CN117683832A - Polysialic acid fermentation medium and application thereof - Google Patents
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Abstract
The invention discloses a polysialic acid fermentation medium and application thereof, and relates to the technical field of biological fermentation. The culture medium is divided into three parts, namely a basic culture medium, a nutritional component and a protective component, wherein the nutritional component comprises Chinese yam, N-acetylglucosamine and microelements, and can provide necessary nutritional components for escherichia coli on one hand and improve the resistance of the escherichia coli to bacteriophage on the other hand; the protective component comprises glycerin and dopamine, and can destroy the complete form of phage and the specific adsorption in the process of phage, so that the probability of phage infection to escherichia coli is reduced.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a polysialic acid fermentation medium and application thereof.
Background
N-acetylneuraminic acid (N-acetylneuraminic acid, neu5 Ac), also known as sialic acid, is a derivative of neuraminic acid, and is widely found in animals, plants, microorganisms, and natural substances. Sialic acid can make saliva feel smooth, and has the function of inducing invasion of bacteria and preventing invasion of bacteria. Sialic acid is also a receptor for influenza virus and also a constituent of brain, and thus has a certain effect in the treatment of influenza, neurological diseases, inflammation, tumors, and the like.
Polysialic acid (PSA) is a homopolymer of N-acetylneuraminic acid (Neu 5 Ac) linked by alpha-2, 8 and/or alpha-2, 9 bonds, and is the major component of capsular polysaccharides of a few bacteria. Both polysialic acid and sialic acid play an important role in many physiological and pathological processes.
At present, the production method of polysialic acid is mainly a microbial fermentation method, and bacillus subtilis and escherichia coli are two microbial strains commonly used for preparing polysialic acid through fermentation. The bacillus subtilis does not contain Neu5Ac and a synthetic path, genetic modification is needed to construct engineering bacteria, the construction of the engineering bacteria has the defects of high cost, long time consumption and the like, and mutation or transgenic treatment is used as an emerging biotechnology means, so that the bacillus subtilis has the problems of immaturity and uncertainty, and the obtained product is accompanied with biosafety. The escherichia coli can be fermented to prepare the polysialic acid without genetic modification, and has the advantages of wide sources, low growth requirements, low cost and the like, so the escherichia coli is still the preferred strain for preparing the polysialic acid by fermentation.
The paper mentions that the recombinant bacillus subtilis is used for efficiently synthesizing N-acetylneuraminic acid by a whole cell transformation method, and escherichia coli is easy to be infected by phage in the fermentation process, so that the difficulty of industrial production is increased. In the prior art, production means for improving the yield of polysialic acid prepared by a microbial fermentation method are concentrated on construction and continuous transformation of engineering bacteria, and a method for improving the production efficiency of polysialic acid by fermenting common bacteria by improving fermentation conditions is freshly reported.
The invention patent CN108588152B discloses a polysialic acid fermentation medium, a production method of polysialic acid and a polysialic acid product, wherein trace elements and CTAB (cetyltrimethylammonium bromide) are added into the medium. However, the invention does not solve the problem that it is susceptible to phage infection when fermentation is performed using E.coli.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a polysialic acid fermentation medium and application thereof. According to the invention, components such as yam, glycerol and dopamine are added on the basis of a basic polysialic acid fermentation culture medium, and the culture medium is divided into three parts, namely a basic culture medium, a nutritional component and a protective component. Wherein, the nutrition component can provide additional nutrition for the escherichia coli during fermentation, thereby improving the antiviral capability of the escherichia coli; the protective substance has anti-phage activity, and can be used as a protective substance to destroy phage and protect Escherichia coli after being added into culture medium, thereby improving yield of polysialic acid.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, a fermentation medium is provided, comprising a basal medium, a nutritional component and a protective component, wherein the weight ratio of the basal medium, the nutritional component and the protective component is (4-6): (1-2): (1.5-4);
the basic culture medium consists of the following raw materials in parts by weight: 10-20 parts of glucose, 1-5 parts of sodium pyruvate, 4-5 parts of magnesium sulfate, 0.1-6 parts of dipotassium hydrogen phosphate, 2-6 parts of ammonium chloride and 0.5-2 parts of corn steep liquor dry powder;
the nutritional components consist of the following raw materials: 0.5-4 parts of Chinese yam, 10-20 parts of N-acetylglucosamine and 0.8-1.2 parts of trace element additive;
the protective component consists of the following raw materials: 3-5 parts of glycerol and 1-5 parts of dopamine.
