CN115125278B

CN115125278B - Feed supplement liquid for producing polysialic acid and preparation method of polysialic acid

Info

Publication number: CN115125278B
Application number: CN202211023824.1A
Authority: CN
Inventors: 宗剑飞; 张小凤; 肖卫华
Original assignee: Shandong Synthetic Vision Biotechnology Co ltd
Current assignee: Shandong Synthetic Vision Biotechnology Co ltd
Priority date: 2022-08-25
Filing date: 2022-08-25
Publication date: 2022-11-18
Anticipated expiration: 2042-08-25
Also published as: CN115125278A; WO2024041228A1

Abstract

The invention relates to the technical field of microbial fermentation, in particular to a feed supplement liquid for producing polysialic acid and a preparation method of the polysialic acid. By investigating the dynamic behavior of NaCl stress on the production of polysialic acid by fermentation of escherichia coli, the invention provides a production method for improving the yield of polysialic acid by feeding NaCl, wherein the feeding amount of NaCl fed-batch feeding is 20-40 g/L of fermentation medium, and the feeding amount is reached 4-6 h before the end of fermentation, so that the improvement on the yield of polysialic acid is particularly remarkable. Furthermore, the invention provides a NaCl-containing feed liquid and application thereof in production of polysialic acid by fed-batch fermentation of escherichia coli. The production method is favorable for improving the yield of the polysialic acid and is favorable for ethanol precipitation and separation of the polysialic acid after fermentation.

Description

Feed supplement liquid for producing polysialic acid and preparation method of polysialic acid

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a feed supplement liquid for producing polysialic acid and a preparation method of the polysialic acid.

Background

Polysialic acid (PSA) is a polyanionic linear exopolysaccharide consisting of alpha- (2, 8) and/or alpha- (2, 9) -linked sialic acid and having a degree of polymerization of about 8 to 400. The polysialic acid has important physiological and pharmacological effects, and has potential application value in the aspects of drug delivery systems and drug macromolecule modification. For example, polysialic acid can promote nerve development and regeneration, and can also replace polyethylene glycol to be used for modifying macromolecular drugs such as protein and the like to improve the half-life period of the drug; can also be used as a framework material of a gel drug release system to delay the in vivo release speed of the drug. In addition, sialic acid can be produced by acid hydrolysis of polysialic acid, and is an important intermediate for preparing sialic acid by fermentation. Sialic acid has been approved as a new food material in countries/regions of the European Union, japan, malaysia, singapore, china, etc.

Polysialic acid is mainly prepared by fermentation of escherichia coli, including escherichia coli K1, escherichia coli K12, escherichia coli K92, escherichia coli K235, etc. (GONZ _ LEZ-clemate,et al. High production of polysialic acid [Neu5Ac alpha(2-8)-Neu5Ac alpha(2-9)]n by Escherichia coli K92 grown in a chemically defined medium. Regulation of temperature[J]biological Chemistry Hoppe-Seyler, 1990, 371 (2): 1101-1106;). The optimal carbon and nitrogen sources required for the synthesis of polysialic acid by different E.coli vary slightly. Various carbon sources can enter escherichia coli cells through a phosphoenolpyruvate transferase system or a NanT transporter, polysialic acid is synthesized through complex biochemical reactions and a polysaccharide assembly way, and the generated polysialic acid is finally transported to the outside of cells through KpsS, kpsD and the like. In the de novo synthesis of polysialic acid by E.coli, the genes involved in the synthesis of polysialic acid includekpsF、kpsM、kpsTEtc., but the mechanism of initiation of polysialic acid synthesis in E.coli is not known (Pavelka MS,et al. Identification of two genes, kpsM and kpsT, in region 3 of the polysialic acid gene cluster of Escherichia coli K1.[J]. Journal of Bacteriology, 1991, 173(15):4603-4610；Rohr TE, et al. Structure and biosynthesis of surface polymers containing polysialic acid in Escherichia coli[J]journal of Biological Chemistry, 1980, 255 (6): 2332-2342.). Various natural Escherichia coli strains or Escherichia coli mutagenized strains have been used for fermentative production of polysialic acid, and Escherichia coli K235 has been studied more extensively in recent years (CN 101195661A, CN112553120A, etc.).

The process for preparing polysialic acid by utilizing escherichia coli fermentation has more reports. In the published literature, carbon sources for the fermentative production of polysialic acid include glucose, glycerol, sorbitol, xylose and the like; organic and inorganic nitrogen sources include: corn steep liquor starch, yeast extract, tryptone, L-proline, aspartic acid, asparagine, NH ₄ Cl、(NH ₄ ) ₂ SO ₄ And the like. The combination of different carbon and nitrogen sources affects the synthesis of polysialic acid. Wherein the carbon source is sorbitol which is relatively common; the nitrogen source is selected from corn steep liquor starch, yeast extract, tryptone, NH ₄ Cl、(NH ₄ ) ₂ SO ₄ Is more commonly used. Coli grows well when glucose is used as a carbon source, but acetate produced by metabolism lowers the pH of the culture solution, thereby promoting degradation of polysialic acid or inhibiting growth of the cells. When xylose is used as the carbon source,the pyruvate content in the culture is higher (Wu JR,et al. Dipotassium phosphate improves the molecular weight stability of polysialic acid in Escherichia coli K235 culture broth[J]bioresource Technology: biomass, bioenergy, biowales, conversion Technologies, biotransformations, production Technologies, 2018, 247, 30-35; effect of the acetic and potassium ion Strength of Asahando et al on Polysialic acid Synthesis and Process molecular Mass variation [ J]Food and fermentation industry, 2017, 43 (7): 6.).

On the basis of screening and optimizing a large amount of carbon sources, nitrogen sources and the like of a culture medium, batch fermentation and fed-batch fermentation of polysialic acid are compared in the field, and fed-batch fermentation is found to be beneficial to improving the polysialic acid fermentation yield. Fed-batch fermentation can overcome many defects of batch fermentation, such as delaying bacterial aging and the like; the ingredients of the supplement include carbon source, nitrogen source, water and other substances (Cao Cowar. Microbiol. Engineering [ M ]. Beijing: 2 nd edition 2007).

On the basis, in order to further improve the polysialic acid fermentation yield, in the field, the dynamic regulation and control of polysialic acid fermentation culture conditions are also widely researched in recent years, and the dynamic regulation and control of temperature, pH and stirring speed in the fermentation culture process are included. It has been found that phased control of the above criteria is also beneficial in increasing polysialic acid fermentation yield (Zheng ZY,et al. A new polysialic acid production process based on dual-stage pH control and fed-batch fermentation for higher yield and resulting high molecular weight product[J]. Applied Microbiology & Biotechnology, 2013, 97(6):2405-2412；Zhan X, et al. Production of polysialic acid from fed-batch fermentation with pH control[J]biochemical Engineering Journal, 2002, 11 (2-3): 201-204; study on preparation of polysialic acid from Liu jin Long by microbial fermentation method [ D]South of the Yangtze university, 2011; CN 109182423A). In addition, stress of sodium pyruvate and hydrogen peroxide is also beneficial to improve the fermentation yield of polysialic acid (CN 104046671A; lichun, et al]The Chinese food journal 2015 (7): 6). But at present polySialic acid fermentation yields are still low and need to be further improved.

Disclosure of Invention

In order to solve the technical problems, the invention researches the kinetic characteristics of the polysialic acid produced by fermenting escherichia coli under the NaCl stress condition, improves the process for producing the polysialic acid by fermenting the escherichia coli on the basis of the existing polysialic acid fermentation process, and further improves the fermentation yield of the polysialic acid.

One of the technical schemes of the invention provides a fed-batch fermentation production method for producing polysialic acid by using escherichia coli. Compared with the publicly reported polysialic acid fed-batch fermentation production method, the publicly reported fed-batch fermentation production method has the advantages that the fed-batch ingredients in the fermentation culture process are mainly carbon sources or a mixture of the carbon sources and nitrogen sources; the typical characteristics of the fed-batch fermentation production method are as follows: the components fed during the fermentation culture process also contain NaCl, so that the fermentation yield of the polysialic acid is further improved.

Further, naCl may be fed continuously or intermittently. In a preferred scheme, the NaCl is fed-batch fed. In another preferred scheme, the NaCl is fed continuously.

Further, when feeding materials, the feeding amount of NaCl is as follows: 20g/L fermentation medium-40 g/L fermentation medium. In a preferred embodiment, the amount of NaCl fed is: 20g/L fermentation medium to 35g/L fermentation medium. The term "fermentation medium", also known in the art as "fed-batch fermentation basal medium", refers to the medium added to the fermentor prior to the start of the fermentation culture. Based on published reports, it will be appreciated by those skilled in the art that in fed-batch fermentation, the total volume of the fermentation broth will typically not exceed 70% of the volume of the fermentor and the volume of fermentation medium will typically be 60% of the volume of the fermentor. I.e.the feed volume does not usually exceed 10% of the fermenter volume. In other words, the amount of NaCl fed can be expressed as the concentration of NaCl in the fermentation broth, depending on the volume of the fermentation medium and the final volume of the fermentation broth at the end of the fermentation. In fed-batch fermentation, the fermentation broth in the fermentor consists of a fermentation medium, an inoculated seed solution, a feed solution, and the like. When the volume of the fermentation medium is V1 (unit: L), the volume of the fermentation liquid in the fermentation tank is V2 (unit: L), and the NaCl supplement amount calculated on the basis of the fermentation medium is C1 (unit: g/L), the NaCl supplement amount expressed as the fermentation liquid is C2 (unit: g/L) = (V1 xC 1)/V2.

