CN109161576B - Method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis - Google Patents
Method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis Download PDFInfo
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Abstract
A method for promoting fermentation of Bacillus subtilis to produce N-acetylneuraminic acid belongs to the field of fermentation of Bacillus subtilis to produce N-acetylneuraminic acid. The method for promoting the fermentation production of the N-acetylneuraminic acid by the bacillus subtilis comprises the steps of inoculating the bacillus subtilis capable of producing the N-acetylneuraminic acid by fermentation into a fermentation culture medium for fermentation culture, and adding schizochytrium limacinum powder in the fermentation culture process. The method for promoting the fermentation of the bacillus subtilis to produce the N-acetylneuraminic acid can effectively improve the yield of the N-acetylneuraminic acid.
Description
Technical Field
The invention relates to the technical field of production of N-acetylneuraminic acid by fermentation of bacillus subtilis, and in particular relates to a method for promoting production of N-acetylneuraminic acid by fermentation of bacillus subtilis.
Background
N-acetylneuraminic acid (N-acetylneuraminic acid) Neu5Ac is the first contact site for cell information transmission, and the molecular structure of the N-acetylneuraminic acid is diverse, so that Neu5Ac participates in multiple physiological processes such as cell recognition, signal transduction, tumorigenesis and fertilization, Neu5Ac can also regulate the anti-inflammatory activity of IgG, enhance the immunity of infants, influence the integrity, permeability and activity of nerve cells and promote the development of the brains of infants, and therefore the production of N-acetylneuraminic acid attracts more attention and research.
N-acetylneuraminic acid has great economic value, and a plurality of production methods such as extraction from natural raw materials, chemical synthesis and the like exist, but the methods all have the problems of high cost, rigorous process, low yield and the like. The microbial fermentation method is also a method for preparing N-acetylneuraminic acid for more research, has the advantages of low cost, simple operation and the like, mainly utilizes escherichia coli engineering bacteria for fermentation to obtain polysialic acid and N-acetylneuraminic acid at present, and some strains in bacillus, such as bacillus subtilis, are GRAS-certified safe strains, are also important host bacteria due to the advantages of no pathogenicity, no easy bacteriophage pollution and the like, and are widely applied to industrial production. Therefore, the use of the constructed bacillus engineering bacteria for producing food, medicine and other additive materials and materials, such as N-acetylneuraminic acid, has high safety and great application prospect, but the fermentation yield of the N-acetylneuraminic acid produced by the fermentation of bacillus genetic engineering bacteria, such as bacillus subtilis, is low, and the use requirements are difficult to meet.
Disclosure of Invention
The invention aims to provide a method for promoting the fermentation production of N-acetylneuraminic acid by bacillus subtilis, which can effectively improve the yield of the N-acetylneuraminic acid.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
A method for promoting Bacillus subtilis to ferment and produce N-acetylneuraminic acid comprises inoculating Bacillus subtilis capable of fermenting and producing N-acetylneuraminic acid into a fermentation culture medium for fermentation culture, and adding Schizochytrium limacinum powder during the fermentation culture.
Further, in the preferred embodiment of the present invention, the fermentation medium comprises glycerol 10-30 g/L, tryptone 2-6 g/L, potassium dihydrogen phosphate 4-8 g/L, ammonium sulfate 2-4 g/L, magnesium sulfate heptahydrate 0.5-2 g/L, and IPTG1-3 mmol/L.
Further, in the preferred embodiment of the present invention, the above-mentioned schizochytrium limacinum powder is added between 0-72h of fermentation culture.
Further, in the preferred embodiment of the present invention, the amount of the powder of the schizochytrium limacinum is 1-4g per 1L of the fermentation medium.
Further, in the preferred embodiment of the invention, the schizochytrium limacinum powder is added between 20 h and 24h of the fermentation culture, and the addition amount is 2-2.5 g/L of the fermentation culture medium.
