WO2022135173A1 - 一株大肠埃希氏菌及其在产聚唾液酸中的应用 - Google Patents

一株大肠埃希氏菌及其在产聚唾液酸中的应用 Download PDF

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WO2022135173A1
WO2022135173A1 PCT/CN2021/136947 CN2021136947W WO2022135173A1 WO 2022135173 A1 WO2022135173 A1 WO 2022135173A1 CN 2021136947 W CN2021136947 W CN 2021136947W WO 2022135173 A1 WO2022135173 A1 WO 2022135173A1
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fermentation
polysialic acid
medium
escherichia coli
solution
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French (fr)
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张天萌
刘英杰
戚明
刘金钊
王景
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华熙生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H7/00—Compounds containing non-saccharide radicals linked to saccharide radicals by a carbon-to-carbon bond
    • C07H7/02—Acyclic radicals
    • C07H7/033—Uronic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

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  • the invention relates to a strain of Escherichia coli and its application in producing polysialic acid, and also relates to a fermentation process for improving the production of polysialic acid by using the strain, belonging to the technical field of biological fermentation.
  • PSA Polysialic acid
  • PSA is a linear polymer linked by ⁇ -2,8 and/or ⁇ -2,9 glycosidic bonds as monomers of N-acetylneuraminic acid.
  • PSA is the end of glycoprotein and glycolipid sugar chain that exists on the surface of some mammalian and pathogenic bacteria cells in the form of capsule. It can be used as a sustained-release material for protein drugs and a scaffold material in nerve repair surgery.
  • Patent CN 111733092 A provides a method for the production of polysialic acid by fermentation, the yield of polysialic acid obtained by continuous fermentation for 20h is 10g/L, and the fermentation rate per unit time is 0.5g/L ⁇ h. This method shortens the production time and reduces the cost, but pure oxygen is introduced into the fermentation process for half of the time, which increases the production cost on the one hand, and more importantly, increases the safety hazard in the production of the enterprise.
  • the purpose of the present invention is to provide a strain of Escherichia coli (Escherichia coli), when the bacterial strain is fermented to produce polysialic acid, it has been verified to have a faster fermentation speed and a higher total fermentation yield, which solves the problem in the prior art.
  • the fermentation production of polysialic acid has the problems of unstable fermentation level, low polysialic acid yield and long fermentation time.
  • the present invention provides a strain of Escherichia coli H03A2190830, which has been preserved in the China Center for Type Culture Collection (CCTCC for short) on November 6, 2019, and the address of the preservation unit is: Wuchang, Wuhan City, Hubei province Luojiashan Wuhan University, the deposit number is CCTCC NO: M2019900.
  • the strain of the present invention is obtained by screening from soil. After identification, the 16Sr DNA sequence of the strain is shown in SEQ ID NO: 1, and the taxonomic name and Latin name are Escherichia coli.
  • the present invention also provides the application of the above-mentioned Escherichia coli H03A2190830CCTCC NO: M2019900 in the production of polysialic acid, the strain can improve the production efficiency of polysialic acid, improve the total output of polysialic acid, and has good prospects for industrial application.
  • polysialic acid is produced in the fermentation broth, preferably, the concentration of polysialic acid in the fermentation broth is above 15.9 g/L.
  • the fermentation medium comprises: carbon source, nitrogen source, inorganic salts, vitamins and trace elements, preferably, 1L fermentation medium contains 25-35g of carbon source, 8-12g of nitrogen source, and 5.5-11.5g of inorganic salt , vitamins 0.8-1.2mL and trace elements 0.8-1.2mL.
  • the present invention provides a method for producing polysialic acid.
  • the method uses the above-mentioned Escherichia coli H03A2190830 CCTCC NO: M2019900 as strain to ferment a fermentation liquid containing polysialic acid.
  • the process method and conditions used can be selected from the process methods and conditions disclosed in the prior art and commonly used.
  • the present invention provides a preferred method for producing polysialic acid.
  • the seed liquid is inoculated into the fermentation medium for initial fermentation, and when the sugar is exhausted, the sugar is added in flow and the fermentation is continued until the fermentation is completed.
  • the fermentation broth was obtained at the end.
  • the fermentation medium contains: carbon source, nitrogen source, inorganic salts, vitamins and trace elements; further preferably, 1 L fermentation medium contains 25-35 g of carbon source, 8-12 g of nitrogen source, and 5.5 g of inorganic salt. -11.5g, vitamins 0.8-1.2mL, trace elements 0.8-1.2mL; most preferably, the fermentation medium is: corn steep liquor dry powder 8-12g/L, potassium dihydrogen phosphate 5-10g/L, glucose monohydrate (Single sterilization) 25-35g/L, magnesium sulfate 0.5-1.5g/L, vitamin solution (sterilization by film) 0.8-1.2mL/L, trace element solution (sterilization by film) 0.8-1.2mL/L , the water balance.
  • the components and compositions of the vitamin solution are: 0.3-0.7 g/L of thiamine, 2.8-3.6 g/L of calcium pantothenate, 0.004-0.008 g/L of biotin, and the balance of water.
  • the components and compositions in the trace element solution are: FeSO 4 ⁇ 7H 2 0 40-60g/L, MnSO 4 ⁇ H 2 O 6-8g/L, CuSO 4 ⁇ 5H 2 0 1-1.5g/L, ZnSO 4 ⁇ 7H 2 O 7-10g/L, balance of water.
  • the dissolved oxygen and pH in the fermenter rise significantly.
  • the obvious rise of dissolved oxygen and pH means that when a certain fermentation time is reached, dissolved oxygen and pH have a gradient rise compared with the previous fermentation time, and the rise is more than 5%.
  • the pH of the system is controlled to be 7-7.5, and after 20h of fermentation, the pH of the system is controlled to be 6.2-6.5.
  • the fermentation temperature was maintained at 35-37°C throughout the fermentation period.