Based on published reports, the skilled person will know that the feed rate depends on feed volume, concentration of aqueous solution for feed, etc. The higher the concentration of the fed-batch aqueous solution is, the smaller the feeding volume and the slower the feeding speed is. And vice versa. In the fed-batch fermentation process, the components required to be fed-batch are added into water to prepare an aqueous solution, and the feeding is carried out in the form of the aqueous solution in the fermentation culture process. The term "aqueous feeding solution" refers to an aqueous solution for feeding prepared from the components requiring feeding and water. Wherein when the aqueous solution for feeding contains a carbon source, it is customarily called as a feed solution or a feed solution. When different feeding components are fed separately, the "aqueous feeding solution" is usually divided into feeding solution and other aqueous feeding solutions, such as aqueous pH regulator solution, and H ₂ O ₂ Aqueous solution, naCl aqueous solution. Based on the difference of feeding modes, the difference of feeding speeds, the difference of feeding ingredients, and the like, in the same fermentation culture process, a plurality of types of 'aqueous solutions for feeding' can be prepared to respectively perform feeding, for example, a component for adjusting the pH value is prepared into a single aqueous solution, a carbon source is prepared into a single aqueous solution, and the two are respectively subjected to feeding operation. This is a routine operation in the art. It follows that the term "feed solution" when used in the context of the present invention particularly refers to "aqueous feed solution" comprising a carbon source. When the term "fed aqueous solution" is used in this context, it refers to feed solutions and other fed aqueous solutions.

NaCl is not an energy source substance of Escherichia coli, and can be absorbed but not metabolized. I.e. the fermentation broth is cumulative for NaCl. Based on this, in a preferred embodiment, the NaCl feeding rate is: the accumulated supplement amount of NaCl 4-8 h before the fermentation culture is finished is 20-40 g/L of fermentation medium. Further preferably, the NaCl feeding rate is: the accumulated supplement amount of NaCl 4-8 h before the fermentation culture is finished is 20-35 g/L of fermentation medium. Still further preferably, the NaCl feed rate is: the accumulated supplement amount of NaCl 4-6 h before the fermentation culture is finished is 20-35 g/L of fermentation medium. Still further preferably, the NaCl feeding rate is: the accumulated supplement amount of NaCl within 9-10 h before the fermentation culture is finished is 5-15 g/L of fermentation medium; the accumulated supplement amount of NaCl 4-6 h before the fermentation culture is finished is 20-35 g/L of fermentation medium.

In the reported method for producing polysialic acid by fed-batch fermentation, the ingredients of fed-batch fermentation include carbon source, nitrogen source, pH regulator (such as NaOH, ammonia water, liquid ammonia), hydrogen peroxide, etc. In most of the methods disclosed and reported therein, a carbon source and a nitrogen source are added into water to prepare an aqueous solution as a feed liquid, i.e., the carbon source and the nitrogen source are synchronously supplemented. In some published reports, the composition of the fed batch feed does not include a nitrogen source. In the reported polysialic acid fed-batch fermentation production method, a carbon source and a nitrogen source are fed in a batch feeding mode or a continuous feeding mode. The pH regulator (such as NaOH and ammonia water) is used for regulating and controlling the pH of the fermentation liquor at different stages, and fed-batch feeding is carried out in an intermittent fed-batch mode, and the fed-batch feeding is usually not synchronously fed-batch with a carbon source and the like. In the published report, hydrogen peroxide is fed in a batch mode. NaCl can be fed in batch or continuously, so that NaCl can be used together with the above-mentioned feeding components and feeding mode. For example, naCl and a carbon source or NaCl, a carbon source and a nitrogen source can be added into water, so that synchronous feeding is convenient (an intermittent feeding mode or a continuous feeding mode can be adopted); or preparing NaCl and hydrogen peroxide into an aqueous solution for synchronous fed-batch (intermittent fed-batch mode can be adopted); or preparing aqueous solution synchronous flow feeding materials from NaCl, a carbon source and hydrogen peroxide or preparing aqueous solution synchronous flow feeding materials from NaCl, the carbon source, the nitrogen source and the hydrogen peroxide (an intermittent flow feeding mode can be adopted).

As to the aforementioned fed-batch fermentative production method for producing polysialic acid using Escherichia coli, the present invention further provides a fed-batch fermentative production method for polysialic acid, comprising the steps of:

f1: inoculating the escherichia coli seed liquid into a fermentation tank containing a fermentation culture medium;

f2: carrying out fermentation culture under the fermentation conditions of 32-42 ℃, pH6.4-8.0, stirring speed of 75-700 rpm and ventilation volume of 0.5-2 vvm;

f3: feeding materials into the fermentation tank in the fermentation culture process of the step F2; the feed supplement comprises the following components: naCl and carbon source. The NaCl feed and feed rate were as described above. The carbon source of the fed-batch material is at least one of glucose, xylose, glycerol and sorbitol.

In a preferable scheme, the fermentation culture temperature of the step F2 is 34-42 ℃, the pH value is 6.4-7.1, and the stirring speed is 150-700 rpm. In another preferred embodiment, the composition of the feed stream in step F3 further comprises a nitrogen source. The nitrogen source of the feed supplement is selected from corn steep liquor starch, yeast extract, tryptone, L-proline, aspartic acid, asparagine, NH ₄ Cl、(NH ₄ ) ₂ SO ₄ At least one of (1). In a further preferred embodiment, the carbon source fed in the step F3 is selected from one of glucose, xylose and sorbitol; the nitrogen source of the fed batch is selected from NH ₄ Cl, L-proline, (NH) ₄ ) ₂ SO ₄ And asparagine. Based on published reports, it will be appreciated by those skilled in the art that the selection of carbon and nitrogen sources in the composition of the feed stream is related to the metabolic properties of E.coli itself, e.g., xylose, L-proline as carbon and nitrogen source for E.coli K1, respectively, is beneficial for increasing polysialic acid yield, glucose and (NH) ₄ ) ₂ SO ₄ Respectively used as a carbon source and a nitrogen source of Escherichia coli K1, wherein the yield of the polysialic acid is slightly lower than that of the combination of xylose and L-proline; the glucose and the L-proline are respectively used as a carbon source and a nitrogen source of the Escherichia coli K92, so that the polysialic acid yield is improved. And Escherichia coli K235, escherichia coli C8, escherichia coli SA8, escherichia coli CASOV-8, etc. with glucose or sorbitol as carbon source, NH ₄ Cl or (NH) ₄ ) ₂ SO ₄ The nitrogen source is favorable for improving the yield of the polysialic acid.

Further, in the step F3, the concentration of the carbon source in the feed supplement liquid used in the feed supplement is 20 g/L-800 g/L; the concentration of the nitrogen source in the feed liquid used in the feed-batch process is 12 g/L-300 g/L. Further preferably, in the step F3, the concentration of the carbon source in the feeding liquid used in the feeding is 60 g/L-800 g/L carbon source; the concentration of the nitrogen source in the feed liquid used in the feed-batch process is 50 g/L-300 g/L.

Further, the feed of step F3 may further comprise a pH regulator and/or hydrogen peroxide. The pH regulator is selected from one of NaOH, ammonia water and liquid ammonia.

In the step F3, the concentration ranges of the carbon source and the nitrogen source in the feed liquid used in the fed-batch process are the conventional ranges in the production of polysialic acid by fermentation of Escherichia coli in the art. As mentioned above, the rate of feeding the feed stream depends on the volume of feed, the concentration of feed solution, etc. The higher the feed liquid concentration is, the smaller the required feed volume is, and the slower the required feed rate is. The skilled person is capable of making reasonable selection of the feeding time, feeding speed, feeding concentration, feeding amount, etc. of the carbon source and nitrogen source according to the consumption rate of the carbon source and nitrogen source during the fermentation culture. In a fed-batch fermentation production method for producing polysialic acid using E.coli, the concentrations of carbon and nitrogen sources, the feeding rate, the feeding time, and the like in a typical and preferred part of the feed solution can be found in, but not limited to, the following documents:

(1) Wu JR, et al. Dipotassium phosphate improves the molecular weight stability of polysialic acid in Escherichia coli K235 culture broth[J]. Bioresource Technology: Biomass, Bioenergy, Biowastes, Conversion Technologies, Biotransformations, Production Technologies, 2018, 247:30-35.

