Huperzine A high-producing strain and method for producing huperzine A by fermenting same

Abstract

The invention discloses a Colletotrichum gloeosporioidesisolate for producing a huperzine A and a method for producing huperzine A by fermenting the same, belonging to the technical field of biology. The Colletotrichum gloeosporioidesisolate YLJ-13 for producing the huperzine A is preserved in the China Center for Type Culture Collection on July 17, 2010, and the preservation number is CCTCC No. M2010181. The strain provided by the invention is separated and purified from huperzia crispate through pressure screening, and the strain is identified as a new variety of Colletotrichum gloeosporioidesisolate through a molecular biological means. The strain is fermented in an improved potato liquid culture medium, and huperzine A is obtained from the fermentation liquid. Huperzine A obtained through fermentation productioncan be secreted to be out of cell, the improved potato liquid culture medium is used as a precursor of biological transformation, and up to 199.6 mg / L huperzine A can be obtained from each liter of culture system. The strain provided by the invention is used for producing huperzine A. The existing rare medicinal huperziaceae plant resources can be protected, but also the shortage situation of huperzine A medication demand can be alleviated.

Description

technical field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a high-yielding endophytic bacterium of huperzine A—Colletotrichum gloeosporioides isolate YLJ-13, the separation, purification and pressure screening of the strain, and the utilization of the strain A method for producing huperzine A through high-yield fermentation of strains. Background technique

[0002] Huperzine A is a specific drug for the treatment of Alzheimer's disease. The aging of the world's population is increasing, and the clinical drug supply of Huperzine A has been seriously insufficient. At present, there are four sources of huperzine A: ① Extraction and separation from plants. It is known that Huperzine Aceae plants containing huperzine A are the main plant sources for obtaining huperzine A, but their self-reproduction ability is low and natural growth The cycle is as long as 10 to 15 years, and it is difficult to survive artificially cultivating Huperda...

Examples

Embodiment 1

[0048] Examples of the present invention are shown below. These examples are not intended to limit the scope of the invention. Example 1, Orthogonal method to determine the optimal culture conditions for the production of Huperzine A by fermentation of G. glyospora YLJ-13

[0049] On the basis of the initial medium, 10-30g of sucrose was added to each liter of potato liquid culture system, the speed of the shaker was 100-150r / min, and the culture was shaken at a constant temperature of 24-34°C for 3-7 days, and the huperzine A in the fermentation liquid was determined. accumulated amount.

[0050] Table 2 Orthogonal test header

[0051]

[0052] Table 3 Orthogonal test design table and results

[0053]

[0054] Table 4 Orthogonal test results analysis table

[0055]

[0056] The results of orthogonal test analysis show that:

[0057] (1) The best ratio for fermentation of this strain is A 2 B 2 C 3 D. 2 , that is, the dosage of sucrose is 20g / L, the culture t...

Embodiment 2

[0059] Embodiment 2, shaking flask fermentation produces huperzine A:

[0060] 1. Culture medium and strains

[0061] Bacterial classification: glyospora anthracnose YLJ-13 of the present invention;

[0062] Preservation and activation medium on slant: improved PDA medium;

[0063] Fermentation medium: improved potato liquid medium;

[0064] 2. Fermentation culture

[0065] Fermentation culture condition: the bacterial classification activated of the present invention is got mycelium, inoculates 0.01% mycelium by weight in 5L fermentation bottle, and pH value is natural value (5.80), ferments under the following conditions:

[0066] Sucrose dosage: 100g

[0067] Shaker temperature: 29°C±1°C

[0068] Speed: 130 rpm

[0069] Fermentation cycle: 96 hours

[0070] The fermentation results are as follows: filter the mycelium, obtain the filtrate, vacuumize and concentrate under reduced pressure to obtain the fermentation concentrate, and analyze the target substance and Hupe...