  • the stirring speed is controlled to be 400-500 rpm, and the ventilation volume is 1.5-2 vvm.
  • the stirring speed and the ventilation rate are adjusted to keep the dissolved oxygen at 20-50% of the dissolved oxygen at the beginning of the fermentation (ie, the fermentation start time).
  • the method includes:
  • the seed liquid is inoculated into the fermentation medium for initial fermentation, and when the sugar is exhausted, the sugar is added to continue the fermentation until the fermentation ends to obtain a fermentation liquid;
  • the fermentation medium comprises: carbon source, nitrogen source, inorganic salts, vitamins and trace elements, further preferably, 1L fermentation medium contains 25-35g of carbon source, 8-12g of nitrogen source, 5.5-11.5g of inorganic salt, 0.8-1.2mL of vitamin, and 0.8-12g of trace element.
  • the fermentation medium is: corn steep liquor dry powder 8-12g/L, potassium dihydrogen phosphate 5-10g/L, glucose monohydrate 25-35g/L, magnesium sulfate 0.5-1.5g/L , vitamin solution 0.8-1.2mL/L, trace element solution 0.8-1.2mL/L, water balance; wherein, the components and compositions in vitamin solution are: thiamine 0.3-0.7g/L, calcium pantothenate 2.8-3.6 g/L, biotin 0.004-0.008g/L; the components and compositions in the trace element solution are: FeSO 4 ⁇ 7H 2 0 40-60g/L, MnSO 4 ⁇ H 2 O 6-8g/L, CuSO 4 ⁇ 5H 2 0 1-1.5g/L, ZnSO 4 ⁇ 7H 2 O 7-10g/L;
  • the pH of the control system is 7-7.5, and after 20h of fermentation, the pH of the system is controlled to be 6.2-6.5; preferably, the temperature is maintained at 35-37°C throughout the fermentation time.
  • the method includes:
  • the fermentation medium comprises: carbon source, nitrogen source, inorganic salts, vitamins and trace elements
  • 1L fermentation medium contains 25-35g carbon source, 8-12g nitrogen source, 5.5-11.5g inorganic salt, 0.8-1.2mL vitamin and 0.8-1.2mL trace element 1.2mL/L
  • the fermentation medium is: corn steep liquor 8-12g/L, potassium dihydrogen phosphate 5-10g/L, glucose monohydrate 25-35g/L, magnesium sulfate 0.5-1.5g /L, vitamin solution 0.8-1.2mL/L, trace element solution 0.8-1.2mL/L, water balance; wherein, the components and compositions in vitamin solution are: thiamine 0.3-0.7g/L, calcium pantothenate 2.8 -3.6g/L, bio
  • the pH of the control system is 7-7.5, and after 20h of fermentation, the pH of the control system is 6.2-6.5; preferably, the temperature is maintained at 35-37°C throughout the fermentation time;
  • 60-75wt% glucose solution is added at a rate of 8-15g/L ⁇ h; preferably, after the sugar is added, the fermentation is continued for 24-27 hours.
  • the method includes:
  • the fermentation medium comprises: carbon source, nitrogen source, inorganic salts, vitamins and trace elements
  • 1L fermentation medium contains 25-35g carbon source, 8-12g nitrogen source, 5.5-11.5g inorganic salt, 0.8-1.2mL vitamin and 0.8-1.2mL trace element 1.2mL
  • the fermentation medium is: corn steep liquor dry powder 8-12g/L, potassium dihydrogen phosphate 5-10g/L, glucose monohydrate 25-35g/L, magnesium sulfate 0.5-1.5g/L , vitamin solution 0.8-1.2mL/L, trace element solution 0.8-1.2mL/L, water balance; among them, the components and compositions in vitamin solution are: thiamine 0.3-0.7g/L, calcium pantothenate 2.8-3.6 g/L, bio
  • the pH of the control system is 7-7.5, and after 20h of fermentation, the pH of the control system is 6.2-6.5; preferably, the temperature is maintained at 35-37°C throughout the fermentation time;
  • the stirring speed is 400-500 rpm, and the ventilation rate is 1.5-2 vvm; after the sugar is added, the stirring speed and ventilation are adjusted to keep the dissolved oxygen at 20% of the dissolved oxygen at the beginning of the fermentation. %-50%.
  • the seed solution can be obtained by means of bacterial activation and seed culture, including:
  • Escherichia coli (Escherichia coli) H03A2190830CCTCC NO: M2019900 is inoculated into the primary seed medium to obtain a primary seed culture solution;
  • the primary seed culture liquid is inoculated into the secondary seed medium to obtain the secondary seed culture liquid, that is, the seed liquid.
  • first-grade seed liquid was obtained by culturing at 35-37° C. and 200-220 rpm for 16-18 h.
  • both the primary seed medium and the secondary seed medium contain tryptone, yeast extract and sodium chloride, preferably, each 1L of the primary seed medium and the secondary seed medium contains 8-12g of peptone, 4-6g of yeast extract and 8-12g of sodium chloride.
  • the components of the primary seed medium and the secondary seed medium are: tryptone 8-12g/L, yeast extract 4-6g/L, sodium chloride 8-12g/L, water balance .
  • the preservation number is CCTCC NO: M2019900
  • the present invention also provides a method for producing sialic acid, the method comprising the step of converting polysialic acid into sialic acid.
  • the polysialic acid is prepared according to the above-mentioned method for producing polysialic acid.
  • polysialic acid After the polysialic acid is obtained, the polysialic acid is converted into sialic acid according to the manner disclosed in the prior art.
  • polysialic acid can be converted to sialic acid by acid hydrolysis or enzymatic hydrolysis.
  • the specific operation mode can refer to the method reported in the prior art.