Preferably, the trace element additive comprises:
MnCl 2 0.5-1 g/L、CoCl 2 0.15-0.25 g/L、CrCl 3 0.05-0.1 g/L and ZnSO 4 0.05-1.2 g/L。
In a second aspect of the invention there is provided the use of a polysialic acid fermentation medium as described above in the fermentative preparation of polysialic acid.
Preferably, the method for preparing polysialic acid by fermentation comprises the following steps:
inoculating Escherichia coli into the fermentation medium according to any one of claims 1-2, and fermenting.
Preferably, the inoculation amount of the escherichia coli is 1-1.5% of the volume of the fermentation medium.
Preferably, the fermentation conditions are: fermenting at 35-40deg.C and pH of 6.0-6.6 for 65-70 hr.
Preferably, the gas flux of the fermentation is 0.5-2vvm, and the oxygen dissolution amount is 26-30%.
The invention has the beneficial effects that:
(1) The invention divides the culture medium into three parts of basic culture medium, nutrient component and protective component. Wherein, the nutrition component can provide additional nutrition for the escherichia coli during fermentation, thereby improving the antiviral capability of the escherichia coli; the protective substance has anti-phage activity and can be used as a protective substance to destroy phage and protect Escherichia coli after being added into the culture medium.
(2) The nutrient components and the protective components in the culture medium can promote the production of polysialic acid, and the three components can play a synergistic effect when used together, so that the production efficiency of polysialic acid can be further improved while phage erosion is resisted.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background art, in the prior art, the preparation method of polysialic acid is only a microbial fermentation method, and escherichia coli is easily infected by phage in the fermentation process, so that the yield of the prepared polysialic acid is low, and the industrial production is not easy.
Based on the above, the invention provides a fermentation medium which can reduce the damage probability of phage to escherichia coli to a certain extent when being applied to fermentation preparation of polysialic acid. The culture medium is divided into three parts, namely a basic culture medium, a nutritional component and a protective component, wherein the nutritional component comprises Chinese yam, N-acetylglucosamine and microelements, and can provide necessary nutritional components for escherichia coli on one hand and improve the resistance of the escherichia coli to bacteriophage on the other hand; the protective component comprises glycerin and dopamine, and can destroy the complete form of phage and the specific adsorption in the process of phage, so that the probability of phage infection to escherichia coli is reduced.
In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
In the following examples and comparative examples, E.coli was purchased from the China center for type culture collection, strain No. CICC 10899, and T4 phage was purchased from the China center for type culture collection, strain No.: cctccc AB 2015375.
The method for activating the escherichia coli comprises the following steps:
(1) Inoculating escherichia coli into a liquid LB culture medium according to an inoculating ratio of 1:800, culturing for 12 hours at 37 ℃ and 220rpm to obtain a primary activated seed culture solution;
(2) The primary activated seed culture solution was prepared at 1:6, inoculating the strain to fresh liquid LB medium again, and culturing at 37 ℃ and 220rpm for 2 hours to obtain activated escherichia coli.
The liquid LB culture medium comprises the following components: 10g/L tryptone, 10g/L sodium chloride, 5g/L yeast powder.
The trace element additives in the following examples and comparative examples include: mnCl 2 1 g/L、CoCl 2 0.25 g/L、CrCl 3 0.1 g/L and ZnSO 4 1.2 g/L。
Example 1: preparation of polysialic acid.