(2) Juan in Zhou Asia, purification of polysialic acid in fermentation broth, and amplification study [ D ] Jiangnan university, 2013.

(3) Zhan Xingbei, zhengzhiyong, zhuli, et al, influence of pH value on polysialic acid batch fermentation and fed-batch fermentation [ J ] reported by Wuxi university of California food and Biotechnology, 2001.

(4) Lechun, et al. Influence of sodium pyruvate in combination with fed-batch fermentation on sialic acid production [ J ] Chinese food bulletin 2015 (7): 6.

(5) Liu JL, et al. A new strategy to enhance polysialic acid production by controlling sorbitol concentration in cultivation of Escherichia coli K235[J]African Journal of Biotechnology, 2012, 9 (16) the DOI of this document is: 10.1186/1475-2859-9-23.

(6) Yan Xia. Study of Escherichia coli K235 carbon source metabolism regulating polysialic acid biosynthesis [ D ]. University of Jiangnan, 2015.

(7) CN104046671A。

The temperature, pH, stirring speed, aeration rate and other parameters of the fermentation culture in the step F2 are in the ranges conventional in the art for producing polysialic acid by fermentation of Escherichia coli. Wherein, the temperature, the pH, the stirring speed and the ventilation capacity can be regulated and controlled in stages, and the following documents can be found in a concrete way:

(1) Liujinlong, research on preparation of polysialic acid by microbial fermentation [ D ] Jiangnan university, 2011.

(3) Influence of pH value on polysialic acid batch fermentation and fed-batch fermentation [ J ] reported by university of Wuxi light industry, food and biotechnology 2001.

(4) Zheng ZY, et al. A new polysialic acid production process based on dual-stage pH control and fed-batch fermentation for higher yield and resulting high molecular weight product[J]. Applied Microbiology & Biotechnology, 2013, 97(6):2405-2412.

(5) Zhan X, et al. Production of polysialic acid from fed-batch fermentation with pH control[J]. Biochemical Engineering Journal, 2002, 11(2-3):201-204.

Further, the Escherichia coli strain used in the fed-batch fermentation production is selected from any one of Escherichia coli K235 WXJYL-11, escherichia coli K235 WXJ4, escherichia coli K235-JYIII-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 and Escherichia coli C8. Wherein the Escherichia coli K235 WXJYL-11, escherichia coli K235 WXJ4, escherichia coli K235-JYII-74, escherichia coli K235 6E61 and Escherichia coli ATCC13027 are all Escherichia coli K235 or obtained by mutagenesis of Escherichia coli K235E 6E 61. The above strains and their deposit numbers, known methods and media for fermentative production of polysialic acid, etc. can be found in the following documents:

study of fermentation kinetics of Escherichia coli K235 WXJYL-11 (CN 1916010A), escherichia coli K235 WXJ4 (Zhanxianbei, et al. [ J ] polysialic acid ]]An industrial microorganism, 2001, 31 (1): 16-18.), escherichia coli K235-JYII-74 (CN 1896263A; effect of Zhanxianbei et al NTG on the production of polysialic acid by E.coli mutants [ J]Journal of Wuxi university of California, 2002. DOI: CNKI SUN WXQG.0.2002-05-004), escherichia coli K235 6E61 (accession number: CCTCC M208088; the amount of the nitrogen atom in the tin Z, et al. New high-density fermentation method for producing high molecular weight polysialic acid based on the combination fermentation strategy[J]. Applied Microbiology and Biotechnology, 2022, 106(7):2381-2391；Liu JL, et al. An efficient and large-scale preparation process for polysialic acid by Escherichia coli CCTCC M208088[J]biochemical Engineering Journal, 2010, 53 (1): 97-103), E.coli SA-8 (accession number: CGMCC No.5585; CN103361283A; CN 111733092A), escherichia coli CASOV-8 (accession number: CCTCC M2018103; CN108588152A; US10301396B 2), escherichia coli K235 (deposit No. ATCC13027; i.e. plum, et al, the Effect of sodium pyruvate in combination with Fed fermentation on sialic acid production [ J]The Chinese food journal 2015 (7) is 6; CN 104046671A), escherichia coli H03a2190830 (accession number: CCTCC No. M2019900; CN 112553120A), escherichia coli GX124 (Guotin, et al., mutagenesis and fermentation Process study of Escherichia coli having high Polysialic acid production [ C]The thirteenth academic annual meeting of the society of bioengineering of china and the national institute of biotechnology 2019), escherichia coli K1 (accession number: DSM 107164; bartling B, et al. Determination of the structural integrity and stability of polysialic acid during alkaline and thermal treatment[J]Molecules, 2019, 25 (1): 165.), escherichia coli LP 1674 (US 8097437B 2), escherichia coli K92 (accession number: ATCC 35860; the concentration of the Navasa N is controlled,et al. Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92[J]applied Microbiology and Biotechnology, 2009, 82 (4): 721-729.), E.coli C8 (Guo L,et alscreening OF polysialic ACID-PRODUCING STRAINS OF studios ON THE SELECTION OF STRAINS PRODUCING COLOMINIC ACID AND CULTURE CONDITIONS AND ACID-PRODUCING CONDITIONS [ J ]]Microbiology, 1998 (2): 103-107).

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

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

The fermentation medium may also be further supplemented with trace elements including, but not limited to, feSO ₄ Or hydrate thereof, cuSO ₄ Or its hydrate, caCl ₂ Or hydrates thereof, K ₂ SO ₄ Or a hydrate thereof, KH ₂ PO ₄ Or NaH ₂ PO ₄ 。

Some typical and preferred fermentation medium formulations can be found in, but are not limited to: CN1916010A; CN104046671A; the Liu JL is a mixture of Liu JL,et al. A new strategy to enhance polysialic acid production by controlling sorbitol concentration in cultivation of Escherichia coli K235[J]african Journal of Biotechnology, 2012, 9 (16) (DOI: 10.1186/1475-2859-9-23); mutagenesis screening and fermentation optimization of high molecular weight polysialic acid producing strains [ J]Food and fermentation industry, 2019, 45 (10): 7; wujinyong, chenxiangsong, yuchao, etc. E.coli ion beam mutagenesis combined with respiratory quotient on-line real-time regulation of high yield polysialic acid [ J]. 2022 (14). Based on published reports, the skilled person will know that the choice of carbon and nitrogen sources in the fermentation medium is related to the metabolic properties of the escherichia coli itself, e.g. xylose, L-proline as carbon and nitrogen source, respectively, of escherichia coli K1 is beneficial for increasing polysialic acid yield; the glucose and the L-proline are respectively used as a carbon source and a nitrogen source of the Escherichia coli K92, so that the polysialic acid yield is improved. And Escherichia coli K235, escherichia coli C8, escherichia coli SA8, escherichia coli CASOV-8, etc. with glucose or sorbitol as carbon source, NH ₄ Cl or (NH) ₄ ) ₂ SO ₄ The nitrogen source is favorable for improving the yield of polysialic acid.

Further, in the step F1, the inoculation amount of the seed solution is 0.05% to 8% by volume percentage.

Further, in the step F1, 4.09 g/L-4.14 g/L of sodium pyruvate is further added into the fermentation tank. The fed-batch components in the step F3 further comprise hydrogen peroxide, and the feeding method of the hydrogen peroxide is shown in CN104046671A, namely feeding and feeding are respectively carried out according to the feeding amount of 2mmol/L fermentation medium, 4mmol/L fermentation medium, 8mmol/L fermentation medium and 8mmol/L fermentation medium in the 5h, 10h, 15h and 20h of fermentation culture. Sodium pyruvate and hydrogen peroxide prove to be beneficial to improving the yield of the polysialic acid produced by fermenting Escherichia coli K235.

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

z1: inoculating escherichia coli to a primary seed culture medium, and culturing for 6-12 h under the conditions of 34-42 ℃, pH 6.4-7.8 and shaking table rotating speed of 150-300 rpm to obtain a primary seed culture solution;

z2: inoculating the primary seed culture solution to a secondary seed culture medium, and culturing for 6-12 h under the conditions of 34-42 ℃, pH 6.4-7.8 and 150-300 rpm to obtain a secondary seed culture solution, namely a seed solution.

Preferably, the primary seed culture medium and the secondary seed culture medium are any one selected from the following culture media: m1: 8-12 g/L of tryptone, 4-10 g/L of yeast extract, 1-12 g/L of NaCl and the balance of water; m2: 10g/L of tryptone, 2-5 g/L of beef extract, 5g/L of NaC and the balance of water; m3: 10g/L of tryptone, 2-5 g/L of beef extract, 5g/L of NaC, 2g/L of yeast extract and the balance of 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 of water. Wherein M1 is a common LB culture medium and an improved culture medium thereof; m2 and M3 are common meat extract peptone culture medium and improved culture medium thereof; LB culture medium and its modified culture medium, meat extract peptone culture medium and its modified culture medium are common basic culture medium widely used in bacterial culture. M4 can be used for Escherichia coli SA-8 (preservation number: CGMCC No. 5585).