  • Microfiltration the polysialic acid fermentation broth obtained by fermentation of Escherichia coli H03A2190830 CCTCC NO: M2019900 is sterilized by microfiltration with a ceramic membrane;
  • Ultrafiltration use an ultrafiltration membrane for ultrafiltration to remove small molecular impurities and salts to obtain a concentrated solution, and the concentration of polysialic acid in the concentrated solution is maintained above 30g/L;
  • Nanofiltration use nanofiltration membrane to concentrate after decolorization, further remove impurities and salt, and collect concentrated solution;
  • Evaporative concentration the concentrated solution is concentrated to a sialic acid concentration of 450-600 g/L at 56°C;
  • the invention self-selects a strain of Escherichia coli with high polysialic acid production efficiency and high yield, and when the strain is used to produce polysialic acid, it can not only shorten the production time, reduce the production cost, but also obtain a high-yield polysialic acid.
  • Polysialic acid has good industrial application prospects.
  • Escherichia coli H03A2190830 has been deposited in the China Center for Type Culture Collection (CCTCC) on November 6, 2019.
  • the address of the deposit is: Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei province, and the deposit number is As: CCTCC NO: M2019900.
  • Fig. 1 is a graph showing the content of polysialic acid in Examples 2-6 of the present invention and Comparative Example 1 under different fermentation times.
  • the concentrations are all concentrations by mass.
  • the content of polysialic acid in the fermentation broth is measured by the resorcinol method, and the steps are as follows:
  • R-reagent Accurately weigh 0.2g of resorcinol and dissolve it in 10mL of water, add 80mL of concentrated hydrochloric acid and 0.25mL of 0.1mol/L copper sulfate solution, and dilute to 100mL with deionized water.
  • Example 1 Strain screening and identification
  • Preliminary screening Weigh 10g of soil into a 500mL beaker, add 200mL of normal saline to dissolve, centrifuge at 5000rpm for 10min, take the supernatant and dilute 10 3 -10 4 times in a gradient, take 200L of the soil and apply it to crystal violet neutral red bile salts. On the agar medium plate, place it in an incubator at 37°C for 18-20h.
  • Re-screening select the colonies that are purple on the crystal violet neutral red bile salt agar medium and have red bile salt precipitation rings around the colonies to prepare a bacterial suspension, and insert it into the fermentation medium according to a certain proportion 37 After culturing at °C for 36 h, the content of polysialic acid was detected by the resorcinol method.
  • Fermentation medium formula: corn steep liquor dry powder 8g/L, potassium dihydrogen phosphate 5g/L, magnesium sulfate 0.5g/L, glucose monohydrate 25g/L, microbial solution 1mL/L, trace element solution 1mL/L.
  • the trace element solution composition is: FeSO 4 ⁇ 7H 2 O 40g/L, MnSO 4 ⁇ H 2 O 6g/L, CuSO 4 ⁇ 5H 2 0 1g/L, ZnSO 4 ⁇ 7H 2 0 7g/L; vitamin solution
  • the composition is: thiamine 0.3g/L, calcium pantothenate 2.8g/L, biotin 0.004g/L.
  • Liquid LB medium formula: yeast extract 5g/L, sodium chloride 10g/L, peptone 10g/L; pH 7.2.
  • Liquid LB medium formula: yeast extract 5g/L, sodium chloride 10g/L, peptone 10g/L; pH 7.2.
  • the composition of the trace element solution is: FeSO 4 ⁇ 7H 2 O 40g/L, MnSO 4 ⁇ H 2 O 6g/L, CuSO 4 ⁇ 5H 2 0 1g/L, ZnSO 4 ⁇ 7H 2 0 7g/L;
  • the vitamin solution is composed of : Thiamine 0.3g/L, calcium pantothenate 2.8g/L, biotin 0.004g/L.
  • the fermentation temperature was controlled at 37°C, and the pH in the fermenter was controlled to be 7.2 with 22% ammonia water during fermentation for 0-20h, and the pH in the fermenter was controlled to be 6.4 after 20h of fermentation.
  • the stirring speed was set to 500 rpm, and the ventilation rate was 2 vvm.
  • the dissolved oxygen and pH increased significantly.
  • 75% glucose monohydrate was added at a rate of 12g/L ⁇ h for 7 hours, and then 75% glucose monohydrate was added at a rate of 8g/L ⁇ h until the fermentation ended; Adjust the rotation speed and ventilation rate to control the dissolved oxygen to be 20-50% of the initial dissolved oxygen at the beginning of fermentation.
  • Polysialic acid was produced according to the method of Example 2, except: fermentation medium formula: corn steep liquor dry powder 10g/L, potassium dihydrogen phosphate 7g/L, magnesium sulfate 1g/L, glucose monohydrate 30g/L, vitamin solution 1mL/L and trace element solution 1mL/L.
  • the composition of the trace element solution is: FeSO 4 ⁇ 7H 2 O 50g/L, MnSO 4 ⁇ H 2 O 7g/L, CuSO 4 ⁇ 5H 2 0 1.25g/L, ZnSO 4 ⁇ 7H 2 0 9g/L;
  • the vitamin solution is composed of : Thiamine 0.5g/L, Calcium pantothenate 3.2g/L, Biotin 0.006g/L.
  • Polysialic acid was produced according to the method of Example 2, except: fermentation medium formula: corn steep liquor dry powder 12g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 1.5g/L, glucose monohydrate 35g/L, vitamins Solution 1mL/L and trace element solution 1mL/L.
  • the composition of trace element solution is: FeSO 4 ⁇ 7H 2 O 60g/L, MnSO 4 ⁇ H 2 O 8g/L, CuSO 4 ⁇ 5H 2 0 1.5g/L, ZnSO 4 ⁇ 7H 2 0 9g/L; vitamin solution composition It is: thiamine 0.7g/L, calcium pantothenate 3.6g/L, biotin 0.008g/L.
  • Polysialic acid was produced according to the method of Example 2, except that when the dissolved oxygen and pH increased significantly, 75% glucose monohydrate was added at a rate of 15g/L ⁇ h, maintained for 7h, and then the rate was changed to 10g /L ⁇ h, until the end of fermentation. After 33 hours of fermentation and culture, the tank was put in the tank, and the content of polysialic acid was detected.
