CN112375795A - Method for producing acetylglucosamine by microbial fermentation - Google Patents
Method for producing acetylglucosamine by microbial fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing acetylglucosamine by microbial fermentation, which comprises the following steps: s1, seed culture, namely inoculating the strain into a seed culture medium for culture to obtain a seed solution; s2, fermentation culture, namely inoculating the seed liquid obtained in the step S1 into a fermentation culture medium for fermentation culture; the fermentation medium is as follows: glucose 6-10g/L, urea 1-3g/L, earthworm liquid 6-10g/L and sodium molybdate 0.01-0.18mg/L, sterilizing at 120 deg.C for 30min, and adjusting pH to 6.8-7.3. According to the invention, the earthworm liquid and sodium molybdate are added into the fermentation culture medium, so that the activity of the N-acetylglucosamine escherichia coli can be improved, the fermentation period is shortened, the fermentation yield is improved, the content of byproducts such as glutamic acid is reduced, and a technical support is provided for the large-scale production of the acetylglucosamine.
Description
Technical Field
The invention relates to the field of microbial fermentation, in particular to a method for producing acetylglucosamine by microbial fermentation.
Background
Acetylglucosamine, also known as N-acetylglucosamine, is an important glucose derivativeA compound (I) is provided. Has a chemical formula of C8H15NO6Molecular weight 221.21, melting point 211 ℃, and is in the form of white crystalline powder in nature. Most of the microorganisms such as animals, plants, bacteria, yeasts and filamentous fungi can synthesize the acetylglucosamine. The carapace of organisms such as shrimps, crabs and shellfish contains abundant acetylglucosamine which is one of important components of cell walls of most bacteria and fungi. Glucosamine obtained after deacetylation of acetylglucosamine is the basic unit of some important polysaccharides in human cells.
The acetyl glucosamine has the functions of diminishing inflammation, resisting tumor and resisting oxidation, is an effective medicament for treating osteoarthritis and rheumatoid arthritis, has important application in the industries of food, chemical industry, medicine and cosmetics, and has wide market prospect.
Currently, the production of acetylglucosamine mainly comprises a chemical method and a biological method. Wherein the chemical method is to produce the acetylglucosamine by hydrolyzing the carapace of the shrimp and crab animals. Although the total cost is lower, the raw material source is limited by seasons and production places, the energy consumption is high, and more waste liquid is generated in the production process, so that the environment is polluted greatly. Biological methods include enzymatic methods and microbiological methods. The production of acetylglucosamine by enzyme method is mainly completed by decomposing chitin under the catalysis of chitinase. This enzyme is present in most organisms, but at a very low level and at a high cost. The microbiological method is mainly used for producing the acetylglucosamine by selecting proper microorganisms and fermenting the microorganisms by taking glucose or molasses and other wastes as main raw materials. The method has less limitation on raw materials, less environmental pollution and larger development space.
CN106191169A discloses a fermentation method for increasing the yield of N-acetylglucosamine, when OD660nm in N-acetylglucosamine fermentation liquor is 28-33, adding beet alkali solution into the fermentation liquor, wherein the mass concentration of the beet alkali solution is 5% -15%, and the speed of the beet alkali solution is 0.1-0.8 g/L.h.
CN109628526A discloses a fermentation method for increasing the yield of N-acetylglucosamine, wherein during the fermentation process, when the OD660nm of the fermentation broth is greater than or equal to 25, the mixed solution of galactose and L-aspartic acid is fed in at a feeding speed of 5mL/h according to the first 5h, and then fed in at a flow rate of 3mL/h until the fermentation is finished, wherein the mass ratio of the galactose to the L-aspartic acid is 25: 3. The invention increases the accumulation concentration of glucosamine by adding L-aspartic acid in galactose to ensure that glmS and gfa1 are over-expressed, and finally improves the yield of N-acetylglucosamine to 120 g/L.
In summary, the existing microbial fermentation production of N-acetylglucosamine still has the phenomena of low yield, low conversion rate and high content of by-products, i.e., acetic acid and glutamic acid, which cause too high production cost and can not meet the requirement of industrial production, and therefore, an effective method for improving the fermentation yield of acetylglucosamine is urgently needed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for producing acetylglucosamine by microbial fermentation.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for producing acetylglucosamine by microbial fermentation comprises the following steps:
s1, seed culture
Inoculating the strain into a seed culture medium for culturing to obtain a seed solution;
s2 fermentation culture
Inoculating the seed liquid obtained in the step S1 into a fermentation culture medium for fermentation culture;
the fermentation medium is as follows: glucose 6-10g/L, urea 1-3g/L, earthworm liquid 6-10g/L and sodium molybdate 0.01-0.18mg/L, sterilizing at 120 deg.C for 30min, and adjusting pH to 6.8-7.3.
