seed liquid.

(3) Add 30L fermentation medium to the 50L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 0.5% (V/V); 37°C, pH 6.4, stirring speed 300rpm, ventilation volume Fermentation culture was carried out for 31 hours under fermentation conditions of 0.5vvm; 6 fed-batch strategies were used:

The first strategy (S11) does not feed NaCl, but feeds liquid K1A1 from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, the total feeding is 20h, and the total feeding volume is 4L.

The second strategy (S12) NaCl is fed at a single time point, and the feeding solution K1B1 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 27 hours after the fermentation started, and the feeding rate was 2L/h. The total feeding volume is 4L.

The third strategy (S13) is NaCl continuous flow feeding. The feeding solution K1C1 is added from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, and the feeding volume is 20h in total, and the feeding volume is 4L.

The fourth strategy (S14) NaCl is fed intermittently, and the feeding liquid K1B1 is added from 8h to 27h after the fermentation starts. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the fermentation started. The feeding speed was 0.5L/h, feeding 1 hour, and the NaCl feeding amount was 5g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.5L/h, the feeding is 1h, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The fifth strategy (S15) uses intermittent feeding of NaCl. Feeding liquid K1B1 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation, the feeding speed was 1.0L/h, the feed was fed for 1 hour, and the NaCl feeding amount was 10g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.0L/h, the feeding is 1h, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The sixth strategy (S16) NaCl is fed intermittently, and the feeding liquid K1B1 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation. The feeding speed was 1.5L/h, feeding 1 hour, and the NaCl feeding amount was 15g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 0.5L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

Calculate the polysialic acid yield (g/L) at the end of fermentation (at the end of 31 hours after the start of fermentation).

Culture medium and culture method of Escherichia coli K92

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 10, yeast extract 5, NaCl 10, the balance is water.

Fermentation medium (g/L): xylose 8, asparagine 9.2, NaCl 1.0, K ₂ SO ₄ 1.0, CaCl ₂ ·6H ₂ O 0.02, MgSO ₄ ·7H ₂ O 0.2, FeSO ₄ ·7H ₂ O 0.001 , CuSO ₄ ·5H ₂ O 0.001, K ₂ HPO ₄ 0.5, NaH ₂ PO ₄ 10.8, and the balance is water.

Feeding solution K92A2 (g/L): xylose 60, asparagine 80;

Feeding solution K92B2 (g/L): xylose 120, asparagine 160;

Feeding solution K92C2 (g/L): xylose 60, asparagine 80, NaCl 150;

NaCl aqueous solution D1 (g/L): 300.

Culture steps and culture conditions:

(1) Inoculate E. coli into the first-level seed culture medium, and culture it for 10 hours at 37°C, pH 7.0, and shaker speed 150 rpm to obtain the first-level seed culture medium.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 3% (V/V), and culture it for 10 hours at 37°C, pH 7.0, and 150 rpm to obtain the secondary seed culture liquid, which is the seed liquid.

(3) Add 30L fermentation medium to the 50L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 0.5% (V/V); 37°C, pH 6.4, stirring speed 200rpm, ventilation volume Fermentation culture was carried out for 31 hours under fermentation conditions of 0.5vvm; 6 fed-batch strategies were used:

The first strategy (S21) does not feed NaCl, but feeds liquid K92A2 from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, the total feeding is 20h, and the total feeding volume is 4L.

The second strategy (S22) NaCl is fed at a single time point, and feeding liquid K92B2 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 27 hours after the fermentation started, and the feeding rate was 2L/h. The total feeding volume is 4L.

The third strategy (S23) is continuous flow feeding of NaCl. Feeding liquid K92C2 is added from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, and the feeding volume is 20h in total, and the feeding volume is 4L.

The fourth strategy (S24) NaCl is fed by intermittent flow. Feed liquid K92B2 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h and the feed is fed for 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation. The feeding speed was 0.5L/h, feeding 1 hour, and the NaCl feeding amount was 5g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.5L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The fifth strategy (S25) NaCl is fed intermittently. The feeding liquid K92B2 is added from 8h to 27h after the fermentation starts. The feeding speed is 100mL/h and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the fermentation started. The feeding speed was 1.0L/h, feeding 1 hour, and the NaCl feeding amount was 10g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.0L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The sixth strategy (S26) NaCl is fed intermittently. The feeding liquid K92B2 is added from 8h to 27h after the fermentation starts. The feeding speed is 100mL/h and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation. The feeding speed was 1.5L/h, feeding 1 hour, and the NaCl feeding amount was 15g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 0.5L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

Calculate the polysialic acid yield (g/L) at the end of fermentation (at the end of 31 hours after the start of fermentation).