Further, in a preferred embodiment of the present invention, the above-mentioned powder of schizochytrium limacinum is obtained by culturing schizochytrium limacinum with a liquid seed culture medium and a liquid fermentation culture medium in this order to obtain schizochytrium limacinum thallus, centrifuging the schizochytrium limacinum thallus, and drying.
Further, in the preferred embodiment of the present invention, the liquid seed culture medium comprises glucose 30-50 g/L, sodium glutamate 20-40 g/L, yeast extract 4-8 g/L, sodium chloride 15-25 g/L, potassium dihydrogen phosphate 4-8 g/L, magnesium sulfate 6-10 g/L.
Furthermore, in the preferred embodiment of the present invention, the liquid fermentation medium comprises glucose 50-70 g/L, sodium glutamate 20-40 g/L, yeast extract 4-8 g/L, sodium chloride 15-25 g/L, potassium dihydrogen phosphate 4-8 g/L, magnesium sulfate 6-10 g/L.
Further, in a preferred embodiment of the present invention, the preservation number of the schizochytrium limacinum is CCTCC NO: m2012494.
Further, in a preferred embodiment of the present invention, the above-mentioned Bacillus subtilis capable of producing N-acetylneuraminic acid by fermentation is constructed by introducing UDP-N-acetylglucosamine epimerase gene, N-acetylneuraminic acid synthase gene and 6-phosphoglucosamine synthase gene into Bacillus subtilis and exogenously expressing them.
The method for promoting the fermentation production of the N-acetylneuraminic acid by the bacillus subtilis has the beneficial effects that: the embodiment of the invention provides a method for promoting bacillus subtilis to produce N-acetylneuraminic acid by fermentation. The method for promoting the fermentation of the bacillus subtilis to produce the N-acetylneuraminic acid can effectively improve the yield of the N-acetylneuraminic acid.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a bar graph comparing the production amounts of N-acetylneuraminic acid prepared according to comparative example, example 1, example 2, example 3 and example 4 of the present invention;
FIG. 2 is a bar graph comparing the production amounts of N-acetylneuraminic acid prepared according to comparative example, example 5, example 6, example 7 and example 8 of the present invention;
FIG. 3 is a bar graph comparing the production amounts of N-acetylneuraminic acid prepared in comparative example, example 9, example 10, example 11 and example 12 of the present invention;
FIG. 4 is a bar graph comparing the production amounts of N-acetylneuraminic acid prepared in examples 2, 6, 10 and 13 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The method for promoting the fermentation production of N-acetylneuraminic acid by the Bacillus subtilis is specifically described below.
The embodiment of the invention provides a method for promoting bacillus subtilis to produce N-acetylneuraminic acid by fermentation, which comprises the following steps of inoculating bacillus subtilis capable of producing N-acetylneuraminic acid by fermentation into a fermentation culture medium for fermentation culture, and adding schizochytrium limacinum powder during the fermentation culture process, preferably adding the schizochytrium limacinum powder between 0h and 72h of the fermentation culture, more preferably adding 1g to 4g of the schizochytrium limacinum powder into 1L of the fermentation culture medium, and further preferably adding the schizochytrium limacinum powder between 20 h and 24h of the fermentation culture, wherein the adding amount of the schizochytrium limacinum powder is 2g to 2.5 g/L of the fermentation culture medium.
The method for promoting the fermentation of the bacillus subtilis to produce the N-acetylneuraminic acid provided by the embodiment of the invention is to add schizochytrium for promoting the fermentation of the bacillus subtilis to produce the N-acetylneuraminic acid in the process of producing the N-acetylneuraminic acid by fermenting the bacillus subtilis capable of producing the N-acetylneuraminic acid, so as to improve the yield of the N-acetylneuraminic acid.