Preferably, the inoculation amount of the seed solution in the step S2 is 10-12% by volume of the fermentation medium.
Preferably, the conditions of the fermentation culture in step S2 are: the pressure of the tank is 0.035-0.045Mpa, the dissolved oxygen is 25-28%, the rotating speed is 150-.
Preferably, the strain in step S1 is N-acetylglucosamine-producing Escherichia coli.
Preferably, the seed culture medium in step S1 is: 12-14g/L glucose, 10-12g/L earthworm liquid, 1-3g/L urea and 6-8g/L dipotassium hydrogen sulfate, sterilizing at 120 ℃ for 30min, and adjusting pH to 6.4-6.5.
Preferably, the seed culture conditions in step S1 are: the pressure of the tank is 0.035-0.045Mpa, the dissolved oxygen is 31-33%, the rotating speed is 150-.
Preferably, the preparation method of the earthworm liquid comprises the following steps:
(1) adding water and cyclodextrin into earthworm to prepare slurry;
(2) and (2) centrifuging the slurry obtained in the step (1) to obtain a supernatant, namely the earthworm liquid.
Further preferably, the earthworms in the step (1) are Eisenia foetida.
Further preferably, the amount of the water added in the step (1) is 5 to 8 times of the weight of the earthworms.
Further preferably, the cyclodextrin in step (1) is added in an amount of 0.03-0.06% by weight of the slurry.
The invention has the beneficial effects that:
(1) according to the invention, the earthworm liquid and sodium molybdate are added into the fermentation culture medium, so that the activity of the N-acetylglucosamine escherichia coli can be improved, the fermentation period is shortened, the fermentation yield is improved, the content of byproducts such as glutamic acid is reduced, and meanwhile, when the mass ratio of the earthworm liquid to the sodium molybdate is 500: 1, the content of glutamic acid as a by-product can be significantly reduced.
(2) The fermentation method provided by the invention is simple in process, short in fermentation period and low in cost, and provides technical support for large-scale production of acetylglucosamine.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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 invention belongs.
And (3) measuring the content of N-acetylglucosamine: high performance liquid chromatography.
And (3) measuring the content of glutamic acid: and (3) taking supernatant after the fermentation liquor is centrifuged, diluting until the concentration of the residual glutamic acid is within 1g/L, and measuring by using a biosensor analyzer.
Example 1
A method for producing acetylglucosamine by microbial fermentation comprises the following steps:
s1, seed culture
Inoculating Escherichia coli producing N-acetylglucosamine into a seed culture medium according to the mass ratio of 4% for culture to obtain a seed solution;
the seed culture medium is as follows: 14g/L glucose, 10g/L earthworm liquid, 3g/L urea and 8g/L dipotassium hydrogen sulfate, sterilizing at 120 ℃ for 30min, and adjusting the pH value to 6.4-6.5;
the seed culture conditions are as follows: the pressure of the tank is 0.035Mpa, the dissolved oxygen is 31 percent, the rotating speed is 150rpm, the temperature is 36 ℃, and the fermentation time is 10 hours;
s2 fermentation culture
Inoculating the seed liquid obtained in the step S1 into a fermentation culture medium according to the volume ratio of 10% to perform fermentation culture;
the fermentation medium is as follows: 6g/L glucose, 1g/L urea, 10g/L earthworm liquid and 0.01mg/L sodium molybdate, sterilizing at 120 deg.C for 30min, and adjusting pH to 6.8-7.3;
the conditions of the fermentation culture are as follows: the pot pressure is 0.035Mpa, the dissolved oxygen is 25 percent, the rotating speed is 150rpm, the temperature is 36 ℃, and the fermentation time is 42 hours;
the preparation method of the earthworm liquid comprises the following steps:
(1) adding Eisenia foetida into water and cyclodextrin to prepare slurry;
the adding amount of the water is 5 times of the weight of the earthworms; the addition amount of the cyclodextrin is 0.06 percent of the weight of the sizing agent;
(2) and (3) centrifuging the slurry obtained in the step (2) to obtain a supernatant, namely the earthworm liquid.
After the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 142.4g/L, and the content of glutamic acid is measured to be 2.2 g/L.
