CN117551177A - Method for improving fermentation yield of echinocandin B - Google Patents
Method for improving fermentation yield of echinocandin B Download PDFInfo
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- CN117551177A CN117551177A CN202410037110.9A CN202410037110A CN117551177A CN 117551177 A CN117551177 A CN 117551177A CN 202410037110 A CN202410037110 A CN 202410037110A CN 117551177 A CN117551177 A CN 117551177A
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- 238000000855 fermentation Methods 0.000 title claims abstract description 145
- 230000004151 fermentation Effects 0.000 title claims abstract description 145
- 108010062092 echinocandin B Proteins 0.000 title claims abstract description 76
- FAUOJMHVEYMQQG-HVYQDZECSA-N echinocandin B Chemical compound C1([C@H](O)[C@@H](O)[C@H]2C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C[C@@H](C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N2)[C@@H](C)O)=O)NC(=O)CCCCCCC\C=C/C\C=C/CCCCC)[C@@H](C)O)=CC=C(O)C=C1 FAUOJMHVEYMQQG-HVYQDZECSA-N 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 30
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims abstract description 132
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 claims abstract description 66
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims abstract description 66
- 239000001963 growth medium Substances 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 28
- 238000012258 culturing Methods 0.000 claims abstract description 26
- 238000011218 seed culture Methods 0.000 claims abstract description 18
- 241000351920 Aspergillus nidulans Species 0.000 claims abstract description 11
- 241001052560 Thallis Species 0.000 claims abstract description 3
- 239000000843 powder Substances 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 244000068988 Glycine max Species 0.000 claims description 10
- 235000010469 Glycine max Nutrition 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 7
- 239000008103 glucose Substances 0.000 claims description 7
- 239000002609 medium Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- QYDYPVFESGNLHU-UHFFFAOYSA-N elaidic acid methyl ester Natural products CCCCCCCCC=CCCCCCCCC(=O)OC QYDYPVFESGNLHU-UHFFFAOYSA-N 0.000 claims description 5
- QYDYPVFESGNLHU-KHPPLWFESA-N methyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC QYDYPVFESGNLHU-KHPPLWFESA-N 0.000 claims description 5
- 229940073769 methyl oleate Drugs 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
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- 239000008272 agar Substances 0.000 claims description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 4
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- 230000015572 biosynthetic process Effects 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000003642 reactive oxygen metabolite Substances 0.000 description 5
- 108010028921 Lipopeptides Proteins 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- 102000004169 proteins and genes Human genes 0.000 description 3
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- 229920001503 Glucan Polymers 0.000 description 2
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- 238000001514 detection method Methods 0.000 description 2
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- 108010064760 Anidulafungin Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- JHVAMHSQVVQIOT-MFAJLEFUSA-N anidulafungin Chemical compound C1=CC(OCCCCC)=CC=C1C1=CC=C(C=2C=CC(=CC=2)C(=O)N[C@@H]2C(N[C@H](C(=O)N3C[C@H](O)C[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N3C[C@H](C)[C@H](O)[C@H]3C(=O)N[C@H](O)[C@H](O)C2)[C@@H](C)O)[C@H](O)[C@@H](O)C=2C=CC(O)=CC=2)[C@@H](C)O)=O)C=C1 JHVAMHSQVVQIOT-MFAJLEFUSA-N 0.000 description 1
- 229960003348 anidulafungin Drugs 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- 230000010224 hepatic metabolism Effects 0.000 description 1
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- 230000014759 maintenance of location Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
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- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
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- 230000009467 reduction Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 1
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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Abstract
The invention discloses a method for improving fermentation yield of echinocandin B, and belongs to the technical field of biology. The method comprises the following steps: (1) slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing for 10-15 days to obtain slant thallus; (2) seed culture: inoculating the bevel thalli into a seed culture medium, and culturing for 2-3 days at the temperature of 25-30 ℃ and the rotating speed of 250rpm to obtain seed liquid; (3) fermentation culture: inoculating the seed liquid into a fermentation culture medium, and performing shaking culture at constant temperature to obtain a fermentation liquid 1; (4) adding gamma-aminobutyric acid: adding gamma-aminobutyric acid solution into the fermentation broth 1, and performing shaking culture at constant temperature to obtain a fermentation broth; (5) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B. The method maintains normal metabolic activity of cells by adding gamma-aminobutyric acid, is beneficial to synthesis of echinocandin B, and improves fermentation yield of echinocandin B.
Description
Technical Field
The invention relates to a method for improving fermentation yield of echinocandin B, and belongs to the technical field of biology.