In a preferred embodiment of the invention, the schizochytrium limacinum powder is obtained by culturing schizochytrium limacinum with a liquid seed culture medium and a liquid fermentation culture medium in sequence, and then centrifuging and drying the schizochytrium limacinum thallus, preferably, the liquid seed culture medium comprises 30-50 g/L of glucose, 20-40 g/L of sodium glutamate, 4-8 g/L0 of yeast extract, 15-25 g/L1 of sodium chloride, 4-8 g/L of potassium dihydrogen phosphate and 6-10 g/L of magnesium sulfate, more preferably, the liquid fermentation culture medium comprises 50-70 g/L of glucose, 20-40 g/L of sodium glutamate, 4-8 g/L of yeast extract, 15-25 g/L of sodium chloride, 4-8 g/L of potassium dihydrogen phosphate and 6-10 g/L of magnesium sulfate, and the liquid fermentation culture medium are prepared with proper components, so that the schizochytrium limacinum can promote propagation of bacillus subtilis to generate N-N fermentation broth.
In a preferred embodiment of the invention, the fermentation medium comprises 10-30 g/L of glycerol, 2-6 g/L of tryptone, 4-8 g/L of monopotassium phosphate, 2-4 g/L of ammonium sulfate, 0.5-2 g/L of magnesium sulfate heptahydrate and 0.3-3 mmol/L of IPTG1-3 mmol/L.
In a preferred embodiment of the invention, the preservation number of the schizochytrium limacinum is CCTCC NO: m2012494. The schizochytrium limacinum has been submitted to China Center for Type Culture Collection (CCTCC) of eight Loica mountain in Wuchang district, Wuhan City, Hubei, with the preservation number of CCTCC NO: m2012494, the Schizochytrium limacinum can better promote the fermentation production of N-acetylneuraminic acid by the Bacillus subtilis capable of producing N-acetylneuraminic acid, and the yield is effectively improved.
In a preferred embodiment of the present invention, Bacillus subtilis capable of producing N-acetylneuraminic acid by fermentation is constructed by introducing UDP-N-acetylglucosamine epimerase gene, N-acetylneuraminic acid synthetase gene and 6-phosphoglucosamine synthetase gene into Bacillus subtilis and exogenously expressing them. In the embodiment of the invention, the bacillus subtilis disclosed in Chinese patent application with the patent number of 2014101026333 and the patent name of 'a bacillus subtilis genetic engineering bacterium for producing N-acetylneuraminic acid and a construction method and application thereof' is preferably adopted, and the bacillus subtilis can be more effectively matched with schizochytrium provided by the embodiment of the invention to efficiently ferment and produce the N-acetylneuraminic acid.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which mainly comprises the following steps:
s11, culturing schizochytrium limacinum, taking schizochytrium limacinum strains preserved by using glycerine tubes from a refrigerating chamber at the temperature of minus 80 ℃, sucking a bacterial liquid of 0.3m L schizochytrium limacinum strains to transfer to a 250m L triangular flask filled with a 50m L liquid seed culture medium after melting, culturing for 48 hours at the temperature of 28 ℃ and at the rotating speed of 180rpm to obtain a primary seed liquid, inoculating the primary seed liquid into a 500m L triangular flask filled with 100ml of liquid seed culture medium according to the inoculation amount of 5 percent by volume percentage, culturing for 96 hours at the temperature of 28 ℃ and at the rotating speed of 180rpm to obtain a fermentation liquid, centrifuging the fermentation liquid obtained after 96 hours of culture for 5 minutes at 5000rpm, washing and centrifuging for three times by using distilled water, removing supernatant, drying the strains in a constant-temperature drying oven to constant weight, grinding into powder, and sieving to obtain schizochytrium limacinum powder.
Wherein, the liquid seed culture medium comprises the following components of glucose 40 g/L, sodium glutamate 30 g/L, yeast extract 6 g/L0, sodium chloride 20 g/L1, potassium dihydrogen phosphate 6 g/L and magnesium sulfate 8 g/L, and the liquid fermentation culture medium comprises the following components of glucose 60 g/L, sodium glutamate 30 g/L, yeast extract 6 g/L, sodium chloride 20 g/L, potassium dihydrogen phosphate 6 g/L and magnesium sulfate 8 g/L.