Embodiment 3

[0072] Embodiment 3, the enlarged fermentation production of huperzine A:

[0073] 1. Culture medium and strains

[0074]Bacterial classification: glyospora anthracnose YLJ-13 of the present invention;

[0075] Incline preservation and activation medium: improved PDA medium;

[0076] Fermentation medium: improved potato liquid medium;

[0077] 2. Fermentation culture

[0078] Fermentation culture condition: the bacterial classification activated of the present invention is got mycelium, adds 0.01% mycelia by weight in 100L fermented liquid, in fermenter, pH value is natural value (5.80), ferments in following condition:

[0079] Sucrose dosage: 2000g

[0080] Tank temperature: 29℃±1℃

[0081] Air volume: 1:0.4

[0082] Speed: 100 rpm

[0083] Tank pressure: 0.04Mpa

[0084] Fermentation cycle: 115 hours ± 5 hours

[0085] The fermentation results are as follows: filter the mycelia, obtain the filtrate, vacuumize and concentrate under reduced pressure to obtain the fer...

technical field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a high-yielding endophytic bacterium of huperzine A—Colletotrichum gloeosporioides isolate YLJ-13, the separation, purification and pressure screening of the strain, and the utilization of the strain A method for producing huperzine A through high-yield fermentation of strains. Background technique

[0002] Huperzine A is a specific drug for the treatment of Alzheimer's disease. The aging of the world's population is increasing, and the clinical drug supply of Huperzine A has been seriously insufficient. At present, there are four sources of huperzine A: ① Extraction and separation from plants. It is known that Huperzine Aceae plants containing huperzine A are the main plant sources for obtaining huperzine A, but their self-reproduction ability is low and natural growth The cycle is as long as 10 to 15 years, and it is difficult to survive artificially cultivating Huperda...

Examples

Embodiment 1

[0048] Examples of the present invention are shown below. These examples are not intended to limit the scope of the invention. Example 1, Orthogonal method to determine the optimal culture conditions for the production of Huperzine A by fermentation of G. glyospora YLJ-13

[0049] On the basis of the initial medium, 10-30g of sucrose was added to each liter of potato liquid culture system, the speed of the shaker was 100-150r / min, and the culture was shaken at a constant temperature of 24-34°C for 3-7 days, and the huperzine A in the fermentation liquid was determined. accumulated amount.

[0050] Table 2 Orthogonal test header

[0051]

[0052] Table 3 Orthogonal test design table and results

[0053]

[0054] Table 4 Orthogonal test results analysis table

[0055]

[0056] The results of orthogonal test analysis show that:

[0057] (1) The best ratio for fermentation of this strain is A 2 B 2 C 3 D. 2 , that is, the dosage of sucrose is 20g / L, the culture t...

Embodiment 2

[0059] Embodiment 2, shaking flask fermentation produces huperzine A:

[0060] 1. Culture medium and strains

[0061] Bacterial classification: glyospora anthracnose YLJ-13 of the present invention;

[0062] Preservation and activation medium on slant: improved PDA medium;

[0063] Fermentation medium: improved potato liquid medium;

[0064] 2. Fermentation culture

[0065] Fermentation culture condition: the bacterial classification activated of the present invention is got mycelium, inoculates 0.01% mycelium by weight in 5L fermentation bottle, and pH value is natural value (5.80), ferments under the following conditions:

[0066] Sucrose dosage: 100g

[0067] Shaker temperature: 29°C±1°C

[0068] Speed: 130 rpm

[0069] Fermentation cycle: 96 hours

[0070] The fermentation results are as follows: filter the mycelium, obtain the filtrate, vacuumize and concentrate under reduced pressure to obtain the fermentation concentrate, and analyze the target substance and Hupe...

Embodiment 3

[0072] Embodiment 3, the enlarged fermentation production of huperzine A:

[0073] 1. Culture medium and strains

[0074]Bacterial classification: glyospora anthracnose YLJ-13 of the present invention;

[0075] Incline preservation and activation medium: improved PDA medium;

[0076] Fermentation medium: improved potato liquid medium;

[0077] 2. Fermentation culture

[0078] Fermentation culture condition: the bacterial classification activated of the present invention is got mycelium, adds 0.01% mycelia by weight in 100L fermented liquid, in fermenter, pH value is natural value (5.80), ferments in following condition:

[0079] Sucrose dosage: 2000g

[0080] Tank temperature: 29℃±1℃

[0081] Air volume: 1:0.4

[0082] Speed: 100 rpm

[0083] Tank pressure: 0.04Mpa

[0084] Fermentation cycle: 115 hours ± 5 hours

[0085] The fermentation results are as follows: filter the mycelia, obtain the filtrate, vacuumize and concentrate under reduced pressure to obtain the fer...

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