Background
Echinocandin B is a cyclic lipopeptid compound, is a precursor substance of anidulafungin, is mainly prepared by fermenting aspergillus nidulans, is an intracellular secondary metabolite, and belongs to the class of antibiotics. It comprises a cyclic hexapeptide core with a lipid side chain that inhibits the synthesis of beta- (1, 3) -D-glucan by a non-competing mechanism of action, resulting in emptying of cell wall glucan, osmotic instability and lysis of fungal cells to exert its antifungal effect. Therefore, the method can be used for preparing the echinocandin antifungal medicines, and the echinocandin antifungal medicines have low toxicity, strong antibacterial effect, no antagonism with other medicines, no liver metabolism and no cross drug resistance, and have become the focus of research in recent years. However, the fermentation yield of echinocandin B is low, and the separation and purification process is complex and difficult, so that the industrial cost is high.
Gamma-aminobutyric acid is an inhibitory neurotransmitter distributed in mammals, mediates more than 40% of inhibitory nerve signals, has important physiological functions, is a non-protein constitutive amino acid, is widely existing in prokaryotes and eukaryotes, and accounts for a considerable proportion of free amino acids in eukaryotic cells.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for improving the fermentation yield of echinocandin B, which maintains normal metabolic activity of cells by adding gamma-aminobutyric acid, is beneficial to synthesis of the echinocandin B and improves the fermentation yield of the echinocandin B.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for increasing fermentation yield of echinocandin B comprising the steps of:
(1) Slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing at 25-30deg.C for 10-15 days to obtain slant thallus;
(2) Seed culture: inoculating the bevel thalli into a seed culture medium, and culturing for 2-3 days at the temperature of 25-30 ℃ and the rotating speed of 250rpm to obtain seed liquid;
(3) Fermentation culture: inoculating the seed liquid into a fermentation culture medium, and performing shaking culture at constant temperature to obtain a fermentation liquid 1;
(4) Gamma-aminobutyric acid was added: adding gamma-aminobutyric acid solution into the fermentation broth 1, and performing shaking culture at constant temperature to obtain a fermentation broth;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Further, in the step (1), the PDA slant culture medium is: potato 200g/L, glucose 20g/L, agar 20g/L, water as solvent and natural pH.
Further, in the step (1), the PDA slant culture is sterilized at 115 ℃ for 30min and then cooled to room temperature for use.
Further, in step (2), the seed culture medium is: 20g/L of soybean cake powder, 10g/L of yeast powder and 10g/L of peptone, wherein the solvent is water, and the pH is natural.
Further, in the step (2), the seed culture is sterilized at 115 ℃ for 30min and then cooled to room temperature for use.
Further, in the step (3), the inoculation amount of the seed liquid is 5-10% of that of the fermentation medium according to the volume percentage.
Further, in the step (3), the composition and the content of the fermentation medium are as follows: 80g/L of methyl oleate, 10g/L of glucose, 8g/L of peptone, 30g/L of soybean cake powder and K 2 HPO 4 ·3H 2 O 5.0g/L，MgSO 4 ·7H 2 O 0.8g/L，MnSO 4 ·H 2 O 0.3g/L，FeSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.5g/L。
Further, in the step (3), the pH of the fermentation medium is 6.0 to 6.8.
Further, in the step (3), the constant temperature shaking culture time is 7 days, which is recorded as the 0 th to 7 th days of the fermentation period.
Further, in the step (4), the constant temperature shaking culture time is 1-5 days, which is recorded as 8-12 days of the fermentation period.
And (3) carrying out constant-temperature shaking culture for 7 days, carrying out constant-temperature shaking culture for 1-5 days in the step (4), and carrying out total constant-temperature shaking culture for 8-12 days in the process of fermenting and producing echinocandin B by aspergillus nidulans. Culturing the step (3) at constant temperature for 7 days, namely, 0 th to 7 th days of a fermentation period, and culturing the step (4) at constant temperature for 1 to 5 days, namely, 8 th to 12 th days of the fermentation period, and simultaneously, the fermentation period is also the later period.
Further, in the step (4), the concentration of the gamma-aminobutyric acid solution is 50-100g/L.
Further, in the step (4), the addition amount of the gamma-aminobutyric acid solution is 1.0 to 5.0% by mass of the fermentation broth 1.
Further, in the step (4), the preparation method of the gamma-aminobutyric acid solution comprises the following steps: dissolving gamma-aminobutyric acid in water, steam sterilizing at 110-120deg.C under 0.08-0.1MPa for 30-40min, and cooling.