Culture medium and culture method of Escherichia coli K235

First-level seed culture medium, second-level seed culture medium (g/L): tryptone 10, beef extract 3, NaCl 5, the balance is water.

Fermentation medium (g/L): sorbitol 10, (NH ₄ ) ₂ SO ₄ 5, K ₂ HPO ₄ 2.5, MgSO ₄ 0.9, tryptone 1.5, the balance is water.

Feeding solution K235A3 (g/L): sorbitol 160, (NH ₄ ) ₂ SO ₄ 80;

Feeding solution K235B3 (g/L): sorbitol 320, (NH ₄ ) ₂ SO ₄ 160;

Feeding solution K235C3 (g/L): sorbitol 160, (NH ₄ ) ₂ SO ₄ 80, NaCl 150;

NaCl aqueous solution D1 (g/L): 300.

Culture steps and culture conditions:

(1) Inoculate Escherichia coli into the first-level seed culture medium, and culture it for 10 hours at 37°C, pH 6.4, and shaker speed 200 rpm to obtain the first-level seed culture medium.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 3% (V/V), and culture it for 12 hours at 37°C, pH 6.4, and 300 rpm to obtain the secondary seed culture liquid, which is the seed liquid.

(3) Add 12L fermentation medium to the 20L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 0.5% (V/V); 37°C, pH 6.4, stirring speed 200rpm, ventilation volume Fermentation and culture were carried out for 31 hours under fermentation conditions of 1vvm; 6 fed-batch strategies were used:

The first strategy (S31) does not feed NaCl, but feeds liquid K235A3 from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, the total feeding volume is 20h, and the total feeding volume is 4L.

The second strategy (S32) NaCl is fed at a single time point, and the feeding liquid K235B3 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 27 hours after the fermentation started, and the feeding rate was 2L/h. The total feeding volume is 4L.

The third strategy (S33) is NaCl continuous flow feeding. The feeding liquid K235C3 is added from 8h to 27h after the start of fermentation. The feeding speed is 200mL/h, and the feeding volume is 20h in total, and the feeding volume is 4L.

The fourth strategy (S34) NaCl is fed intermittently, and the feeding liquid K235B3 is added from 8h to 27h after the start of fermentation. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation. The feeding speed was 0.5L/h, feeding 1 hour, and the NaCl feeding amount was 5g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.5L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The fifth strategy (S35) NaCl is fed intermittently, and the feeding liquid K235B3 is added from 8h to 27h after the fermentation starts. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation, the feeding speed was 1.0L/h, the feed was fed for 1 hour, and the NaCl feeding amount was 10g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 1.0L/h, the feeding is 1h, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

The sixth strategy (S36) NaCl is fed intermittently, and the feeding liquid K235B3 is added from 8h to 27h after the fermentation starts. The feeding speed is 100mL/h, and the feeding is 20h. NaCl aqueous solution D1 was added 22 hours after the start of fermentation. The feeding speed was 1.5L/h, feeding 1 hour, and the NaCl feeding amount was 15g/L fermentation medium; NaCl aqueous solution D1 was added 27 hours after the fermentation started. The fed feeding speed is 0.5L/h, the feeding is 1 hour, and the cumulative feeding amount of NaCl is 20g/L fermentation medium.

Calculate the polysialic acid yield (g/L) at the end of fermentation (at the end of 31 hours after the start of fermentation).

3. Results

The polysialic acid yields produced by the fermentation of E. coli K1, E. coli K92, and E. coli K235 under different NaCl feeding strategies are shown in Figure 4, Figure 5, and Figure 6 respectively. Among them, S11, S21, and S31 are NaCl-free feeding strategies; S12, S22, and S32 are NaCl feeding strategies at a single time point 5 hours before the end of fermentation; S13, S23, and S33 are NaCl continuous flow feeding strategies; S14~ S16, S24~S26, and S34~S36 are strategies for intermittent NaCl feeding. There are certain differences in the polysialic acid produced by different strains and different NaCl feeding strategies. Among them, the polysialic acid yield of NaCl intermittent feeding is slightly higher than the polysialic acid yield of NaCl fed at a single time point. The yield of polysialic acid with continuous NaCl fed feeding is ≤ NaCl fed at a single time point, but compared with feeding without NaCl, the yield of polysialic acid is still increased by 7.47% (E. coli K1) and 7.61% (E. coli) respectively. K92), 7.02% (E. coli K235).