S12, culturing the bacillus subtilis, inoculating the strain of the bacillus subtilis preserved at the temperature of 80 ℃ below zero to a solid L B culture medium, culturing overnight at the temperature of 37 ℃, scraping a ring of flat strains to a 100m L triangular flask with a 10m L liquid L B culture medium, and performing constant-temperature shaking culture at the temperature of 37 ℃ and the rotation speed of 200rpm for 5 hours to obtain a strain.
Wherein the solid L B culture medium comprises tryptone 10 g/L, yeast powder 5 g/L, sodium chloride 10 g/L, agar powder 20 g/L, and liquid L B culture medium comprises tryptone 10 g/L, yeast powder 5 g/L, and sodium chloride 10 g/L.
S13, inoculating the seed solution with the inoculation amount of 4% by volume into a 250m L triangular flask with a 50m L fermentation medium, culturing for 72h at 37 ℃ and 200rpm, and adding 1 g/L g of the schizochytrium limacinum powder at the 0h of culture (namely, inoculation).
Wherein the fermentation medium comprises 10-30 g/L of glycerol, 2-6 g/L of tryptone, 4-8 g/L of monopotassium phosphate, 2-4 g/L of ammonium sulfate, 0.5-2 g/L of magnesium sulfate heptahydrate and 1-3 mmol/L of IPTG (isopropyl thiogalactoside).
Example 2
The embodiment of the invention provides a method for promoting fermentation production of N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 2 g/L of the schizochytrium limacinum powder is added in the 0h (namely, at the inoculation) of culture.
Example 3
The embodiment of the invention provides a method for promoting fermentation production of N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 3 g/L g of the schizochytrium limacinum powder is added in the 0h (namely, at the inoculation) of culture.
Example 4
The embodiment of the invention provides a method for promoting fermentation production of N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 4 g/L of the schizochytrium limacinum powder is added in the 0h (namely, at the inoculation) of culture.
Example 5
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 1 g/L g of the schizochytrium limacinum powder is added in the 24 th hour of culture.
Example 6
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 2 g/L g of schizochytrium limacinum powder is added in the 24 th hour of culture.
Example 7
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 3 g/L g of schizochytrium limacinum powder is added in the 24 th hour of culture.
Example 8
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 4 g/L g of schizochytrium limacinum powder is added in the 24 th hour of culture.
Example 9
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 1 g/L g of the schizochytrium limacinum powder is added in the 36 th hour of culture.
Example 10
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 2 g/L g of schizochytrium limacinum powder is added in the 36 th hour of culture.
Example 11
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 3 g/L g of schizochytrium limacinum powder is added in the 36 th hour of culture.
Example 12
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 4 g/L g of schizochytrium limacinum powder is added in the 36 th hour of culture.
Example 13
The embodiment of the invention provides a method for promoting fermentation of bacillus subtilis to produce N-acetylneuraminic acid by using schizochytrium limacinum, which is the same as the step in the embodiment 1, except that 2 g/L g of schizochytrium limacinum powder is added in the 48 th hour of culture.
Comparative example
The comparative example provides a method for producing N-acetylneuraminic acid by using bacillus subtilis through fermentation, which mainly comprises the following steps:
s21, culturing the bacillus subtilis, inoculating the strain of the bacillus subtilis preserved at the temperature of 80 ℃ below zero to a solid L B culture medium, culturing overnight at the temperature of 37 ℃, scraping a ring of flat strains to a 100m L triangular flask with a 10m L liquid L B culture medium, and performing constant-temperature shaking culture at the temperature of 37 ℃ and the rotation speed of 200rpm for 5 hours to obtain a strain.
Wherein the solid L B culture medium comprises tryptone 10 g/L, yeast powder 5 g/L, sodium chloride 10 g/L, agar powder 20 g/L, and liquid L B culture medium comprises tryptone 10 g/L, yeast powder 5 g/L, and sodium chloride 10 g/L.
S22, inoculating the seed liquid with the inoculum size of 4% by volume into a 250m L triangular flask with 50m L fermentation medium, and culturing at 37 ℃ and 200rpm for 72 h.