The preparation method comprises the following steps: the activated escherichia coli is inoculated to the polysialic acid fermentation medium, and the inoculation amount is 1.25% of the volume of the polysialic acid fermentation medium. The fermentation conditions are as follows: fermenting at 37deg.C and pH of 6.5 for 70 hr with air flux of 1vvm and oxygen dissolution of 28%.
The polysialic acid fermentation medium in the embodiment comprises a basic medium, a nutritional component and a protective component according to the weight ratio of 5:1.5:2, the proportion of the components is as follows; wherein the basic culture medium consists of the following raw materials in parts by weight: 15 parts of glucose, 2 parts of sodium pyruvate, 4.5 parts of magnesium sulfate, 4 parts of dipotassium hydrogen phosphate, 4 parts of ammonium chloride and 1 part of corn steep liquor dry powder; the nutritional components consist of the following raw materials: 2 parts of Chinese yam, 15 parts of N-acetylglucosamine and 1 part of trace element additive; the protective component consists of the following raw materials: 4 parts of glycerol and 3 parts of dopamine.
Example 2: preparation of polysialic acid.
The preparation method comprises the following steps: the activated escherichia coli is inoculated to the polysialic acid fermentation medium, and the inoculation amount is 1% of the volume of the polysialic acid fermentation medium. The fermentation conditions are as follows: fermenting at 35deg.C and pH of 6.0 for 65 hr with gas flux of 0.5vvmv and oxygen dissolution of 26%.
The polysialic acid fermentation medium in the embodiment comprises a basic medium, a nutritional component and a protective component according to the weight ratio of 4:1: 1.5; wherein the basic culture medium consists of the following raw materials in parts by weight: 10 parts of glucose, 1 part of sodium pyruvate, 4 parts of magnesium sulfate, 0.1 part of dipotassium hydrogen phosphate, 2 parts of ammonium chloride and 0.5 part of corn steep liquor dry powder; the nutritional components consist of the following raw materials: 0.5 part of Chinese yam, 10 parts of N-acetylglucosamine and 0.8 part of trace element additive; the protective component consists of the following raw materials: 3 parts of glycerol and 1 part of dopamine.
Example 3: preparation of polysialic acid.
The preparation method comprises the following steps: the activated escherichia coli is inoculated to the polysialic acid fermentation medium, and the inoculation amount is 1.5% of the volume of the polysialic acid fermentation medium. The fermentation conditions are as follows: fermenting at 40deg.C and pH of 6.6 for 70 hr with gas flux of 2vvm and oxygen dissolution of 30%.
The polysialic acid fermentation medium in the embodiment comprises a basic medium, a nutritional component and a protective component according to the weight ratio of 6:2:4, the proportion composition of the components is as follows; wherein the basic culture medium consists of the following raw materials in parts by weight: 20 parts of glucose, 5 parts of sodium pyruvate, 5 parts of magnesium sulfate, 6 parts of dipotassium hydrogen phosphate, 6 parts of ammonium chloride and 2 parts of corn steep liquor dry powder; the nutritional components consist of the following raw materials: 4 parts of Chinese yam, 20 parts of N-acetylglucosamine and 1.2 parts of trace element additive; the protective component consists of the following raw materials: 5 parts of glycerol and 5 parts of dopamine.
Comparative example 1: preparation of polysialic acid.
Polysialic acid was prepared according to the procedure of example 1, except that the polysialic acid fermentation medium of this comparative example contained no nutritional and protective components.
Comparative example 2: preparation of polysialic acid.
Polysialic acid was prepared according to the procedure of example 1, except that the polysialic acid fermentation medium of this comparative example contained no protective component.
Comparative example 3: preparation of polysialic acid.
Polysialic acid was prepared according to the procedure of example 1, except that the polysialic acid fermentation medium of this comparative example contained no nutritional components.