4. Conclusion

The NaCl feeding strategy for polysialic acid produced by E. coli fermentation can choose intermittent feeding or continuous feeding. Intermittent feeding is slightly better than continuous feeding.

Example 3 Effect of temperature and pH control combined with NaCl fed feeding on the yield of polysialic acid produced by E. coli K235 fermentation

1. Strains

Escherichia coli K235 (deposit number: ACTT13027).​

2. Culture medium and culture methods

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 10, beef extract 3, yeast extract 2, NaCl 5, the balance is water.

Fermentation medium (g/L): sorbitol 60, (NH ₄ ) ₂ SO ₄ 5, K ₂ HPO ₄ ·3H ₂ O 5, MgSO ₄ 0.9, tryptone 1.5, the balance is water.

Feeding solution K235C4 (g/L): sorbitol 600, (NH ₄ ) ₂ SO ₄ 50, NaCl 200;

Culture steps and culture conditions:

(1) Inoculate E. coli into the first-level seed culture medium, and culture it for 12 hours at 37°C, pH 7.0, and shaker speed 150 rpm to obtain the first-level seed culture medium.

(2) Inoculate the primary seed culture liquid into the secondary seed culture medium at an inoculation volume of 4% (V/V), and culture it for 12 hours at 37°C, pH 7.0, and 200 rpm to obtain the secondary seed culture liquid, that is, the seed liquid.

(3) Add 12L fermentation medium to the 20L fermentation tank, and inoculate the seed liquid into the fermentation tank containing the fermentation medium at an inoculation amount of 4% (V/V); fermentation culture is carried out under the fermentation conditions of stirring speed 300rpm and ventilation volume 2vvm 24h. Feeding solution K235C4 was added from 6h to 20h after the start of fermentation. The feeding speed was 80mL/h, and the feeding volume was 15h in total. The feeding volume was 1.2L. The fermentation temperatures were selected as 34°C, 37°C, and 41°C. The pH value is selected as 6.4, 7.1, 8.0 (when the pH is lower than the selected value 0.4~0.6, add NaOH to increase it to the selected value, such as 6.4, 7.1, 8.0). Use the orthogonal design function of SPSS software to generate an orthogonal experiment table, conduct experiments according to the orthogonal experiment table, and determine the polysialic acid difference rate at the end of 24 hours under different conditions. Analysis of variance (univariate analysis in general linear models. LSD method was used for multiple comparisons between groups) was used in SPSS software to examine the effects of temperature and pH on the yield of polysialic acid.

3. Results and conclusions

This experiment examined the effects of temperature and pH on the yield of polysialic acid when the NaCl feeding amount and feeding speed were the same. According to the factor level setting of 2 factors and 3 levels, SPSS software generated an orthogonal experiment table and conducted 9 experiments. The results of orthogonal experiments are shown in Table 1. As shown in Table 2, within the selected factor level range and other fermentation culture conditions, the effect of temperature on the yield of polysialic acid is not significant (p≥0.05), and the effect of pH on the yield of polysialic acid is significant (p< 0.05). Multiple comparisons were made on the yield of polysialic acid under different pH conditions. As shown in Table 3, the yield of polysialic acid under pH 6.4 was higher than that at pH 7.1 and pH 8.0 (p<0.05).