The cultivation temperature, pH, and the rotation speed of the rocking platform in the aforementioned steps Z1 and Z2 are the conventional ranges in the art for producing polysialic acid by fermentation using Escherichia coli. Some typical and preferred primary seed culture media, secondary seed culture media and culture conditions can be found in, but not limited to, CN108588152A, CN109136308A.

The yeast extract is also referred to as yeast extract powder, and the tryptone is also referred to as tryptone.

The above technical solution provides a fed-batch fermentation production method for producing polysialic acid using Escherichia coli, which may further comprise a step F4 of extracting and separating polysialic acid from the fermentation broth. Compared with the public reported polysialic acid fermentation production method, the fed-batch fermentation production method for producing polysialic acid by using the escherichia coli provided by the first technical scheme and the second technical scheme of the invention does not introduce new impurities. Thus, the polysialic acid can be isolated from the fermentation broth in step F4 by extraction, as is conventional in the art. Specifically, centrifugation is carried out at 3000 rpm-8000 rpm for 10 min-30 min, and then thallus in the fermentation liquor is removed by filtration (see CN 1896263A); or filtering with 50-300 nm ceramic membrane to remove thallus from the fermentation liquid (CN 109369730A); saturated CaCl may be used ₂ Removal of phosphate from fermentation broth (see Levenson. St. Sialic acid fermentation control and isolation and purification Studies [ D ]]South of the Yangtze university, 2005); can be obtained by ethanol precipitation or ultrafiltration and concentration combined with ethanol precipitation (see J extraction of sialic acid from E.coli fermentation broth]The journal of the Chinese pharmaceutical industry, 2003, 34 (001): 8-10.) or ethanol precipitation in combination with cetylpyridinium chloride complexation to isolate polysialic acid in the fermentation broth (see: purification of polysialic acid from fermentation broth and scale-up study [ D]South of the Yangtze university, 2013.); removing protein by adopting a Sevag method, a trichlorotrifluoroethane method, a trichloroacetic acid method or a centrifugation method (centrifugation at 15000 rpm-20000 rpm for 10 min-30 min); the effect of NTG on the yield of polysialic acid from E.coli mutants can be removed by dialysis, electrodialysis or ultrafiltration (see: CN111386350A; zhan-North, legionella, wujiarong, et al]The journal of food and biotechnology, 2002, 21 (5): 456-459. When polysialic acid is separated from fermentation broth by ethanol precipitation, naCl is fed to fed-batch fermentation method for producing polysialic acid using Escherichia coli (CN 101195661A; CN113005161A; yudanfeng, polysialic acid and ethanol solution) in the method according to one or both of the first and second aspects of the present inventionStudy of sialic acid extraction Process [ D]South of the Yangtze river university, 2008; study on preparation of polysialic acid by Liujinlong and microbial fermentation method [ D]University of south of the Yangtze river, 2011; purification Process and amplification study of polysialic acid in fermentation broth of Juan in Ardisia]South of the Yangtze university, 2013; ) (ii) a Decolorizing with activated carbon when fermentation medium contains MgSO ₄ When the sulfate is barium chloride or Ca (OH) ₂ Removing sulfate by precipitation.

On the basis of the fed-batch fermentation production method for producing polysialic acid by using escherichia coli, the second technical scheme of the invention provides a fed-batch fermentation liquid for producing polysialic acid by using escherichia coli, wherein the fed-batch fermentation liquid contains a carbon source or a carbon source and a nitrogen source, and the flowing and adding liquid also contains NaCl; the concentration of NaCl in the flowing liquid is 150 g/L-200 g/L.

Furthermore, the feed liquid contains 20 g/L-800 g/L of carbon source, or 20 g/L-800 g/L of carbon source and 12 g/L-300 g/L of nitrogen source.

Furthermore, the feed liquid contains 60 g/L-800 g/L of carbon source, or 60 g/L-800 g/L of carbon source and 50 g/L-300 g/L of nitrogen source.

Further, the carbon source in the feed liquid is selected from at least one of glucose, glycerol, sorbitol and xylose; the nitrogen source in the feed liquid is selected from corn steep liquor starch, yeast extract, tryptone, L-proline, asparagine and NH ₄ Cl、(NH ₄ ) ₂ SO ₄ At least one of (1). In a preferred scheme, the carbon source in the feed liquid is selected from one of glucose, sorbitol and xylose; the nitrogen source in the feed liquid is selected from L-proline, asparagine and NH ₄ Cl、(NH ₄ ) ₂ SO ₄ To (3) is provided.

Has the beneficial effects that:

(1) The fed-batch fermentation production method for producing the polysialic acid by using the escherichia coli improves the yield of polysialic acid fermentation by adding NaCl in a fed-batch manner in the fermentation culture stage.

(2) The fed-batch fermentation production method for producing the polysialic acid by using the escherichia coli, provided by the invention, has the advantages that the NaCl fed-batch fermentation production method is fed-batch in the fermentation culture stage, when the polysialic acid is separated by using an ethanol precipitation method, the precipitation speed of the polysialic acid in an ethanol solution can be promoted, and the separation of the polysialic acid in a fermentation liquid by using the ethanol precipitation method is facilitated.

(3) On the premise of meeting the requirement of escherichia coli on a carbon source, naCl is added to facilitate synchronous fed-batch of NaCl, so that the beneficial effect of improving the yield of polysialic acid by NaCl is exerted.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to assist in a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention unduly. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 is a graph showing the kinetics of cell concentration and polysialic acid productivity in Escherichia coli K1 fermentation culture in example 1; wherein the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, PS420 are in turn the polysialic acid yields (g/L) for fermentation media with NaCl supplementation amounts of 0, 15, 20, 25, 30, 35 g/L; wherein the solid lines OD0, OD180, OD240, OD300, OD360 and OD420 are respectively the cell concentrations corresponding to the fermentation medium with NaCl supplement amounts of 0, 15, 20, 25, 30 and 35g/L, that is, the OD values at the wavelength of 600 nm.

FIG. 2 is the dynamic curve diagram of the cell concentration and polysialic acid yield of the Escherichia coli K92 fermentation culture in example 1; wherein the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, PS420 are in turn the polysialic acid yields (g/L) for fermentation media with NaCl supplementation amounts of 0, 15, 20, 25, 30, 35 g/L; wherein the solid lines OD0, OD180, OD240, OD300, OD360 and OD420 are the cell concentrations corresponding to the fermentation medium with NaCl supplement amounts of 0, 15, 20, 25, 30 and 35g/L, that is, the OD value at the wavelength of 600 nm.

FIG. 3 is a graph showing the kinetics of cell concentration and polysialic acid productivity in the fermentation culture of Escherichia coli K235 in example 1; wherein the dotted lines PSA0, PSA180, PSA240, PSA300, PSA360, PS420 are in turn the polysialic acid yields (g/L) for fermentation media with NaCl supplementation amounts of 0, 15, 20, 25, 30, 35 g/L; wherein the solid lines OD0, OD180, OD240, OD300, OD360 and OD420 are the cell concentrations corresponding to the fermentation medium with NaCl supplement amounts of 0, 15, 20, 25, 30 and 35g/L, that is, the OD value at the wavelength of 600 nm.

FIG. 4 is a bar graph of polysialic acid yield of E.coli K1 with different NaCl feeding strategies according to example 2; wherein S11 is not supplemented with NaCl; s12: naCl is fed in a single-point flow and is fed in the 27h, and the NaCl feed amount is 20g/L of the fermentation culture medium; s13: naCl is continuously fed and supplemented, and is fed and added in 8 h-27 h, the concentration of the NaCl is 300g/L, and the supplementing speed of the NaCl is 200mL/h; s14: naCl is fed-batch material, and the accumulated feeding amount of NaCl in the 22 th hour and 27 th hour is 5g/L and 20g/L respectively; s15: naCl is fed-batch material, and the accumulated feeding amount of NaCl in the 22 th hour and 27 th hour is 10g/L and 20g/L respectively; s16: naCl is fed batch, and the cumulative feeding amount of NaCl at 22h and 27h is 15g/L and 20g/L respectively.

FIG. 5 is a bar graph of polysialic acid yield of E.coli K92 in example 2 with different NaCl feeding strategies; wherein S21 is not supplemented with NaCl; s22: adding NaCl as single-point feed supplement, and feeding NaCl in the 27 th hour with the amount of NaCl supplement being 20 g/L; s23: naCl is continuously fed and supplemented, and is fed and added in 8 h-27 h, the concentration of the NaCl is 300g/L, and the supplementing speed of the NaCl is 200mL/h; s24: naCl is fed-batch material, and the cumulative feeding amount of NaCl in 22h and 27h is respectively 5g/L and 20g/L of fermentation culture medium; s25: naCl is fed-batch material, and the accumulated feeding amount of NaCl in the 22 th hour and 27 th hour is 10g/L and 20g/L respectively; s26: naCl is fed-batch, and the cumulative feeding amount of NaCl at 22h and 27h is 15g/L and 20g/L respectively.