The non-protein amino acid gamma-aminobutyric acid is associated with lipopeptides accumulation, while echinocandin B is one of the lipopeptides. In the stage of rapid accumulation of echinocandin B, gamma-aminobutyric acid can be used as a compensation way to maintain TCA cycle, which is favorable for fermentation production of echinocandin B, and a large amount of Reactive Oxygen Species (ROS) appear in cells at the later stage of fermentation, and the accumulation of gamma-aminobutyric acid can determine high resistance of oxidative stress. Therefore, in the process of fermenting and producing the echinocandin B by the aspergillus nidulans, the gamma-aminobutyric acid is added, so that normal metabolic activity of cells can be maintained, and the fermentation yield of the echinocandin B is improved.
Further, in the step (4), the addition time of the gamma-aminobutyric acid solution is 8-11 days of the fermentation period, and the gamma-aminobutyric acid solution is added to the fermentation broth 1.
Further, in the step (4), the addition time of the gamma-aminobutyric acid solution is that the gamma-aminobutyric acid solution is added to the fermentation broth 1 every day in four consecutive days from 8 th to 11 th of the fermentation period.
And in the 8 th to 11 th days of the fermentation period, the gamma-aminobutyric acid solution is added into the fermentation liquid 1 in 1 to 4 days at random, so that the fermentation yield of the echinocandin B can be improved, and the higher the number of days of addition, the higher the yield of the echinocandin B produced by fermenting the aspergillus nidulans.
Furthermore, the conditions of the constant temperature shaking culture are all that the temperature is 25 ℃ and the rotating speed is 220-280rpm.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the method, in the process of producing the echinocandin B by fermenting aspergillus nidulans, gamma-aminobutyric acid is added, so that a large amount of active oxygen in cells in the later period of fermentation can be resisted, normal metabolic activity of cells can be maintained, the fermentation yield of the echinocandin B is improved, and the yield can be improved by about 58%.
2. The method has simple and feasible operation process, no complex steps, labor saving, capability of improving the fermentation yield of the echinocandin B, capability of solving the problem of low fermentation yield of the echinocandin B, contribution to industrial production of the echinocandin B and reduction of production cost.
3. The gamma-aminobutyric acid selected by the method is a non-protein constitutive amino acid and widely exists in prokaryotes and eukaryotes. Gamma-aminobutyric acid is not only associated with lipopeptides accumulation, but also is able to resist the large amounts of Reactive Oxygen Species (ROS) that occur intracellularly in the late stages of fermentation. Echinocandin B is a lipopeptide compound, and in the stage of rapid accumulation of the echinocandin B, gamma-aminobutyric acid can be used as a compensation way to maintain TCA circulation, which is beneficial to fermentation production of the echinocandin B, a large amount of ROS appear in cells in the later period of fermentation, and the accumulation of gamma-aminobutyric acid can determine high resistance of oxidative stress.
Drawings
FIG. 1 is the effect of adding gamma-aminobutyric acid at different stages of fermentation on echinocandin B production;
FIG. 2 is the effect of adding different amounts of gamma-aminobutyric acid on echinocandin B production at the late stage of fermentation (day 8);
FIG. 3 effect of duration of late fermentation addition of gamma-aminobutyric acid on echinocandin B production.