Wherein the fermentation medium comprises 10-30 g/L of glycerol, 2-6 g/L of tryptone, 4-8 g/L of monopotassium phosphate, 2-4 g/L of ammonium sulfate, 0.5-2 g/L of magnesium sulfate heptahydrate and 1-3 mmol/L of IPTG (isopropyl thiogalactoside).
The fermentation broth obtained by culturing the culturing examples 1 to 13 and the comparative examples is repeated by culturing the comparative examples for 9 times, and the obtained data is averaged, wherein the detection method comprises the steps of carrying out high performance liquid chromatography (HP L C) detection on Shimadzu L C-.
As shown in FIG. 1, the yield of N-acetylneuraminic acid produced by the blank control group is 0.78 g/L when the schizochytrium is added at 0h (inoculation) of the fermentation culture, the yield of N-acetylneuraminic acid is kept stable after the yield of N-acetylneuraminic acid is gradually increased along with the gradual increase of the addition amount of the schizochytrium (0, 1, 2, 3, 4 g/L), and the yield of N-acetylneuraminic acid reaches the highest value of 1.2 g/L when 2 g/L schizochytrium powder is added, and is increased by about 58% relative to the control group.
As shown in FIG. 2, the yield of N-acetylneuraminic acid produced by the blank control group is 0.78 g/L when the schizochytrium limacinum is added at the 24 th hour of the fermentation culture, the yield of N-acetylneuraminic acid is kept stable after the yield of N-acetylneuraminic acid is gradually increased with the gradual increase of the addition amount of the schizochytrium limacinum (0, 1, 2, 3, 4 g/L), and the yield of N-acetylneuraminic acid reaches the highest value of 1.41 g/L when 2 g/L schizochytrium limacinum powder is added, and is increased by about 81 percent compared with the control group.
As shown in FIG. 3, the yield of N-acetylneuraminic acid produced by the blank control group is 0.76 g/L when the schizochytrium limacinum is added at the 36 th hour of the fermentation culture, the yield of N-acetylneuraminic acid is kept stable after the yield of N-acetylneuraminic acid is gradually increased with the gradually increased addition amount of the schizochytrium limacinum (0, 1, 2, 3, 4 g/L), and the yield of N-acetylneuraminic acid reaches the highest value of 1.01 g/L when 2 g/L schizochytrium limacinum powder is added, and is increased by about 33 percent compared with the control group.
As shown in FIG. 4, when the schizochytrium is added at 0h, 24h, 36h and 48h of the fermentation culture, 2 g/L of the schizochytrium powder is added at 24h of the fermentation culture, the yield of the N-acetylneuraminic acid is highest, and the increase of the yield of the N-acetylneuraminic acid is gradually reduced along with the addition of the N-acetylneuraminic acid after the addition of the N-acetylneuraminic acid is advanced, and the yield of the N-acetylneuraminic acid reaches 1.40 g/L when the 2 g/L of the schizochytrium powder is added at 24h of the fermentation culture.
In summary, the embodiment of the present invention provides a method for promoting bacillus subtilis to produce N-acetylneuraminic acid by fermentation, the adopted schizochytrium can effectively promote bacillus subtilis to produce N-acetylneuraminic acid by fermentation, and compared with the prior art, the yield of N-acetylneuraminic acid can be increased by 33-81%.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (9)
1. A method for promoting the fermentation production of N-acetylneuraminic acid by bacillus subtilis is characterized in that the bacillus subtilis capable of producing the N-acetylneuraminic acid by fermentation is inoculated into a fermentation culture medium for fermentation culture, and the fungus powder of schizochytrium limacinum is added in the fermentation culture process; the bacillus subtilis capable of producing N-acetylneuraminic acid by fermentation is constructed by introducing a UDP-N-acetylglucosamine epimerase gene, an N-acetylneuraminic acid synthetase gene and a 6-phosphoglucosamine synthetase gene into the bacillus subtilis for exogenous expression.