The above Example 1 demonstrates the effect of NaCl feeding on the yield of polysialic acid under the same fermentation conditions; Example 2 demonstrates the impact of different NaCl feeding strategies on the yield of polysialic acid; Example 3 investigates When the NaCl feeding amount and feeding speed are the same, the effects of temperature and pH on the yield of polysialic acid. In summary, it can be seen from the above examples that in the process of producing polysialic acid by E. coli fed-batch fermentation, NaCl is fed intermittently or continuously, and the feeding amount is 20g/L fermentation medium to 35g/L fermentation medium; The feeding rate is 9h to 10h before the end of fermentation culture. The cumulative feeding amount of NaCl is 5g/L fermentation medium to 15g/L fermentation medium; the cumulative feeding amount of NaCl 4h to 6h before the end of fermentation culture is 20g/L fermentation. Medium ~35g/L fermentation medium can increase the yield of polysialic acid. When the NaCl feeding amount and feeding speed are the same, the temperature in the range of 34°C to 41°C has little effect on the polysialic acid yield. An increase in pH in the pH range of 6.4 to 8.0 can lead to a decrease in the polysialic acid yield. However, as shown in Examples 1 and 2, under the same temperature and pH conditions, NaCl fed feeding is still beneficial to improving the yield of polysialic acid.

Table 1 Orthogonal experiment results

Table 2 Test of inter-subject effects

^a : R-squared = .977 (adjusted R-squared = .955).

Table 3 Multiple comparisons

*:p<0.05.

Industrial applicability

The invention is used in the industrial production of polysialic acid and has industrial practicability.

Sequence Listing Free Content

This invention does not relate to sequence listings.

Claims (16)