Test example 1: determination of polysialic acid yield.
(1) The testing method comprises the following steps:
the fermentation broths obtained after fermentation in examples 1 to 3 and comparative examples 1 to 3 were taken and the content of polysialic acid was measured. Polysialic acid is measured by adopting a high performance liquid phase, and the detection conditions are as follows:
shimadzu Lc-15c; detection column Bio-Rad AMINEX HPX87H Organic Analysis Column (300X 7.8 mm); column temperature 60 ℃; the mobile phase was 6mmol/L sulfuric acid, and the flow rate was 0.6mL/min; the detection wavelength is 210nm.
(2) Test results:
the test results are shown in the table.
Table 1: yield of polysialic acid
As shown in the table above, after fermentation, the polysialic acid content in the fermentation broth obtained in examples 1 to 3 was 20.4g/L, 17.8g/L and 18.2g/L, respectively, which indicates that the polysialic acid yield can be significantly improved after the polysialic acid fermentation medium provided by the invention is used. Meanwhile, as can be seen from comparison of the data of example 1 with the data of comparative examples 1 to 3, the three components in the polysialic acid fermentation medium have a synergistic effect, and the influence of phage infection on escherichia coli is reduced, so that the yield of polysialic acid is improved.
Test example 2: anti-phage assay.
(1) The test method comprises the following steps:
the comparative example was set with 4 treatment groups, 5X 10 5 And (3) jointly inoculating the T4 phage and the escherichia coli into a culture medium for fermentation to prepare polysialic acid, and taking a fermentation liquid after fermentation to determine the number of plaques of the polysialic acid, and judging the infection efficiency of the phage according to the number of the plaques so as to judge the infection resistance of the escherichia coli. The method of determination of plaques refers to the method of detection of phages in liquids disclosed in patent CN 104313182B. The treatment groups of this comparative example were set as follows:
treatment group 1: phage and E.coli were inoculated into the fermentation medium described in example 1 and fermented according to the method of example 1;
treatment group 2: inoculating phage and escherichia coli into the fermentation medium described in the comparative example 1, and fermenting according to the method of the comparative example 1;
treatment group 3: inoculating phage and escherichia coli into the fermentation medium described in the comparative example 2, and fermenting according to the method of the comparative example 2;
treatment group 4: phage and E.coli were inoculated into the fermentation medium described in comparative example 3 and fermented according to the method of comparative example 3.
(2) Test results:
table 2: plaque count for each treatment group
As can be seen from the data in the table, the culture medium is used for preparing polysialic acid through fermentation, and the number of plaques after being connected with phage is far smaller than that of other treatment groups, so that the culture medium can reduce the infection of escherichia coli by phage to a certain extent and improve the antiviral capability of escherichia coli.
The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.
Claims (7)
1. The polysialic acid fermentation medium is characterized by comprising a basic medium, a nutritional component and a protective component, wherein the weight ratio of the basic medium to the nutritional component to the protective component is (4-6): (1-2): (1.5-4);
the basic culture medium consists of the following raw materials in parts by weight: 10-20 parts of glucose, 1-5 parts of sodium pyruvate, 4-5 parts of magnesium sulfate, 0.1-6 parts of dipotassium hydrogen phosphate, 2-6 parts of ammonium chloride and 0.5-2 parts of corn steep liquor dry powder;
the nutritional components consist of the following raw materials: 0.5-4 parts of Chinese yam, 10-20 parts of N-acetylglucosamine and 0.8-1.2 parts of trace element additive;
the protective component consists of the following raw materials: 3-5 parts of glycerol and 1-5 parts of dopamine.
2. The polysialic acid fermentation medium of claim 1 wherein the trace element additive comprises:
MnCl 2 0.5-1 g/L、CoCl 2 0.15-0.25 g/L、CrCl 3 0.05-0.1 g/L and ZnSO 4 0.05-1.2 g/L。
3. Use of a fermentation medium according to any one of claims 1-2 for the fermentative preparation of polysialic acid.
4. Use according to claim 3, wherein the method for the fermentative preparation of polysialic acid comprises the following steps:
inoculating Escherichia coli into the fermentation medium according to any one of claims 1-2, and fermenting.