FIG. 6 is a bar graph of polysialic acid yield of E.coli K235 in example 2 with different NaCl feeding strategies; wherein S31 is not supplemented with NaCl; s32: adding NaCl as single-point feed supplement, and feeding NaCl in the 27 th hour with the amount of NaCl supplement being 20 g/L; s33: naCl is continuously fed and supplemented, and is fed and added in 8 h-27 h, the concentration of the NaCl is 300g/L, and the supplementing speed of the NaCl is 200mL/h; s34: naCl is fed-batch material, and the cumulative feeding amount of NaCl in 22h and 27h is respectively 5g/L and 20g/L of fermentation culture medium; s35: naCl is fed-batch material, and the cumulative feeding amount of NaCl in 22h and 27h is respectively 10g/L and 20g/L of fermentation culture medium; s36: naCl is fed batch, and the cumulative feeding amount of NaCl at 22h and 27h is 15g/L and 20g/L respectively.

Detailed Description

The technical solutions of the present invention are illustrated by the following specific examples, which should not be construed as limiting the scope of the present invention. Unless otherwise indicated, the strains, reagents, equipment used in the following examples were obtained commercially or were prepared using commercially available reagents using procedures conventional in the art. In addition, the strains used in the following examples can also be obtained from the microorganism depository. In the following examples, the solution for feeding and the medium for culture were sterilized in advance.

With the parameters shown in the examples below, it is within the ability of the person skilled in the art to convert the concentration of the aqueous feed solution required for a given amount of feed, the amount of inoculum and the volume of feed accepted for fermenters of different specifications (e.g. 5L fermenter, 20L fermenter, 50L fermenter, 100L fermenter, 200L fermenter, 500L fermenter, etc.). So that several simple deductions or substitutions can be made without departing from the basic principle of the invention. It is also within the ability of those skilled in the art to adjust and vary the amount of inoculum, temperature, rotation speed, pH, aeration, etc., used in the fermentation culture depending on the growth characteristics of the strain without departing from the scope of the invention. Trace elements, such as CaCl, used in the fermentation media of the following examples ₂ 、FeSO ₄ 、CuSO ₄ The growth of the strain is favored, but usually already present in the culture medium raw material, optionally with additional ingredients, which are well known to the person skilled in the art (see Cao Coca, eds. Microbial engineering, beijing: science and technology Press, 2007, 2 nd edition, page 84).

In the following examples, the yield of polysialic acid (g/L, i.e., the concentration of polysialic acid in the fermentation broth at the end of fermentation) and the cell concentration were measured by the resorcinol method and the absorbance method (measuring the OD at a wavelength of 600 nm), respectively. The detailed methods and procedures can be found in the study on the control of sialic acid fermentation and its isolation and purification (Liwenqiang; 2005 Master thesis, university of Jiangnan, see sections 2.2.4.1 and 2.2.4.6 for details). During the determination of the polysialic acid yield, fermentation liquor is sampled, centrifuged at 7000r/min for 15min to remove thalli, and an ultrafiltration membrane (with the molecular weight cutoff of 2 kD) is adopted to remove small molecules. Then, the measurement was carried out by the resorcinol method.

The percentage (%) improvement in sialic acid productivity in the following examples was calculated as (Px-P0)/P0 x100, where P0 is the sialic acid productivity (g/L or mg/L) at the reference point and Px is the sialic acid productivity (g/L or mg/L) at the examined point.

Example 1 kinetic characterization of the fermentative production of polysialic acid in E.coli

1. The influence of the NaCl supplement amount on the growth of the escherichia coli thalli and the sialic acid fermentation yield is examined.

2. Method for producing a composite material

(I) Strain

Escherichia coli K1 (deposit number: DSM 107164), escherichia coli K92 (deposit number: ATCC 35860), and Escherichia coli K235 (deposit number: ACTT 13027).

(II) culture Medium and culture method

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 and the balance of 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 of water.

Feed K11 (g/L): xylose 500, L-proline 300 and the balance of water.

Culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH7.5 and shaking table rotation speed of 250rpm for 6h to obtain first-stage seed culture solution.

(2) Inoculating the primary seed culture solution to a secondary seed culture medium according to the inoculation amount of 2% (V/V), and culturing at 37 deg.C, pH7.5 and 250rpm for 12h to obtain a secondary seed culture solution, i.e. a seed solution.

(3) Adding 12L of fermentation medium into a 20L fermentation tank, and inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 2% (V/V); fermenting and culturing for 24h under the fermentation conditions of 37 ℃, pH6.8, stirring speed of 600rpm and ventilation volume of 1 vvm; continuously adding a material supplementing liquid K11 from 9h to 20h after the fermentation starts, wherein the material supplementing speed is 25mL/h; naCl is added into water to prepare a NaCl aqueous solution for feeding, naCl 0 (namely NaCl is not added into the water), 180g, 240g, 300g, 360g and 420g are respectively added into a fermentation tank at one time in 9h after the fermentation is started, the NaCl feeding volume is 1.36L (the feeding amount of NaCl is 0g/L to 35g/L of fermentation medium; namely 0g/L to 30.22g/L of fermentation broth), and the NaCl aqueous solution for feeding is respectively 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L and 308.82g/L in concentration.

And (4) measuring the yield (g/L) of polysialic acid and the concentration (OD value at 600 nm) of the bacteria at different time points when different NaCl feeding amounts are measured.

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 and the balance of 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 of water.

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

Culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH7.2 and shaking table rotation speed of 180rpm for 12 hr to obtain first-stage seed culture solution.

(2) Inoculating the first-stage seed culture solution to a second-stage seed culture medium according to the inoculation amount of 2% (V/V), and culturing at 37 deg.C, pH7.2, and 180rpm for 8h to obtain a second-stage seed culture solution.

(3) Adding 12L of fermentation medium into a 20L fermentation tank, and inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 2% (V/V); fermenting and culturing for 24h under the fermentation conditions of 37 ℃, pH7.1, stirring speed of 150rpm and ventilation capacity of 2 vvm; continuously adding a feed liquid K921 from 9h to 20h after the fermentation starts, wherein the feed speed is 25mL/h; naCl is added into water to prepare a NaCl aqueous solution for feeding, naCl 0 (namely NaCl is not added into the water), 180g, 240g, 300g, 360g and 420g are respectively added into a fermentation tank at one time in 9h after the fermentation is started, the NaCl feeding volume is 1.36L (the feeding amount of NaCl is 0g/L to 35g/L of fermentation medium; namely 0g/L to 30.22g/L of fermentation broth), and the NaCl aqueous solution for feeding is respectively 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L and 308.82g/L in concentration.

Culture medium and culture method of escherichia coli K235

Primary seed culture medium, secondary seed culture medium (g/L): tryptone 10, beef extract 3, naCl 5 and the balance of 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 and the balance of water.

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

Culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH7.0, and shaking table rotation speed of 250rpm for 12 hr to obtain first-stage seed culture solution.

(2) Inoculating the first-stage seed culture solution to a second-stage seed culture medium according to the inoculation amount of 2% (V/V), and culturing at 37 deg.C, pH7.0, and 250rpm for 6h to obtain a second-stage seed culture solution.

(3) Adding 12L of fermentation medium into a 20L fermentation tank, and inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 2% (V/V); fermenting and culturing for 24h under the fermentation conditions of 37 ℃, pH6.8, stirring speed of 400rpm and ventilation capacity of 2 vvm; continuously adding a feed liquid K2351 in the 9 th to 20 th hours after the fermentation starts, wherein the feed speed is 25mL/h; naCl is added into water, and after the fermentation is started, naCl 0 (namely, naCl is not added into the water), 180g, 240g, 300g, 360g and 420g are respectively added into the fermentation tank at one time in the 9 th hour, the NaCl supplementing volume is 1.36L (the supplementing amount of NaCl is 0g/L to 35g/L of fermentation medium; namely, 0g/L to 30.22g/L of fermentation liquid), namely, the concentrations of NaCl aqueous solution for fed-batch are respectively 0, 132.35g/L, 176.47g/L, 220.59g/L, 264.70 g/L and 308.82g/L.

3. Results

Results of Escherichia coli K1 experiment

The kinetic profile of the production of polysialic acid by fed-batch fermentation of E.coli K1 is shown in FIG. 1. The thallus concentration and the polysialic acid yield in the fermentation liquor of 9h to 24h show a trend of increasing along with the time. Wherein NaCl is supplemented by 0g (OD 0 in the figure) and 180g (OD 180 in the figure), and the thallus concentration curves are basically similar; when 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD 360 in the figure), and 420g (OD 420 in the figure) of NaCl was supplemented, the cell concentration curve was lower than that when 0g of NaCl was supplemented. The growth speed sequence of the thalli is as follows: naCl supplement 0g and NaCl supplement 180g, naCl supplement 240g, naCl supplement 300g, naCl supplement 360g and NaCl supplement 420g.