2. The method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis according to claim 1, wherein the fermentation medium comprises 10-30 g/L of glycerol, 2-6 g/L of tryptone, 4-8 g/L of monopotassium phosphate, 2-4 g/L of ammonium sulfate, 0.5-2 g/L of magnesium sulfate heptahydrate, and 3 mmol/L of IPTG 1.
3. The method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis according to claim 1, wherein the powder of the schizochytrium limacinum is added between 0 and 72 hours of fermentation culture.
4. The method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis according to claim 1, wherein the powder of the schizochytrium limacinum is added in an amount of 1-4g per 1L of the fermentation medium.
5. The method for promoting the fermentation production of N-acetylneuraminic acid by the bacillus subtilis according to claim 1, wherein the powder of the schizochytrium limacinum is added between 20 h and 24h of the fermentation culture, and the addition amount is 2-2.5 g/L of the fermentation culture medium.
6. The method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis according to claim 1, wherein the schizochytrium limacinum fungal powder is obtained by culturing schizochytrium limacinum with a liquid seed culture medium and a liquid fermentation culture medium in sequence to obtain schizochytrium limacinum thallus, centrifuging the schizochytrium limacinum thallus and drying.
7. The method for promoting fermentation of Bacillus subtilis to produce N-acetylneuraminic acid according to claim 6, wherein the liquid seed culture medium comprises 30-50 g/L of glucose, 20-40 g/L of sodium glutamate, 4-8 g/L of yeast extract, 15-25 g/L of sodium chloride, 4-8 g/L of monopotassium phosphate and 6-10 g/L of magnesium sulfate.
8. The method for promoting fermentation of Bacillus subtilis to produce N-acetylneuraminic acid according to claim 6, wherein the liquid fermentation medium comprises 50-70 g/L of glucose, 20-40 g/L of sodium glutamate, 4-8 g/L of yeast extract, 15-25 g/L of sodium chloride, 4-8 g/L of monopotassium phosphate and 6-10 g/L of magnesium sulfate.
9. The method for promoting fermentation production of N-acetylneuraminic acid by bacillus subtilis according to claim 1, wherein the preservation number of the schizochytrium limacinum is CCTCC NO: m2012494.
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| CN103923869A (en) * | 2014-03-19 | 2014-07-16 | 武汉中科光谷绿色生物技术有限公司 | Bacillus subtilis gene engineering bacterial producing Neu5Ac, construction method and application thereof |
| CN104017846B (en) * | 2014-06-04 | 2016-09-28 | 中国科学院等离子体物理研究所 | A kind of method that the bandit's of utilization formula fermented abandoned dreg of Crypthecodinium cohnii ATCC30772 prepares high yield natamycin |
| CN104041827B (en) * | 2014-06-30 | 2016-06-15 | 南京极燕生物科技有限公司 | A kind of microcapsule containing sialic acid and DHA and preparation method thereof |
| CN104805139B (en) * | 2015-03-20 | 2018-03-16 | 湖北产业技术创新与育成中心 | A kind of method added purple ball algae and improve Mortierella alpina fermenting and producing arachidonic acid yield |
| CN105331670B (en) * | 2015-12-04 | 2018-12-04 | 广东润科生物工程股份有限公司 | Split the method for pot algae Yu the hidden dinoflagellate mixed fermentation of Kou Shi |
| CN107523504A (en) * | 2016-06-21 | 2017-12-29 | 嘉必优生物技术（武汉）股份有限公司 | Schizochytrium limacinum mutant strain |
| CN106119323B (en) * | 2016-07-11 | 2019-02-26 | 珀莱雅化妆品股份有限公司 | A kind of fermentation culture method that sodium bacillus subtilis lipopeptide yield can be improved |
| CN106591398A (en) * | 2017-01-23 | 2017-04-26 | 中国科学院合肥物质科学研究院 | Method for obtaining SA by using biodiesel by-product crude glycerol to perform high added-value conversion |
| CN108498523B (en) * | 2017-02-24 | 2023-06-20 | 上海交通大学 | Preparation method and application of ganglioside derivative containing unsaturated fatty acid chain |
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