Example 2 a method for producing acetylglucosamine by microbial fermentation, comprising the steps of: s1, seed culture
Inoculating Escherichia coli producing N-acetylglucosamine into a seed culture medium according to the mass ratio of 4% for culture to obtain a seed solution;
the seed culture medium is as follows: 12g/L glucose, 12g/L earthworm liquid, 1g/L urea and 6g/L dipotassium hydrogen sulfate, sterilizing at 120 ℃ for 30min, and adjusting the pH value to 6.4-6.5;
the seed culture conditions are as follows: the pot pressure is 0.045Mpa, the dissolved oxygen is 33 percent, the rotating speed is 180rpm, the temperature is 38 ℃, and the fermentation time is 7.5 hours;
s2 fermentation culture
Inoculating the seed liquid obtained in the step S1 into a fermentation culture medium according to the volume ratio of 12% to perform fermentation culture;
the fermentation medium is as follows: 10g/L glucose, 3g/L urea, 6g/L earthworm liquid and 0.018mg/L sodium molybdate, sterilizing at 120 deg.C for 30min, and adjusting pH to 6.8-7.3;
the conditions of the fermentation culture are as follows: the pot pressure is 0.045Mpa, the dissolved oxygen is 28 percent, the rotating speed is 180rpm, the temperature is 38 ℃, and the fermentation time is 30 hours;
the preparation method of the earthworm liquid comprises the following steps:
(1) adding Eisenia foetida into water and cyclodextrin to prepare slurry;
the adding amount of the water is 8 times of the weight of the earthworms; the addition amount of the cyclodextrin is 0.03 percent of the weight of the sizing agent;
(2) and (3) centrifuging the slurry obtained in the step (2) to obtain a supernatant, namely the earthworm liquid.
After the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 138.7g/L, and the content of glutamic acid is measured to be 2.8 g/L.
Example 3
A method for producing acetylglucosamine by microbial fermentation comprises the following steps:
s1, seed culture
Inoculating Escherichia coli producing N-acetylglucosamine into a seed culture medium according to the mass ratio of 4% for culture to obtain a seed solution;
the seed culture medium is as follows: 13/L glucose, 11g/L earthworm liquid, 1.5g/L urea and 7g/L dipotassium hydrogen sulfate, sterilizing at 120 ℃ for 30min, and adjusting the pH value to 6.4-6.5;
the seed culture conditions are as follows: the pot pressure is 0.038Mpa, the dissolved oxygen is 32 percent, the rotating speed is 160rpm, the temperature is 37 ℃, and the fermentation time is 8.6 hours;
s2 fermentation culture
Inoculating the seed liquid obtained in the step S1 into a fermentation culture medium according to the volume ratio of 11% to perform fermentation culture;
the fermentation medium is as follows: sterilizing 7g/L glucose, 2g/L urea, 7g/L earthworm liquid and 0.014mg/L sodium molybdate at 120 deg.C for 30min, and adjusting pH to 6.8-7.3;
the conditions of the fermentation culture are as follows: the pot pressure is 0.042Mpa, the dissolved oxygen is 26 percent, the rotating speed is 160rpm, the temperature is 37 ℃, and the fermentation time is 38 hours;
the preparation method of the earthworm liquid comprises the following steps:
(1) adding Eisenia foetida into water and cyclodextrin to prepare slurry;
the adding amount of the water is 6 times of the weight of the earthworms; the addition amount of the cyclodextrin is 0.04 percent of the weight of the slurry;
(2) and (3) centrifuging the slurry obtained in the step (2) to obtain a supernatant, namely the earthworm liquid.
After the fermentation, the content of N-acetylglucosamine in the fermentation liquor is 149.8g/L, and the content of glutamic acid is 1.4 g/L.
Example 4
This example differs from example 3 in that the fermentation medium is: sterilizing glucose 8g/L, urea 1.5g/L, Lumbricus liquid 8g/L and sodium molybdate 0.016mg/L at 120 deg.C for 30min, and pH 6.8-7.3;
after the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 147.3g/L, and the content of glutamic acid is measured to be 1.7 g/L.
Comparative example 1
This comparative example differs from example 3 in that: the fermentation medium does not contain sodium molybdate; after the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 116.5g/L, and the content of glutamic acid is measured to be 3.6 g/L.
Comparative example 2
This comparative example differs from example 3 in that: the content of sodium molybdate in the fermentation medium is 0.02 mg/L; after the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 117.7g/L, and the content of glutamic acid is measured to be 3.4 g/L.
Comparative example 3
This comparative example differs from example 3 in that: the earthworm liquid in the fermentation culture medium is 12g/L, and the sodium molybdate is 0.009 mg/L; after the fermentation, the content of N-acetylglucosamine in the fermentation liquor is measured to be 118.6g/L, and the content of glutamic acid is measured to be 3.1 g/L.