 A feeding liquid for producing polysialic acid, containing a carbon source of 20g/L to 800g/L, characterized in that the feeding liquid contains NaCl; the concentration range of NaCl in the feeding liquid is 150g/L ~200g/L; the carbon source is selected from at least one of glucose, glycerol, sorbitol, and xylose. The feeding liquid according to claim 1, characterized in that the feeding liquid contains 12g/L to 300g/L nitrogen source, and the nitrogen source is selected from the group consisting of corn steep starch, yeast extract, tryptone, L- At least one of proline, asparagine, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ . The feeding liquid according to claim 2, wherein the concentration range of the carbon source in the feeding liquid is 60g/L to 800g/L, and the concentration range of the nitrogen source in the feeding liquid is 50g/L. ~300g/L. The feeding liquid according to claim 3, characterized in that the carbon source in the feeding liquid is selected from one of glucose, sorbitol and xylose; the nitrogen source in the feeding liquid is selected from L -One of proline, asparagine, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ . A fed-batch fermentation production method using Escherichia coli to produce polysialic acid, characterized in that the fed-batch fermentation production method includes the following steps: F1: Inoculate the E. coli seed liquid into the fermentation tank containing fermentation medium; F2: Fermentation culture is carried out under the fermentation conditions of 32℃~42℃, pH 6.4~8.0, stirring speed 75rpm~700rpm, and ventilation volume 0.5vvm~2vvm; F3: During the fermentation culture process of step F2, feed materials are fed into the fermentation tank; the ingredients of the fed feed materials include: NaCl and carbon source; the feeding amount of NaCl is 20g/L fermentation medium ~ 40g/L Fermentation medium; the feeding rate of NaCl is: the cumulative feeding amount of NaCl 4h ~ 8h before the end of fermentation culture is 20g/L fermentation medium ~ 40g/L fermentation medium; the carbon source of fed feeding At least one selected from the group consisting of glucose, xylose, glycerol, and sorbitol; the NaCl is fed by continuous flow or intermittent flow. The fed-batch fermentation production method according to claim 5, characterized in that the feeding amount of NaCl is 20g/L fermentation medium to 35g/L fermentation medium; the feeding speed of NaCl is, The cumulative feed amount of NaCl 4h to 8h before the end of fermentation culture is 20g/L fermentation medium to 35g/L fermentation medium. The fed-batch fermentation production method according to claim 6, characterized in that the feeding speed of NaCl is, and the cumulative feeding amount of NaCl 9h~10h before the end of fermentation culture is 5g/L fermentation medium~15g /L fermentation medium; the cumulative feed amount of NaCl 4h to 6h before the end of fermentation culture is 20g/L fermentation medium to 35g/L fermentation medium. The fed-batch fermentation production method according to claim 5, characterized in that in step F3, the ingredients of the fed feed include a nitrogen source, and the nitrogen source of the fed feed is selected from the group consisting of corn steep starch, yeast extract, At least one of tryptone, L-proline, aspartic acid, asparagine, NH ₄ Cl, (NH ₄ ) ₂ SO ₄ . The fed-batch fermentation production method according to claim 8, characterized in that the fermentation culture temperature in step F2 is 34°C~42°C, the pH is 6.4~7.1, and the stirring speed is 150rpm~700rpm; and supplementary flow is added in step F3. The carbon source of the feed is selected from one of glucose, xylose and sorbitol; the nitrogen source of the fed feed in step F3 is selected from NH ₄ Cl, L-proline, (NH ₄ ) ₂ SO ₄ and asparagine. One of the above; in step F3, the concentration of the carbon source in the feeding liquid used during fed feeding is 20g/L ~ 800g/L; the concentration of the nitrogen source in the feeding liquid used during fed feeding is 12g/L. ~300g/L. The fed-batch fermentation production method according to claim 9, characterized in that, in step F3, the concentration of the carbon source in the feeding liquid used during fed feeding is 60g/L ~ 800g/L; fed feeding The concentration of nitrogen source in the feeding solution used is 50g/L ~ 300g/L. The fed-batch fermentation production method according to claim 9, wherein the E. coli strain used in the fed-batch fermentation production is selected from the group consisting of E. coli K235 WXJYL-11, E. coli K235 WXJ4, and E. coli K235- JYII-74, Escherichia coli K235 6E61, Escherichia coli SA-8, Escherichia coli CASOV-8, Escherichia coli ATCC13027, Escherichia coli H03A2190830, Escherichia coli GX124, Escherichia coli K1, Escherichia coli LP 1674, Escherichia coli K92, Escherichia coli C8 any kind. The fed-batch fermentation production method according to claim 9, characterized in that the fermentation medium in step F1 contains carbon source, nitrogen source, K ₂ HPO ₄ or K ₂ HPO ₄ hydrate 0.5g/L to 27g /L, MgSO ₄ or MgSO ₄ hydrate 0.15g/L ~ 1.5g/L; the carbon source of the fermentation medium is selected from at least one of sorbitol, glycerol, glucose and xylose; the fermentation medium The nitrogen source is selected from at least one of NH ₄ Cl, (NH ₄ ) ₂ SO ₄ , corn steep starch, yeast extract, tryptone, L-proline, aspartic acid, and asparagine. The fed-batch fermentation production method according to claim 12, characterized in that the carbon source of the fermentation medium is selected from at least one of the following components: sorbitol 10g/L~60g/L, glucose 7g/L , xylose 8g/L ~ 15g/L; the nitrogen source of the fermentation medium is selected from at least one of the following components: (NH ₄ ) ₂ SO ₄ 5g/L, yeast extract 10g/L, tryptone 1.5g /L～10g/L, L-proline 17g/L～19g/L, asparagine 9g/L～12g/L. The fed-batch fermentation production method according to claim 9, characterized in that the fermentation medium contains at least one of the following components: FeSO ₄ or its hydrate, CuSO ₄ or its hydrate, CaCl ₂ or its hydrate Hydrate, K ₂ SO ₄ or its hydrate, KH ₂ PO ₄ or NaH ₂ PO ₄ . The fed-batch fermentation production method according to claim 9, characterized in that the preparation method of the seed liquid in step F1 includes the following steps: Z1: Inoculate Escherichia coli into the first-level seed culture medium, culture it at 34°C to 42°C, pH 6.4-7.8, and shaker speed 150rpm-300rpm for 6h-12h to obtain the first-level seed culture medium; Z2: Inoculate the primary seed culture liquid into the secondary seed culture medium, and cultivate it for 6 to 12 hours at 34°C to 42°C, pH 6.4 to 7.8, and 150rpm to 300rpm to obtain the secondary seed culture liquid, that is, the seed liquid. The fed-batch fermentation production method according to claim 15, characterized in that the first-level seed culture medium and the second-level seed culture medium are selected from any one of the following culture media: M1: Tryptone 8g/L~12g/L, yeast extract 4g/L~10g/L, NaCl 1g/L~12g/L, the balance is water; M2: tryptone 10g/L, beef extract 2g/L~5g/L, NaC1 5g/L, the balance is water; M3: Tryptone 10g/L, beef extract 2g/L~5g/L, NaC1 5g/L, yeast extract 2g/L, the balance is water; M4: Glucose 25g/L, (NH ₄ ) ₂ SO ₄ 5g/L, tryptone 52g/L, K ₂ HPO ₄ 20g/L, MgSO ₄ 0.4g/L, and the balance is water.