When NaCl feed was used at 240g (PSA 240), 300g (PSA 300), 360g (PSA 360), 420g (PSA 420), polysialic acid yield was significantly higher between 12h and 16h than when NaCl feed was used at 0g. Compared with 0g of NaCl feed, the polysialic acid yield at the 16 th hour is improved by 4.78-7.35% when 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) of NaCl feed is added; the polysialic acid yield is improved by about 8.19 to 19.30 percent at the 14 th hour; the polysialic acid yield is improved by about 9.20 to 21.84 percent at the 12 th hour; and the yield of polysialic acid is improved by a percentage higher than that of the polysialic acid at the 12 th hour under the same NaCl supplement amount. The polysialic acid yield at this stage (12 h to 16 h) was in the order from high to low: 420g of NaCl supplement is larger than or equal to 360g of NaCl supplement, 300g of NaCl supplement, 240g of NaCl supplement, 180g of NaCl supplement and approximately equal to 0g of NaCl supplement. Wherein the percentage of sialic acid yield increase of 1.20%, 12.64%, 21.84% and 21.84% compared to 0g NaCl feed was observed at 12h, 240g (PSA 240), 300g (PSA 300), 360g (PSA 360) and 420g (PSA 420) of NaCl feed. And (4) narrowing the yield difference of the polysialic acid at different NaCl feeding amounts from 18h to 20h. At 22 h-24 h, the polysialic acid yield of 360g and 420g of NaCl supplemented material is lower than that of 0g of NaCl supplemented material.

(II) Escherichia coli K92 test results

The kinetic profile of the production of polysialic acid by fed-batch fermentation of E.coli K92 is shown in FIG. 2. The concentration of the thalli in the fermentation liquor is in the trend of rising first and then falling after 9h to 24h, and the OD values at the wavelength of 22h to 24h 600nm are reduced slightly; polysialic acid productivity increased with time. Wherein NaCl is supplemented by 0g (OD 0 in the figure) and 180g (OD 180 in the figure), and the thallus concentration curves are basically similar; when 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD 360 in the figure), and 420g (OD 420 in the figure) of NaCl was added, the cell concentration curve was lower than that when 0g of NaCl was added. The growth speed sequence of the thalli is as follows: naCl feed supplement 0g is approximately equal to NaCl feed supplement 180g, naCl feed supplement 240g, naCl feed supplement 300g, naCl feed supplement 360g, and NaCl feed supplement 420g.

When NaCl feed was used at 240g (PSA 240), 300g (PSA 300), 360g (PSA 360), 420g (PSA 420), polysialic acid yield was significantly higher between 12h and 16h than when NaCl feed was used at 0g. Compared with 0g of NaCl feed, the polysialic acid yield at the 16h is improved by 1.37-4.79% when 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) of NaCl feed is added; the polysialic acid yield is improved by about 8.25 to 17.53 percent at the 14 th hour; the polysialic acid yield is improved by about 8.16 to 16.33 percent at the 12 th hour; and the 14h polysialic acid yield is improved by a percentage higher than that of the 12h polysialic acid yield when the NaCl supplement amount is equal. The polysialic acid yield at this stage (12 h to 16 h) was in the order from high to low: 420g of NaCl supplement, 360g of NaCl supplement, 300g of NaCl supplement, 240g of NaCl supplement, 180g of NaCl supplement and 0g of NaCl supplement. Wherein the percentage increase of sialic acid yield was 8.25%, 12.37%, 15.46% and 17.53% when NaCl was supplemented at 14h, 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 supplemented at 0g. The yield of polysialic acid in 420g and 360g of NaCl supplemented material is successively lower than that in 0g of NaCl supplemented material in 18 h-24h, and the difference is enlarged along with the time.

(III) results of Escherichia coli K235 experiment

The kinetic profile of the production of polysialic acid by fed-batch fermentation of E.coli K235 is shown in FIG. 3. The thallus concentration and polysialic acid yield in the fermentation liquor of 9 h-24 h show a trend of increasing along with the time. Wherein NaCl is supplemented by 0g (OD 0 in the figure) and 180g (OD 180 in the figure), and the thallus concentration curves are basically similar; when 240g (OD 240 in the figure), 300g (OD 300 in the figure), 360g (OD 360 in the figure), and 420g (OD 420 in the figure) of NaCl was added, the cell concentration curve was lower than that when 0g of NaCl was added. The growth speed sequence of the thalli is as follows: naCl feed supplement 0g is approximately equal to NaCl feed supplement 180g, naCl feed supplement 240g, naCl feed supplement 300g, naCl feed supplement 360g, and NaCl feed supplement 420g.

The polysialic acid yield is obviously higher than that of NaCl supplemented material 0g at 12 h-18 h when NaCl supplemented material is 240g (PSA 240 in the figure), 300g (PSA 300 in the figure), 360g (PSA 360 in the figure), 420g (PSA 420 in the figure). Compared with 0g of NaCl feed, the polysialic acid yield at 18h is improved by 2.61-5.44% when 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) of NaCl feed is added; the polysialic acid yield at the 16 th hour is improved by 5.15 to 12.06 percent; the 14h polysialic acid yield is improved by about 10.70-21.85%; the polysialic acid yield is improved by about 7.38-18.20% at the 12 th hour; and the 14h polysialic acid yield is improved by a percentage higher than that of the 12h polysialic acid yield when the NaCl supplement amount is equal. The polysialic acid yield at this stage is in order from high to low: 420g of NaCl supplement, 360g of NaCl supplement, 300g of NaCl supplement, 240g of NaCl supplement, 180g of NaCl supplement and 0g of NaCl supplement. Wherein the percentage increase of sialic acid yield at 14h was 10.70%, 14.96%, 17.60% and 21.85% compared to 0g NaCl feed when 240g (PSA 240), 300g (PSA 300), 360g (PSA 360) and 420g (PSA 420) were added. At 20 h-24 h, the polysialic acid yield of 240g, 300g, 360g and 420g of NaCl supplement is obviously lower than that of 0g of NaCl supplement.

4. Conclusion

In the process for preparing the polysialic acid by the fed-batch fermentation of escherichia coli K1, escherichia coli K92 and escherichia coli K235, the feeding amount of fed-batch feeding NaCl is 0-15 g/L of fermentation medium, and the influence of the fermentation medium on the growth of escherichia coli and the yield of sialic acid fermentation is not obvious; the yield of polysialic acid can be improved when the supplement amount of the fed-batch supplement NaCl is 20-35 g/L of fermentation medium, namely 17.27-30.22 g/L of fermentation broth. Wherein the polysialic acid yield is obviously improved 4-8 hours after the NaCl fed-batch is finished. The polysialic acid yield is improved to the maximum extent 4-6 h after the NaCl fed-batch is finished. Wherein the yield of the polysialic acid of the Escherichia coli K1 is improved to the maximum extent 4h after NaCl fed-batch; the yield of the polysialic acid of Escherichia coli K92 and Escherichia coli K235 is improved to the maximum extent after 6 hours of NaCl fed-batch.

Example 2 feed strategy Studies of NaCl flow-fed feed

1. The influence of NaCl intermittent fed-batch and continuous fed-batch on the sialic acid fermentation yield of the escherichia coli cells is examined when the volume of the fed-batch is equal and the feeding speed of the carbon source and the nitrogen source is equal. On the basis of the study related to example 1, a fermentation medium with a NaCl feed amount of 20g/L was selected, and fermentation was continued for 4 hours after the end of the NaCl feed stream.

2. Method of producing a composite material

(ii) Strain

(II) culture Medium and culture method

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 and the balance of 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 of water.

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

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

feed solution K1C1 (g/L): 100 parts of xylose, 60 parts of L-proline and 150 parts of NaCl;

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

culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH6.8 and shaking table rotation speed of 200rpm for 8 hr to obtain first-stage seed culture solution.

(2) Inoculating the primary seed culture solution to a secondary seed culture medium according to the inoculation amount of 3% (V/V), and culturing at 37 deg.C, pH6.8 and 200rpm for 12h to obtain a secondary seed culture solution, i.e. a seed solution.

(3) Adding 30L fermentation medium into a 50L fermentation tank, inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 0.5% (V/V); fermenting and culturing for 31h under the fermentation conditions of 37 ℃, pH6.4, stirring speed of 300rpm and ventilation capacity of 0.5 vvm; the fed-batch materials adopt 6 strategies:

in the 1 st strategy (S11), naCl is not fed in a fed-batch manner, a fed-batch liquid K1A1 is fed in 8 h-27 h after the start of fermentation, the speed of the fed-batch is 200mL/h, the fed-batch time is 20h, and the total fed-batch volume is 4L.

In the 2 nd strategy (S12), naCl is fed-batch at a single time point, the fed-batch liquid K1B1 is fed-batch at the speed of 100mL/h for 8h to 27h after the start of fermentation, and the fed-batch is fed for 20h. And feeding NaCl aqueous solution D1 at the 27 th hour after the fermentation is started, wherein the feeding speed is 2L/h. The total feed volume was 4L.