Comparative example 4
This comparative example differs from example 3 in that: the preparation process of the earthworm liquid does not add cyclodextrin. After the fermentation is finished, the content of N-acetylglucosamine in the fermentation liquor is measured to be 121.4g/L, and the content of glutamic acid is measured to be 2.9 g/L.
Comparative example 5
This comparative example differs from example 3 in that: the seed culture medium does not contain earthworm liquid; after the fermentation is finished, the content of N-acetylglucosamine in the fermentation liquor is measured to be 120.4g/L, and the content of glutamic acid is measured to be 3.4 g/L.
The yield of N-acetylglucosamine obtained by fermentation in embodiments 1 to 4 of the present invention is 138.7 to 149.8g/L, and the content of glutamic acid as a byproduct is 1.4 to 2.8g/L, which indicates that, in the present invention, adding earthworm liquid and sodium molybdate to a fermentation medium can improve the activity of Escherichia coli producing N-acetylglucosamine, shorten the fermentation period, improve the fermentation yield, and reduce the content of glutamic acid and other byproducts, and at the same time, it is found that, when the mass ratio of earthworm liquid to sodium molybdate is 500: 1, the content of glutamic acid as a by-product can be significantly reduced.
Meanwhile, the fermentation method disclosed by the invention is simple in process, short in fermentation period and low in cost, and provides technical support for large-scale production of the N-acetylglucosamine.
The present invention has been further described with reference to specific embodiments, which are only exemplary and do not limit the scope of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for producing acetylglucosamine by microbial fermentation is characterized by comprising the following steps:
s1, seed culture
Inoculating the strain into a seed culture medium for culturing to obtain a seed solution;
s2 fermentation culture
Inoculating the seed liquid obtained in the step S1 into a fermentation culture medium for fermentation culture;
the fermentation medium is as follows: glucose 6-10g/L, urea 1-3g/L, earthworm liquid 6-10g/L and sodium molybdate 0.01-0.018mg/L, sterilizing at 120 deg.C for 30min, and adjusting pH to 6.8-7.3.
2. The method of claim 1, wherein the seed solution is inoculated in step S2 in an amount of 10-12% by volume of the fermentation medium.
3. The method according to claim 1, wherein the conditions of the fermentation culture in step S2 are: the pressure of the tank is 0.035-0.045Mpa, the dissolved oxygen is 25-28%, the rotating speed is 150-.
4. The method of claim 1, wherein the bacterial species in step S1 is N-acetylglucosamine-producing escherichia coli.
5. The method of claim 4, wherein the seed medium in step S1 is: 12-14g/L glucose, 10-12g/L earthworm liquid, 1-3g/L urea and 6-8g/L dipotassium hydrogen sulfate, sterilizing at 120 ℃ for 30min, and adjusting pH to 6.4-6.5.
6. The method of claim 4, wherein the seed culture conditions are: the pressure of the tank is 0.035-0.045Mpa, the dissolved oxygen is 31-33%, the rotating speed is 150-.
7. The method according to any one of claims 1 to 6, wherein the earthworm liquid is prepared by:
(1) adding water and cyclodextrin into earthworm to prepare slurry;
(2) and (2) centrifuging the slurry obtained in the step (1) to obtain a supernatant, namely the earthworm liquid.
8. The method according to claim 7, wherein the earthworms in step (1) are Eisenia foetida.
9. The method of claim 7, wherein the water is added in an amount of 5 to 8 times the weight of the earthworms in step (1).
10. The method of claim 7, wherein the cyclodextrin of step (1) is added in an amount of 0.03-0.06% by weight of the slurry.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570565A (en) * | 2008-04-30 | 2009-11-04 | 珠海博康药业有限公司 | Functional component prepared from earthworms for promoting growth of microorganisms and improving culture units and preparation method thereof |
| CN104059872A (en) * | 2014-07-16 | 2014-09-24 | 华东理工大学 | High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570565A (en) * | 2008-04-30 | 2009-11-04 | 珠海博康药业有限公司 | Functional component prepared from earthworms for promoting growth of microorganisms and improving culture units and preparation method thereof |
| CN104059872A (en) * | 2014-07-16 | 2014-09-24 | 华东理工大学 | High-yield N-acetylglucosamine metabolic engineering bacterium, as well construction method and applications thereof |
Non-Patent Citations (1)
| Title |
|---|
| LONG LIU等: "Microbial production of glucosamine and N-acetylglucosamine: advances and perspectives", 《APPL MICROBIOL BIOTECHNOL》 * |
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Application publication date: 20210219 |