  • Polysialic acid was produced according to the method of Example 2, except that after fermenting and culturing for 30 h, the tank was put in a tank, and the content of polysialic acid was detected.
  • the basal medium used is glucose 25g/L, ammonium sulfate 5g/L, casein peptone 15g/L, dipotassium hydrogen phosphate 20g/L, magnesium sulfate 0.4g/L, after mixing, steam sterilization. After reaching 121°C, the tank pressure was maintained at 0.09 MPa and sterilized for 30 minutes. The sterilization process requires continuous stirring at 200 rpm. After cooling, 2 ml of Escherichia coli CCTCC NO:M2019900 seed liquid was inoculated into 200 ml of basal medium, and cultured at 37°C for 12 hours to obtain a seed culture liquid.
  • the flow rate of glucose is started, and the flow rate maintains the concentration of glucose in the fermentation liquid to be 2-3 g/liter in the first 6 hours, the fermentation temperature is 37 ° C, and the pH is 25% ammonia with a mass percentage concentration.

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Abstract

提供了一株大肠埃希氏菌H03A2190830，该菌株的保藏号为CCTCC NO：M2019900。还提供了使用该大肠埃希氏菌生产聚唾液酸的方法，以及使用该方法制得的聚唾液酸生产唾液酸的方法。采用该菌株生产聚唾液酸时，不仅能缩短生产时间，降低生产成本，还能得到较高产量的聚唾液酸，具有很好的工业应用前景。

Description

一株大肠埃希氏菌及其在产聚唾液酸中的应用 技术领域
本发明涉及一株大肠埃希氏菌及其在产聚唾液酸中的应用，还涉及一种采用该菌种提高聚唾液酸产量的发酵工艺，属于生物发酵技术领域。
背景技术
聚唾液酸(Polysialic acid，PSA)是以N-乙酰神经氨酸为单体，以α-2,8和/或α-2,9糖苷键连接的线性聚合物。PSA是以荚膜的形式存在于一些哺乳动物和致病性细菌细胞表面的糖蛋白和糖脂糖链末端，可用作蛋白药物的缓释材料和神经修复手术中的支架材料。
目前，生产聚唾液酸的方法只有微生物发酵法。随着近几年聚唾液酸在医药领域应用潜力的不断发现，细菌合成聚唾液酸的文献和报道逐渐增多，提高聚唾液酸的发酵水平的研究也逐步开展。报道的聚唾液酸的产量大多集中在10g/L左右，难以满足市场需求，因此提高聚唾液酸的发酵水平是高校和企业的研究方向。
此外，现阶段微生物发酵法制备聚唾液酸的发酵时间也较长，例如江苏集萃工业生物技术研究有限公司经7个批次连续发酵130h，实现聚唾液酸总产量为48.1g/L，但长时间的连续发酵增加了企业生产成本。专利CN 111733092 A提供了一种发酵法生产多聚唾液酸的方法，该方法连续发酵20h得到的聚唾液酸的产量为10g/L，单位时间发酵速度为0.5g/L·h。这一方法缩短了生产时间，降低了成本，但是发酵过程中有一半的时间都是通入纯氧，一方面增加了生产成本，更重要的是增加了企业生产的安全隐患。
发明内容
本发明的目的是提供一株大肠埃希氏菌(Escherichia coli)，该菌种发酵生产聚唾液酸时，经过验证具有较快的发酵速度和较高的发酵总产量，解决了现有技术中发酵生产聚唾液酸存在的发酵水平不稳定、聚唾液酸产量低、发酵时间长的问题。
本发明提供了一株大肠埃希氏菌(Escherichia coli)H03A2190830，该菌株已于2019年11月6日保藏于中国典型培养物保藏中心(简称CCTCC)，保藏单位地址为：湖北省武汉市武昌珞珈山武汉大学，保藏号为CCTCC NO：M2019900。
进一步的，本发明菌株是从土壤中筛选得到，经过鉴定，该菌株的16Sr DNA序列如SEQ ID NO：1所示，分类命名及拉丁学名为大肠埃希氏菌(Escherichia coli)。
本发明还提供了上述大肠埃希氏菌(Escherichia coli)H03A2190830CCTCC NO：M2019900在生产聚唾液酸中的应用，该菌株能够提高聚唾液酸的生产效率，提高聚唾液酸的总产量，具有很好的工业化应用前景。
进一步的，在发酵液中产生聚唾液酸，优选的，聚唾液酸在发酵液中的浓度在15.9g/L以上。
进一步的，发酵培养基包含：碳源、氮源、无机盐、维生素和微量元素，优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL和微量元素0.8-1.2mL。