And (3) NaCl in the strategy (S13) is continuous fed-batch, the fed-batch liquid K1C1 is fed-batch after the 8 th to 27 th hours after the fermentation is started, the speed of the fed-batch is 200mL/h, the fed-batch is 20 hours, and the fed-batch volume is 4L.

In the 4 th strategy (S14), naCl is fed in batch, the fed liquid K1B1 is fed in batch at the 8 th-27 th hour after the fermentation is started, the feeding speed is 100mL/h, and the feeding is 20 hours. Feeding NaCl aqueous solution D1 at 22h after the start of fermentation, wherein the feeding speed is 0.5L/h, the feeding speed is 1h, and the NaCl feeding amount is 5g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20 g/L.

And (5) NaCl in the strategy (S15) is fed intermittently, the fed liquid K1B1 is fed at the speed of 100mL/h for 8h to 27h after the fermentation is started, and the fed liquid is fed for 20h. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation is started, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the NaCl feeding amount is 10g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20g/L of fermentation medium.

NaCl in the 6 th strategy (S16) is fed batch, the fed batch liquid K1B1 is fed batch in 8 h-27 h after the fermentation is started, the fed batch speed is 100mL/h, and the fed batch time is 20h. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation is started, wherein the feeding speed is 1.5L/h, the feeding is 1h, and the NaCl feeding amount is 15g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 0.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20g/L of fermentation medium.

The polysialic acid yield (g/L) was measured at the end of fermentation (at the end of 31h 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, and the balance of 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 of water.

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

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

feed K92C2 (g/L): xylose 60, asparagine 80, naCl 150;

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

culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH7.0 and shaking table rotation speed of 150rpm for 10 hr to obtain first-stage seed culture solution.

(2) Inoculating the first-stage seed culture solution to a second-stage seed culture medium according to the inoculation amount of 3% (V/V), and culturing at 37 deg.C, pH7.0 and 150rpm for 10h to obtain a second-stage seed culture solution, i.e. a seed solution.

(3) Adding 30L of fermentation medium into a 50L fermentation tank, and inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 0.5% (V/V); fermenting and culturing for 31h under the fermentation conditions of 37 ℃, pH6.4, stirring speed of 200rpm and ventilation volume of 0.5 vvm; the fed-batch materials adopt 6 strategies:

in the 1 st strategy (S21), naCl is not fed in a fed-batch manner, the fed-batch liquid K92A2 is fed in 8 h-27 h after the fermentation is started, the speed of the fed-batch is 200mL/h, the fed-batch time is 20h, and the total fed-batch volume is 4L.

In the 2 nd strategy (S22), naCl is fed-batch at a single time point, the fed-batch liquid K92B2 is fed-batch at the speed of 100mL/h for 8h to 27h after the start of fermentation, and the fed-batch is fed-batch for 20h. And feeding NaCl aqueous solution D1 at the 27 th hour after the fermentation is started, wherein the feeding speed is 2L/h. The total feed volume was 4L.

NaCl in the 3 rd strategy (S23) is fed continuously, the feeding liquid K92C2 is fed 8 h-27 h after the fermentation starts, the feeding speed is 200mL/h, the feeding time is 20h, and the feeding volume is 4L.

In the 4 th strategy (S24), naCl is fed in batch, the fed liquid K92B2 is fed in 8 h-27 h after the fermentation is started, the feeding speed is 100mL/h, and the feeding is 20h. Feeding NaCl aqueous solution D1 at 22h after the start of fermentation, wherein the feeding speed is 0.5L/h, the feeding speed is 1h, and the NaCl feeding amount is 5g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20 g/L.

And (5) NaCl in the strategy (S25) is fed intermittently, the fed liquid K92B2 is fed at the 8 th-27 th hour after the fermentation is started, the fed speed is 100mL/h, and the fed liquid is fed for 20 hours. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation is started, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the NaCl feeding amount is 10g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20 g/L.

In the 6 th strategy (S26), naCl is fed in batch, the fed liquid K92B2 is fed in 8 h-27 h after the fermentation is started, the feeding speed is 100mL/h, and the feeding is 20h. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation starts, wherein the feeding speed is 1.5L/h, the feeding time is 1h, and the NaCl feeding amount is 15g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 0.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20g/L of fermentation medium.

Culture medium and culture method of escherichia coli K235

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

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

Feed liquid 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) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH6.4, and shaking table rotation speed of 200rpm for 10 hr to obtain first-stage seed culture solution.

(2) Inoculating the first-stage seed culture solution to a second-stage seed culture medium according to the inoculation amount of 3% (V/V), and culturing at 37 deg.C, pH6.4 and 300rpm for 12h to obtain a second-stage seed culture solution, i.e. a seed solution.

(3) Adding 12L fermentation medium into 20L fermentation tank, inoculating seed liquid into fermentation tank containing fermentation medium according to 0.5% (V/V) inoculation amount; fermenting and culturing for 31h under the fermentation conditions of 37 ℃, pH6.4, stirring speed of 200rpm and ventilation volume of 1 vvm; the fed-batch materials adopt 6 strategies:

in the 1 st strategy (S31), naCl is not fed in a fed-batch manner, a fed-batch liquid K235A3 is fed in 8 h-27 h after the fermentation is started, the speed of the fed-batch is 200mL/h, the fed-batch time is 20h, and the total fed-batch volume is 4L.

In the 2 nd strategy (S32), naCl is fed-batch at a single time point, the fed-batch liquid K235B3 is fed-batch at the speed of 100mL/h for 8h to 27h after the start of fermentation, and the fed-batch is fed-batch for 20h. And feeding NaCl aqueous solution D1 at the 27 th hour after the fermentation is started, wherein the feeding speed is 2L/h. The total feed volume was 4L.

And (3) NaCl in the strategy (S33) is continuous fed-batch, the fed-batch liquid K235C3 is fed-batch after the 8 th to 27 th hours after the fermentation is started, the speed of the fed-batch is 200mL/h, the fed-batch is 20 hours, and the fed-batch volume is 4L.

In the 4 th strategy (S34), naCl is fed in batch, the feeding liquid K235B3 is fed in 8 h-27 h after the fermentation starts, the feeding speed is 100mL/h, and the feeding is 20h. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation starts, wherein the feeding speed is 0.5L/h, the feeding time is 1h, and the NaCl feeding amount is 5g/L of the fermentation culture medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20 g/L.

And (5) NaCl in the strategy (S35) is fed intermittently, the fed liquid K235B3 is fed at the speed of 100mL/h for 8h to 27h after the fermentation starts, and the fed liquid is fed for 20h. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation is started, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the NaCl feeding amount is 10g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 1.0L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20g/L of fermentation medium.

In the 6 th strategy (S36), naCl is fed in batch, the fed liquid K235B3 is fed in the 8 th to 27 th hours after the fermentation is started, the feeding speed is 100mL/h, and the feeding is 20 hours. Feeding NaCl aqueous solution D1 at the 22 th hour after the fermentation is started, wherein the feeding speed is 1.5L/h, the feeding is 1h, and the NaCl feeding amount is 15g/L of fermentation medium; feeding NaCl aqueous solution D1 at 27h after the start of fermentation, wherein the feeding speed is 0.5L/h, the feeding is 1h, and the accumulated NaCl feeding amount is 20g/L of fermentation medium.

3. Results

The yield of polysialic acid produced by fermenting Escherichia coli K1, escherichia coli K92 and Escherichia coli K235 under different NaCl feeding strategies is shown in figures 4, 5 and 6 respectively. Wherein S11, S21 and S31 are a NaCl-free feeding strategy; s12, S22 and S32 are strategies of NaCl single-time point feeding 5h before the end of fermentation; s13, S23 and S33 are strategies of NaCl continuous flow feeding; S14-S16, S24-S26 and S34-S36 are NaCl intermittent flow feeding strategies. Polysialic acid of different strains under different NaCl feeding strategies has certain difference. Wherein the polysialic acid yield of the NaCl intermittent fed-batch feed is slightly higher than that of the NaCl single-time-point feed. The polysialic acid yield of the NaCl continuous flow feeding material is less than or equal to that of the NaCl single-time point feeding material, but compared with the NaCl-free feeding material, the polysialic acid yield is still respectively improved by 7.47 percent (Escherichia coli K1), 7.61 percent (Escherichia coli K92) and 7.02 percent (Escherichia coli K235).

4. Conclusion

The NaCl feeding strategy of the polysialic acid produced by the fermentation of the escherichia coli can select intermittent feeding or continuous feeding, wherein the intermittent feeding is slightly superior to the continuous feeding.

Example 3 Effect of temperature and pH control in combination with NaCl feed streams on the production of polysialic acid by fermentation of E.coli K235

1. Strain of bacillus

Escherichia coli K235 (accession number: ACTT 13027).

2. Culture medium and culture method

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

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

Culture steps and culture conditions:

(1) Inoculating Escherichia coli in the first-stage seed culture medium, and culturing at 37 deg.C, pH7.0 and shaking table rotation speed of 150rpm for 12 hr to obtain first-stage seed culture solution.