5. The use according to claim 4, wherein the escherichia coli is inoculated in an amount of 1-1.5% by volume of the polysialic acid fermentation medium.
6. The use according to claim 4, wherein the fermentation conditions are: fermenting at 35-40deg.C and pH of 6.0-6.6 for 65-70 hr.
7. The use according to claim 4, wherein the fermentation has a gas flux of 0.5-2vvm and an oxygen dissolution of 26-30%.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588152A (en) * | 2018-05-14 | 2018-09-28 | 中国科学院合肥物质科学研究院 | Poly sialic acid fermentation medium, the production method of poly sialic acid and poly sialic acid product |
| CN109182423A (en) * | 2018-09-26 | 2019-01-11 | 武汉中科光谷绿色生物技术有限公司 | Promote the method for Escherichia coli fermentation production poly sialic acid |
| CN111733101A (en) * | 2020-06-29 | 2020-10-02 | 嘉必优生物技术（武汉）股份有限公司 | Polysialic acid fermentation medium and method for producing polysialic acid by fermenting escherichia coli |
| US20200332325A1 (en) * | 2017-11-21 | 2020-10-22 | Jennewein Biotechnologie Gmbh | Process for purification of a sialic acid from a fermentation broth |
| CN114276937A (en) * | 2021-12-28 | 2022-04-05 | 江苏神华药业有限公司 | Method for fermenting paecilomyces hepiali by using Chinese yam as carbon source |
| CN115299438A (en) * | 2022-08-04 | 2022-11-08 | 云南民族大学 | Application of dopamine in anti-phage |
-
2024
- 2024-02-04 CN CN202410153069.1A patent/CN117683832B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20200332325A1 (en) * | 2017-11-21 | 2020-10-22 | Jennewein Biotechnologie Gmbh | Process for purification of a sialic acid from a fermentation broth |
| CN108588152A (en) * | 2018-05-14 | 2018-09-28 | 中国科学院合肥物质科学研究院 | Poly sialic acid fermentation medium, the production method of poly sialic acid and poly sialic acid product |
| CN109182423A (en) * | 2018-09-26 | 2019-01-11 | 武汉中科光谷绿色生物技术有限公司 | Promote the method for Escherichia coli fermentation production poly sialic acid |
| CN111733101A (en) * | 2020-06-29 | 2020-10-02 | 嘉必优生物技术（武汉）股份有限公司 | Polysialic acid fermentation medium and method for producing polysialic acid by fermenting escherichia coli |
| CN114276937A (en) * | 2021-12-28 | 2022-04-05 | 江苏神华药业有限公司 | Method for fermenting paecilomyces hepiali by using Chinese yam as carbon source |
| CN115299438A (en) * | 2022-08-04 | 2022-11-08 | 云南民族大学 | Application of dopamine in anti-phage |
Non-Patent Citations (3)
| Title |
|---|
| BAI-XUE LIN 等: ""Polysialic acid biosynthesis and production inEscherichia coli: current state and perspectives"", 《APPL MICROBIOL BIOTECHNOL》, vol. 100, 17 October 2015 (2015-10-17), pages 1 - 8, XP035870370, DOI: 10.1007/s00253-015-7019-x * |
| 吴亚迪 等: ""噬菌体保藏方法研究综述"", 《聊城大学学报》, vol. 36, no. 6, 31 December 2023 (2023-12-31), pages 86 - 93 * |
| 施明雨 等: ""聚唾液酸高产菌株的高通量诱变选育及发酵工艺优化"", 《生物加工过程》, vol. 20, no. 3, 31 May 2022 (2022-05-31), pages 292 - 298 * |
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