本发明提供了一种生产聚唾液酸的方法，该方法以上述大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900为菌种发酵生产含有聚唾液酸的发酵液。
进一步的，在使用上述大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900进行发酵生产时，所用的工艺方法和条件可以从现有技术中公开和普通使用的工艺方法和条件中进行选择。
进一步的，本发明提供了一种优选的生产聚唾液酸的方法，在发酵时，将种子液接种至发酵培养基中，进行初始发酵，待糖分耗尽时，流加糖分继续发酵，直至发酵结束得到发酵液。
优选的，所述发酵培养基包含：碳源、氮源、无机盐、维生素和微量元素；进一步优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL，微量元素0.8-1.2mL；最优选的，所述发酵培养基为：玉米浆干粉8-12g/L，磷酸二氢钾5-10g/L，一水葡萄糖(单独灭菌)25-35g/L，硫酸镁0.5-1.5g/L，维生素溶液(过膜除菌)0.8-1.2mL/L，微量元素溶液(过膜除菌)0.8-1.2mL/L，水余量。其中，维生素溶液中各成分及组成为：硫胺素0.3-0.7g/L，泛酸钙2.8-3.6g/L，生物素0.004-0.008g/L，水余量。微量元素溶液中各成分及组成为：FeSO 4·7H 20 40-60g/L，MnSO 4·H 2O 6-8g/L，CuSO 4·5H 20 1-1.5g/L，ZnSO 4·7H 2O 7-10g/L，水余量。
进一步的，当糖分耗尽时，发酵罐中溶氧和pH明显上升。溶氧和pH明显上升指的是，达到某一发酵时间时，溶氧和pH与前一发酵时间相比有梯度式上升，上升在5％以上。
进一步的，在发酵0-20h时，控制体系pH为7-7.5，发酵20h之后，控制体系pH为6.2-6.5。
进一步的，整个发酵期间保持发酵温度为35-37℃。
进一步的，待糖分耗尽时，以8-15g/L·h的速率补加60-75wt％的葡萄糖溶液，以流加的方式补加。从开始流加糖分时计，再发酵24-27h。
进一步的，从发酵起始到流加糖分之间的初始发酵阶段，控制搅拌转速为400-500rpm，通气量为1.5-2vvm。待流加糖分后，调整搅拌转速和通气量保持溶氧量为发酵刚开始(即发酵起始时间)时溶氧量的20-50％。
进一步的，所述方法包括：
发酵时，将种子液接种至发酵培养基中，进行初始发酵，待糖分耗尽时，流加糖分继续发酵，直至发酵结束得到发酵液；优选的，所述发酵培养基包含：碳源、氮源、无机盐、维生素和微量元素，进一步优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL，微量元素0.8-1.2mL，最优选的，所述发酵培养基为：玉米浆干粉8-12g/L，磷酸二氢钾5-10g/L，一水葡萄糖25-35g/L，硫酸镁0.5-1.5g/L，维生素溶液0.8-1.2mL/L，微量元素溶液0.8-1.2mL/L，水余量；其中，维生素溶液中各成分及组成为：硫胺素0.3-0.7g/L，泛酸钙2.8-3.6g/L，生物素0.004-0.008g/L；微量元素溶液中各成分及组成为：FeSO 4·7H 20 40-60g/L，MnSO 4·H 2O 6-8g/L，CuSO 4·5H 20 1-1.5g/L，ZnSO 4·7H 2O 7-10g/L；
发酵0-20h时，控制体系pH为7-7.5，发酵20h之后，控制体系pH为6.2-6.5；优选的，整个发酵时间保持温度为35-37℃。
进一步的，所述方法包括：
发酵时，将种子液接种至发酵培养基中，进行初始发酵，待糖分耗尽时，流加糖分继续发酵，直至发酵结束得到发酵液；优选的，所述发酵培养基包含:碳源、氮源、无机盐、维生素和微量元素，进一步优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL和微量元 素0.8-1.2mL/L，最优选的，所述发酵培养基为：玉米浆干粉8-12g/L，磷酸二氢钾5-10g/L，一水葡萄糖25-35g/L，硫酸镁0.5-1.5g/L，维生素溶液0.8-1.2mL/L，微量元素溶液0.8-1.2mL/L，水余量；其中，维生素溶液中各成分及组成为：硫胺素0.3-0.7g/L，泛酸钙2.8-3.6g/L，生物素0.004-0.008g/L；微量元素溶液中各成分及组成为：FeSO 4·7H 20 40-60g/L，MnSO 4·H 2O 6-8g/L，CuSO 4·5H 20 1-1.5g/L，ZnSO 4·7H 2O 7-10g/L；
发酵0-20h时，控制体系pH为7-7.5，发酵20h之后，控制体系pH为6.2-6.5；优选的，整个发酵时间保持温度为35-37℃；
待糖分耗尽时，以8-15g/L·h的速率流加60-75wt％的葡萄糖溶液；优选的，流加糖分后，再发酵24-27h。
进一步的，所述方法包括：
发酵时，将种子液接种至发酵培养基中，进行初始发酵，待糖分耗尽时，流加糖分继续发酵，直至发酵结束得到发酵液；优选的，所述发酵培养基包含:碳源、氮源、无机盐、维生素和微量元素，进一步优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL和微量元素0.8-1.2mL，最优选的，所述发酵培养基为：玉米浆干粉8-12g/L，磷酸二氢钾5-10g/L，一水葡萄糖25-35g/L，硫酸镁0.5-1.5g/L，维生素溶液0.8-1.2mL/L，微量元素溶液0.8-1.2mL/L，水余量；其中，维生素溶液中各成分及组成为：硫胺素0.3-0.7g/L，泛酸钙2.8-3.6g/L，生物素0.004-0.008g/L；微量元素溶液中各成分及组成为：FeSO 4·7H 20 40-60g/L，MnSO 4·H 2O 6-8g/L，CuSO 4·5H 20 1-1.5g/L，ZnSO 4·7H 2O 7-10g/L；