(2) Inoculating the primary seed culture solution to a secondary seed culture medium according to the inoculation amount of 4% (V/V), and culturing at 37 deg.C, pH7.0 and 200rpm for 12h to obtain a secondary seed culture solution, i.e. a seed solution.

(3) Adding 12L of fermentation medium into a 20L fermentation tank, and inoculating the seed solution into the fermentation tank containing the fermentation medium according to the inoculation amount of 4% (V/V); the stirring speed is 300rpm, and the ventilation volume is 2vvm, and the fermentation culture is carried out for 24 hours. Feeding the feed liquid K235C4 in 6-20 h after the fermentation is started, wherein the feeding speed is 80mL/h, the feeding time is 15h in total, and the feeding volume is 1.2L. The fermentation temperature is selected to be 34 deg.C, 37 deg.C, 41 deg.C. The pH value is selected to be 6.4, 7.1, 8.0 (when the pH value is lower than the selected value by 0.4-0.6, naOH is added to adjust the pH value to the selected value, such as 6.4, 7.1, 8.0). And (3) generating an orthogonal experimental table by adopting an orthogonal design function of SPSS software, testing according to the orthogonal experimental table, and determining the polysialic acid difference rate at the end of 24h under different conditions. The influence of temperature and pH on polysialic acid yield was examined in the SPSS software using analysis of variance (univariate analysis in general linear model. Multiple comparisons between groups using the LSD method).

3. Results and conclusions

The experiment investigates the influence of temperature and pH on the polysialic acid yield when the NaCl feeding amount and the feeding speed are the same. Based on the factor level setting of the 2-factor 3 level, the SPSS software generated an orthogonal experiment table and performed 9 experiments. The results of the orthogonality experiment are shown in table 1. As shown in Table 2, the effect of temperature on polysialic acid yield was insignificant (p.gtoreq.0.05) and the effect of pH on polysialic acid yield was significant (p < 0.05) over a selected range of factor levels and other fermentation culture conditions. Multiple comparisons of polysialic acid yields at different pH conditions were performed, as shown in Table 3, with polysialic acid yields at pH6.4 higher than at pH7.1 and pH 8.0 (p < 0.05).

The above example 1 shows the effect of NaCl feed stream on polysialic acid yield improvement under the same fermentation conditions; example 2 demonstrates the effect of different NaCl feeding strategies on polysialic acid yield; example 3 the effect of temperature and pH on polysialic acid production was examined when NaCl feed and feed rate were the same. In summary, the above examples show that, in the process of producing polysialic acid by Escherichia coli fed-batch fermentation, naCl adopts intermittent feeding or continuous feeding, and the feeding amount is 20g/L to 35g/L of fermentation medium; the feeding speed is that the accumulated feeding amount of NaCl for 9-10 h before the end of the fermentation culture is 5-15 g/L of fermentation culture medium; the accumulated supplement amount of NaCl 4-6 h before the fermentation culture is finished is 20-35 g/L of fermentation culture medium, and the polysialic acid yield can be improved. Wherein the NaCl feeding amount and the feeding speed are the same, the influence of the temperature in the range of 34-41 ℃ on the polysialic acid yield is small, and the increase of the pH value in the range of 6.4-8.0 can cause the reduction of the polysialic acid yield. However, as shown in examples 1 and 2, the NaCl fed-batch feed is still beneficial to improve the polysialic acid yield under the same temperature and pH conditions.

TABLE 1 results of orthogonal experiments

TABLE 2 examination of the Effect between subjects

^a R side =.977 (adjust R side =.955).

TABLE 3 multiple comparisons

*. p< 0.05。

Claims

1. A fed-batch fermentative production process for producing polysialic acid using escherichia coli, comprising the steps of:

f1: inoculating the escherichia coli seed solution into a fermentation tank containing a fermentation culture medium; the fermentation medium contains carbon source, nitrogen source and K ₂ HPO ₄ Or K ₂ HPO ₄ Hydrate 0.5-27 g/L, mgSO ₄ Or MgSO 2 ₄ Hydrate is 0.15 g/L-1.5 g/L; the carbon source of the fermentation medium is at least one selected from sorbitol, glycerol, glucose and xylose; the nitrogen source of the fermentation medium is selected from NH ₄ Cl、(NH ₄ ) ₂ SO ₄ At least one of corn steep liquor starch, yeast extract, tryptone, L-proline, aspartic acid and asparagine;

f3: feeding materials into the fermentation tank in the fermentation culture process of the step F2; the feed supplement comprises the following components: naCl and carbon source; the supplement amount of NaCl is 20g/L to 40g/L of fermentation medium; the feeding speed of NaCl is that the accumulated feeding amount of NaCl 4-8 h before the end of the fermentation culture is 20-40 g/L of the fermentation culture medium; the carbon source of the fed-batch material is selected from at least one of glucose, xylose, glycerol and sorbitol; and the NaCl adopts continuous flow feeding or intermittent flow feeding.

2. Fed-batch fermentation production process according to claim 1, characterized in that the NaCl is in a feed amount of 20-35 g/L fermentation medium; the feed supplement speed of the NaCl is that the accumulated feed supplement amount of the NaCl within 4-8 h before the fermentation culture is finished is 20-35 g/L of the fermentation culture medium.

3. Fed-batch fermentation production process according to claim 2, characterized in that the NaCl is fed at a rate such that the cumulative feed amount of NaCl from 9 to 10 hours before the end of the fermentation culture is from 5 to 15g/L of fermentation medium; the accumulated supplement amount of NaCl 4-6 h before the fermentation culture is finished is 20-35 g/L of fermentation medium.

4. Fed-batch fermentation production process according to claim 1, characterized in that the composition of the fed-batch in step F3 comprises a nitrogen source, and the nitrogen source of the fed-batch is selected from corn steep liquor starch, yeast extract, tryptone, L-proline, aspartic acid, asparagine, NH ₄ Cl、(NH ₄ ) ₂ SO ₄ At least one of (a).

5. A fed-batch fermentation production process according to claim 4, wherein the fermentation culture temperature of step F2 is 34-42 ℃, the pH is 6.4-7.1, and the stirring rotation speed is 150-700 rpm; the carbon source of the feeding material in the step F3 is selected from one of glucose, xylose and sorbitol; the nitrogen source of the feed stream in step F3 is selected from NH ₄ Cl, L-proline, (NH) ₄ ) ₂ SO ₄ One of asparagine and asparagine; in the step F3, the concentration of the carbon source in the feed supplement liquid used in the flow feed supplement is 20 g/L-800 g/L; the concentration of the nitrogen source in the feed liquid used in the feed-batch process is 12 g/L-300 g/L.

6. A fed-batch fermentation production method according to claim 5, wherein in step F3, the concentration of the carbon source in the feed liquid used in the fed-batch process is 60g/L to 800g/L; the concentration of the nitrogen source in the feed liquid used in the feed-batch process is 50 g/L-300 g/L.

7. A fed-batch fermentation production process according to claim 5, wherein the E.coli strain used in the fed-batch fermentation production is selected from any one of E.coli K235 WXJYL-11, E.coli K235 WXJ4, E.coli K235-JYII-74, E.coli K235E 61, E.coli SA-8, E.coli CASOV-8, E.coli ATCC13027, E.coli H03A2190830, E.coli GX124, E.coli K1, E.coli LP 1674, E.coli K92, E.coli C8.

8. A fed-batch fermentative production process according to claim 5, wherein the carbon source of the fermentation medium is selected from at least one of the following components: sorbitol 10 g/L-60 g/L, glucose 7g/L, xylose 8 g/L-15 g/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, 10g/L of yeast extract, 1.5-10 g/L of tryptone, 17-19 g/L of L-proline and 9-12 g/L of asparagine.

9. A fed-batch fermentation production process according to claim 5, wherein the fermentation medium contains at least one of the following components: feSO ₄ Or hydrate thereof, cuSO ₄ Or its hydrate, caCl ₂ Or hydrates thereof, K ₂ SO ₄ Or a hydrate thereof, KH ₂ PO ₄ Or NaH ₂ PO ₄ 。

10. Fed-batch fermentation production method according to claim 5, characterized in that the preparation method of the seed liquid of step F1 comprises the following steps:

z1: inoculating escherichia coli into a primary seed culture medium, and culturing for 6-12 h under the conditions of 34-42 ℃, pH 6.4-7.8 and shaking table rotating speed of 150-300 rpm to obtain a primary seed culture solution;

11. A fed-batch fermentation production process according to claim 10, wherein the primary and secondary seed culture media are selected from any one of the following media:

m1: 8-12 g/L tryptone, 4-10 g/L yeast extract, 1-12 g/L NaCl and the balance of water; m2: 10g/L of tryptone, 2-5 g/L of beef extract, 5g/L of NaC and the balance of water;

m3: 10g/L of tryptone, 2-5 g/L of beef extract, 5g/L of NaC, 2g/L of yeast extract and the balance of 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 of water.