发酵0-20h时，控制体系pH为7-7.5，发酵20h之后，控制体系pH为6.2-6.5；优选的，整个发酵时间保持温度为35-37℃；
待糖分耗尽时，以8-15g/L·h的速率流加60-75wt％的葡萄糖溶液；优选的，流加糖分后，再发酵24-27h；
在流加糖分前的初始发酵阶段，搅拌转速为400-500rpm，通气量为1.5-2vvm；待流加糖分后，调整搅拌转速和通气量保持溶氧量为发酵起始时溶氧量的20％-50％。
进一步的，种子液可以采用菌种活化和种子培养的方式获得，包括：
将大肠埃希氏菌(Escherichia coli)H03A2190830CCTCC NO：M2019900接种至一级种子培养基中得到一级种子培养液；
将一级种子培养液接种至二级种子培养基中得到二级种子培养液，即种子液。
进一步的，在35-37℃、200-220rpm下培养16-18h得到一级种子液。
进一步的，在35-37℃、200-220rpm下培养6-8h得到二级种子培养液。
进一步的，一级种子培养基和二级种子培养基中均包含胰蛋白胨、酵母提取物和氯化钠，优选的，每1L一级种子培养基和二级种子培养基中均包含8-12g的蛋白胨、4-6g的酵母提取物和8-12g的氯化钠。
进一步的，包括：
a.将大肠埃希氏菌(Escherichia coli)H03A2190830CCTCC NO：M2019900接种至一级种子培养基中，35-37℃、200-220rpm下培养16-18h，得到一级种子培养液；
b.将一级种子培养液接种至二级种子培养基中，35-37℃、200-220rpm下培养6-8h，得到二级种子培养液，即种子液。
进一步的，所述一级种子培养基和二级种子培养基的成分均为：胰蛋白胨8-12g/L，酵母提取物4-6g/L，氯化钠8-12g/L，水余量。
在本发明某一具体实施方式中，提供了一种具体的聚唾液酸生产方法，如下：
(1)菌种活化
取保藏的大肠埃希氏菌(保藏编号为CCTCC NO：M2019900)，接种至含有50mL的一级种子培养基中，35-37℃，200-220rpm培养16-18h，得到一级种子培养液。
(2)种子培养
取1-2mL一级种子培养液，接种至含有100-200mL的二级种子培养基中，35-37℃，200-220rpm培养6-8h，得到二级种子培养液。
(3)发酵
取二级种子培养液，按照5-8wt％的接种量，接种至含有3L发酵培养基的5L发酵罐中，进行初始发酵，待溶氧和pH明显上升时，以8-15g/L·h的速率补加糖分，继续发酵24-27h；发酵0-20h时控制pH为7.2，发酵20h之后控制pH为6.4；在流加糖分之前的初始发酵阶段，设置转速为500rpm、通气量为2vvm； 在流加糖分后，调节转速和通气量，控制溶氧为发酵最初时间(发酵最开始时间)溶氧量的20％-50％。
本发明还提供了一种生产唾液酸的方法，该方法包括将聚唾液酸转化为唾液酸的步骤。所述聚唾液酸按照上述生产聚唾液酸的方法制得。
在得到聚唾液酸后，根据现有技术中公开的方式，将聚唾液酸转化为唾液酸。例如，可以将聚唾液酸通过酸水解或酶水解转化为唾液酸。具体的操作方式可以参照现有技术中报道的方法。
在本发明某一具体实施方式中，公开了一种聚唾液酸转化为唾液酸的方法，具体如下：
(1)微滤：将大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900发酵得到的聚唾液酸发酵液用陶瓷膜进行微滤除菌；
(2)超滤：使用超滤膜进行超滤，除去小分子杂质和盐分，得到浓缩液，浓缩液中聚唾液酸的浓度保持30g/L以上；
(3)水解：调节浓缩液pH为1.5，83℃水解4-8h，加碱中和至pH 5.5以上，然后过滤；
(4)纳滤：脱色后使用纳滤膜进行浓缩，进一步除去杂质和盐分，收集浓缩液；
(5)蒸发浓缩：56℃下将浓缩液浓缩至唾液酸浓度为450-600g/L；
(6)结晶：调节上述步骤(5)中溶液pH为1.2-1.5之间，0-4℃结晶12-24h；
(7)洗涤干燥：结晶后分离晶体，洗涤，干燥，得唾液酸产品。
本发明具有以下有益效果：
本发明自行筛选了一株聚唾液酸生产效率和产量均较高的大肠埃希氏菌，采用该菌株生产聚唾液酸时，不仅能缩短生产时间，降低生产成本，还能得到较高产量的聚唾液酸，具有很好的工业应用前景。
微生物保藏信息
大肠埃希氏菌(Escherichia coli)H03A2190830，已于2019年11月6日保藏于中国典型培养物保藏中心(简称CCTCC)，保藏单位地址为：湖北省武汉市武昌珞珈山武汉大学，保藏编号为：CCTCC NO：M2019900。
附图说明
图1为本发明实施例2-6和对比例1在不同发酵时间下聚唾液酸含量曲线图。
具体实施方式
下面结合具体的实施例对本发明进行进一步阐述，但本发明的保护范围并不仅限于此。
下述实施例中，如无特别说明，所述浓度均为质量百分浓度。
下述实施例中，发酵液中聚唾液酸的含量采用间苯二酚法测得，步骤如下：
1.R-试剂的配制：准确称量0.2g间苯二酚溶于10mL水中，并加入80mL浓盐酸和0.25mL 0.1mol/L的硫酸铜溶液，用去离子水定容至100mL。
2.标准曲线的绘制：分别吸取0mL，0.2mL，0.4mL，0.6mL，0.8mL，1.0mL，1.2mL，1.4mL，1.6mL，1.8mL的100mg/L的标准聚唾液酸溶液于比色管中，加水至2.0mL，加入2.0mL R-试剂，沸水浴15min，流水冷却后加入5mL正戊醇，剧烈振荡后冷水浴冷却10min，1000rpm离心5min，将有机相转入比色皿中，以水作对照，在580nm处测光密度，绘制聚唾液酸浓度与光密度的标准曲线。
3、样品分析：取一定量的发酵液稀释m倍，用0.22微米滤膜过滤后，吸取2mL于比色管中，加入2.0mL R-试剂，按照步骤2的方法测光密度，测得的光密度由标准曲线查算出样品中聚唾液酸量为n，按照下式得出发酵液中聚唾液酸的含量：
聚唾液酸含量
Figure PCTCN2021136947-appb-000001
实施例1：菌株筛选和鉴定
1、初筛：称取10g土壤于500mL烧杯中，加入200mL生理盐水溶解，5000rpm离心10min，取上层清液梯度稀释10 3-10 4倍，分别取200L涂布到结晶紫中性红胆盐琼脂培养基平板上，放置培养箱中37℃培养18-20h。
2、复筛：挑选结晶紫中性红胆盐琼脂培养基上为紫红色，菌落周围有红色的胆盐沉淀环的菌落制备成菌悬液，将其按照一定比例接入发酵培养基中37℃培养36h，采用间苯二酚法检测聚唾液酸含量，选取吸光值大的菌种作为目标菌种，命名为H03A2190830。
发酵培养基配方：玉米浆干粉8g/L，磷酸二氢钾5g/L，硫酸镁0.5g/L，一水葡萄糖25g/L，微生素溶液1mL/L，微量元素溶液1mL/L。其中，微量元素溶液组成为：FeSO 4·7H 2O 40g/L，MnSO 4·H 2O 6g/L，CuSO 4·5H 20 1g/L，ZnSO 4·7H 20  7g/L；维生素溶液组成为：硫胺素0.3g/L，泛酸钙2.8g/L，生物素0.004g/L。
3、鉴定：将得到的目标菌种H03A2190830送至中国典型微生物保藏中心进行鉴定，其16Sr DNA序列如SEQ ID NO：1所示，分类命名及拉丁学名为大肠埃希氏菌(Escherichia coli)。
实施例2
1、取保藏的大肠埃希氏菌(保藏编号CCTCC NO:M2019900)，接种于50mL液体LB培养基中，200rpm，37℃培养18h，得到一级种子培养液。液体LB培养基配方：酵母提取物5g/L，氯化钠10g/L，蛋白胨10g/L；pH为7.2。
2、取2mL一级种子培养液接种于200mL液体LB培养基，200rpm，37℃培养6h，得到二级种子培养液。液体LB培养基配方：酵母提取物5g/L，氯化钠10g/L，蛋白胨10g/L；pH为7.2。
3、将200mL二级种子培养液接种至装有3L发酵培养基的5L发酵罐中，进行发酵制备聚唾液酸；发酵培养基配方：玉米浆干粉8g/L，磷酸二氢钾5g/L，硫酸镁0.5g/L，一水葡萄糖25g/L，维生素溶液1mL/L和微量元素溶液1mL/L。微量元素溶液组成为：FeSO 4·7H 2O 40g/L，MnSO 4·H 2O 6g/L，CuSO 4·5H 20 1g/L，ZnSO 4·7H 20 7g/L；维生素溶液组成为：硫胺素0.3g/L，泛酸钙2.8g/L，生物素0.004g/L。
4、发酵温度控制在37℃，发酵0-20h时使用22％氨水控制发酵罐内pH为7.2，发酵20h后控制发酵罐内pH为6.4。在流加葡萄糖以前，设置搅拌转速为500rpm，通气量为2vvm，待发酵6h左右时，溶氧和pH出现明显上升。此时，以12g/L·h的速率补加75％的一水葡萄糖，维持7h，然后以8g/L·h的速率补加75％的一水葡萄糖，至发酵结束；流加葡萄糖后，调整转速和通气量控制溶氧量为最开始发酵时的初始溶氧的20-50％。
发酵培养33h后进行放罐，检测聚唾液酸含量。
实施例3
按照实施例2的方法生产聚唾液酸，不同的是：发酵培养基配方：玉米浆干粉10g/L，磷酸二氢钾7g/L，硫酸镁1g/L，一水葡萄糖30g/L，维生素溶液1mL/L和微量元素溶液1mL/L。微量元素溶液组成为：FeSO 4·7H 2O 50g/L，MnSO 4·H 2O7g/L，CuSO 4·5H 20 1.25g/L，ZnSO 4·7H 20 9g/L；维生素溶液组成为：硫胺素0.5g/L， 泛酸钙3.2g/L，生物素0.006g/L。
发酵培养33h后进行放罐，检测聚唾液酸含量。
实施例4
按照实施例2的方法生产聚唾液酸，不同的是：发酵培养基配方：玉米浆干粉12g/L，磷酸二氢钾10g/L，硫酸镁1.5g/L，一水葡萄糖35g/L，维生素溶液1mL/L和微量元素溶液1mL/L。微量元素溶液组成为：FeSO 4·7H 2O 60g/L，MnSO 4·H 2O 8g/L，CuSO 4·5H 20 1.5g/L，ZnSO 4·7H 20 9g/L；维生素溶液组成为：硫胺素0.7g/L，泛酸钙3.6g/L，生物素0.008g/L。
发酵培养33h后进行放罐，检测聚唾液酸含量。
实施例5
按照实施例2的方法生产聚唾液酸，不同的是：待溶氧和pH出现明显上升时，以15g/L·h的速率补加75％的一水葡萄糖，维持7h，然后速率改为10g/L·h，至发酵结束。发酵培养33h后进行放罐，检测聚唾液酸含量。
实施例6
按照实施例2的方法生产聚唾液酸，不同的是：发酵培养30h后进行放罐，检测聚唾液酸含量。
对比例1
参照专利CN 111733092 A中实施例1的方法生产聚唾液酸，不同的是，使用本发明大肠埃希氏菌CCTCC NO:M2019900。步骤为：
(1)所用的基础培养基为葡萄糖25g/L，硫酸铵5g/L，酪蛋白胨15g/L，磷酸氢二钾20g/L，硫酸镁0.4g/L，混合均匀后，通蒸汽灭菌。至121℃后，维持罐压0.09MPa，灭菌30分钟。灭菌过程需用200rpm连续搅拌。待冷却后，将2ml大肠埃希氏菌CCTCC NO:M2019900种子液接种到200ml基础培养基中，37℃培养12小时获得种子培养液。
(2)将200ml种子培养液接种于3L含上述基础培养基的发酵罐中，通空气，采用氨水将pH控制在6.6-6.8，37℃下进行发酵培养，并流加碳源。
(3)在接种种子培养液后开始流加葡萄糖，前6小时流加速度维持葡萄糖在发酵液中的浓度为2-3克/升，发酵温度37℃，用质量百分比浓度为25％氨水将pH控制在6.6-6.8，6小时后，通纯氧，纯氧与空气体积比为0.75：1，温度 仍然控制在37℃，pH控制在6.6-6.8；10小时后，纯氧与空气体积比为1：1，pH控制为6.3-6.5，温度由37℃降为34℃；在6小时到发酵12小时流加速度维持葡萄糖在发酵液中的浓度为5-8克/升；从12小时起开始降低糖浓度，使葡萄糖在发酵液中的浓度为2-3克/升，17小时后停加葡萄糖；发酵17小时后采用质量百分比浓度为0.5％的Ca(OH) 2溶液调节pH值8.0-8.5，停止搅拌，并降温至20℃，此时停止纯氧，改用无菌空气，维持通气2小时，到20小时结束发酵过程。
发酵结束后，检测聚唾液酸含量。
在实施例2-6和对比例1的发酵过程中，每隔一段时间取固定的发酵液，对其中的聚唾液酸含量进行检测，结果如图1所示。从图中可以看出，随着发酵时间的延长，聚唾液酸的含量不断提高。
实施例2-6及对比例1所得聚唾液酸含量如下表1所示。
表1
  发酵时间(h) 聚唾液酸含量(g/L) 发酵速度(g/L·h)
实施例2 33 16.7 0.506
实施例3 33 19.3 0.585
实施例4 33 18.1 0.548
实施例5 33 17.6 0.533
实施例6 30 15.9 0.530
对比例1 20 11.1 0.555
CN 111733092 A 20 10 0.500
从表1可以看出，使用实施例3的发酵培养基时，聚唾液酸的含量更高，发酵速度最快。从对比例1的数据与专利CN 111733092 A中数据对比可以看出，在同等的发酵条件和发酵时间下，本发明大肠埃希氏菌产聚唾液酸的速度和总产量明显提高，效果优于专利CN 111733092 A中的菌种。从实施例和对比例1的数据对比可以看出，本发明工艺方法也具有一定的提高生产速度的作用。

Claims (15)

  1. 一株大肠埃希氏菌(Escherichia coli)H03A2190830，其特征是：保藏号为CCTCC NO：M2019900。
  2. 根据权利要求1所述的大肠埃希氏菌(Escherichia coli)H03A2190830，其特征是：其16Sr DNA序列如SEQ ID NO：1所示。
  3. 权利要求1或2所述的大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900在生产聚唾液酸中的应用。
  4. 根据权利要求3所述的应用，其中，在发酵液中产生聚唾液酸，优选的，聚唾液酸在发酵液中的浓度在15.9g/L以上。
  5. 根据权利要求3或4所述的应用，发酵培养基包含：碳源、氮源、无机盐、维生素和微量元素，优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL和微量元素0.8-1.2mL。
  6. 一种生产聚唾液酸的方法，其特征是：以权利要求1或2所述的大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900为菌种发酵生产含有聚唾液酸的发酵液。
  7. 根据权利要求6所述的方法，其特征是：发酵时，将种子液接种至发酵培养基中，进行初始发酵，待糖分耗尽时，流加糖分继续发酵，直至发酵结束得到发酵液；优选的，所述发酵培养基包含：碳源、氮源、无机盐、维生素和微量元素；进一步优选的，在1L发酵培养基中含有碳源25-35g，氮源8-12g，无机盐5.5-11.5g，维生素0.8-1.2mL和微量元素0.8-1.2mL；最优选的，所述发酵培养基为：玉米浆干粉8-12g/L，磷酸二氢钾5-10g/L，一水葡萄糖25-35g/L，硫酸镁0.5-1.5g/L，维生素溶液0.8-1.2mL/L，微量元素溶液0.8-1.2mL/L，水余量；其中，维生素溶液中各成分及组成为：硫胺素0.3-0.7g/L，泛酸钙2.8-3.6g/L，生物素0.004-0.008g/L；微量元素溶液中各成分及组成为：FeSO 4·7H 2O 40-60g/L，MnSO 4·H 2O 6-8g/L，CuSO 4·5H 2O 1-1.5g/L，ZnSO 4·7H 2O 7-10g/L。
  8. 根据权利要求6或7所述的方法，其特征是：发酵0-20h时，控制体系pH为7-7.5，发酵20h之后，控制体系pH为6.2-6.5；优选的，整个发酵时间保持温度为35-37℃。
  9. 根据权利要求6-8中任一项所述的方法，其特征是：待糖分耗尽时，以8-15g/L·h的速率流加60-75wt％的葡萄糖溶液；优选的，流加糖分后，再发酵24-27h。
  10. 根据权利要求6-9中任一项所述的方法，其特征是：在流加糖分前的初始发酵阶段，搅拌转速为400-500rpm，通气量为1.5-2vvm；待流加糖分后，调整搅拌转速和通气量保持溶氧量为发酵起始时溶氧量的20％-50％。
  11. 根据权利要求6-10中任一项所述的方法，其特征是：种子液的获取方式为：
    a.将大肠埃希氏菌(Escherichia coli)H03A2190830 CCTCC NO：M2019900接种至一级种子培养基中得到一级种子培养液；
    b.将一级种子培养液接种至二级种子培养基中得到二级种子培养液，即种子液。
  12. 根据权利要求11所述的方法，其特征是：在35-37℃、200-220rpm下培养16-18h得到一级种子液。
  13. 根据权利要求11或12所述的方法，其特征是：在35-37℃、200-220rpm下培养6-8h得到二级种子培养液。
  14. 根据权利要求11-13中任一项所述的方法，其特征是：一级种子培养基和二级种子培养基中均包含胰蛋白胨、酵母提取物和氯化钠，优选的，每1L一级种子培养基和二级种子培养基中均包含8-12g的蛋白胨、4-6g的酵母提取物和8-12g的氯化钠。
  15. 一种生产唾液酸的方法，包括将聚唾液酸转化为唾液酸的步骤，其特征是：所述聚唾液酸按照权利要求6-14中任一项所述的生产聚唾液酸的方法制得。
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