Detailed Description
Example 1
A method for increasing fermentation yield of echinocandin B comprising the steps of:
(1) Slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing at 25deg.C for 10 days to obtain slant thallus;
(2) Seed culture: inoculating the bevel bacteria into a seed culture medium, and culturing for 3 days at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain a seed liquid, wherein the seed culture medium is: 20g/L of soybean cake powder, 10g/L of yeast powder and 10g/L of peptone, wherein the solvent is water, and the pH is natural;
(3) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to 10% of inoculation amount, and culturing for 7 days at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain a fermentation liquid 1, wherein the fermentation culture medium comprises the following components in percentage by weight: 80g/L of methyl oleate, 10g/L of glucose, 8g/L of peptone, 30g/L of soybean cake powder and K 2 HPO 4 ·3H 2 O 5.0g/L，MgSO 4 ·7H 2 O 0.8g/L，MnSO 4 ·H 2 O 0.3g/L，FeSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.5g/L,500mL conical flask volume is 100mL;
(4) Gamma-aminobutyric acid was added: on the 8 th day of fermentation, adding 5mL of gamma-aminobutyric acid solution with the concentration of 100g/L into the fermentation broth 1, and continuously culturing at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain the fermentation broth;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Example 2
A method for increasing fermentation yield of echinocandin B comprising the steps of:
(1) Slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing at 25deg.C for 10 days to obtain slant thallus;
(2) Seed culture: inoculating the bevel bacteria into a seed culture medium, and culturing for 3 days at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain a seed liquid, wherein the seed culture medium is: 20g/L of soybean cake powder, 10g/L of yeast powder and 10g/L of peptone, wherein the solvent is water, and the pH is natural;
(3) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to 10% of inoculation amount, and culturing for 7 days at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain a fermentation liquid 1, wherein the fermentation culture medium comprises the following components in percentage by weight: 80g/L of methyl oleate, 10g/L of glucose, 8g/L of peptone, 30g/L of soybean cake powder and K 2 HPO 4 ·3H 2 O 5.0g/L，MgSO 4 ·7H 2 O 0.8g/L，MnSO 4 ·H 2 O 0.3g/L，FeSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.5g/L,500mL conical flask volume is 100mL;
(4) Gamma-aminobutyric acid was added: on the 9 th day of fermentation, adding 5mL of gamma-aminobutyric acid solution with the concentration of 100g/L into the fermentation liquid 1, and continuously culturing at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain fermentation liquid;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Example 3
A method for increasing fermentation yield of echinocandin B comprising the steps of:
(1) Slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing at 25deg.C for 10 days to obtain slant thallus;
(2) Seed culture: inoculating the bevel bacteria into a seed culture medium, and culturing for 3 days at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain a seed liquid, wherein the seed culture medium is: 20g/L of soybean cake powder, 10g/L of yeast powder and 10g/L of peptone, wherein the solvent is water, and the pH is natural;
(3) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to an inoculum size of 5%, and culturing for 7 days at a temperature of 25 ℃ and a rotating speed of 250rpm to obtain a fermentation liquid 1, wherein the fermentation culture medium comprises the following components in percentage by weight: 80g/L of methyl oleate, 10g/L of glucose, 8g/L of peptone, 30g/L of soybean cake powder and K 2 HPO 4 ·3H 2 O 5.0g/L，MgSO 4 ·7H 2 O 0.8g/L，MnSO 4 ·H 2 O 0.3g/L，FeSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.5g/L,500mL conical flask volume is 100mL;
(4) Gamma-aminobutyric acid was added: on the 8 th day of fermentation, adding 3mL of gamma-aminobutyric acid solution with the concentration of 100g/L into the fermentation liquid 1, and continuously culturing at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain fermentation liquid;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Experimental example 1 effect of addition of gamma-aminobutyric acid on production of echinocandin B at different periods of fermentation
Steps (1) - (2) are the same as steps (1) - (2) of example 1.
(3) Fermentation culture: inoculating seed solution into 11 control groups and 11 treatment groups according to 10% inoculum size, wherein each group comprises 3 repeated fermentation media, the volume of 500mL conical bottled liquid is 100mL, and culturing at 25deg.C and rotation speed of 250rpm for 7 days to obtain fermentation liquid 1;
(4) Gamma-aminobutyric acid was added: adding 5mL of gamma-aminobutyric acid solution with the concentration of 100g/L into each bottle of fermentation liquid 1 in the treatment group at the fermentation time of 0-11 days, continuously culturing at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain fermentation liquid, and adding equal amount of clear water in the control group at the fermentation time of 0-11 days;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
(6) Determination of the content of echinocandin B (ECB): shaking the fermentation liquor, taking 1mL of fermentation liquor in a 10mL volumetric flask, using methanol to fix the volume, carrying out ultrasonic treatment for 20-30min, centrifuging for 5min at 12000 Xg, taking the supernatant, filtering with an organic film with the thickness of 0.22 mu m, and then using the supernatant for detecting the spike area of echinocandin B by high performance liquid chromatography.
The calculation method of the echinocandin B concentration comprises the following steps: detecting the peak area of the sample by adopting high performance liquid chromatography, and obtaining the yield of echinocandin B according to a echinocandin B standard curve.
The high performance liquid chromatography detection adopts Agilent high performance liquid chromatograph, and the conditions are as follows: c18 column (Agilent, 4.6 mm. Times.250 mm. Times.5 μm) with methanol as mobile phase: acetonitrile: water = 7:1:2, the flow rate is 1mL/min; ultraviolet detector: the detection wavelength is 222nm, the sample injection amount is 20 mu L, and the column temperature is 35 ℃. The retention time of echinocandin B was about 14.5 min.
The measurement results are shown in figure 1.
As can be seen from FIG. 1, the treatment groups added gamma-aminobutyric acid at days 0-11 of fermentation respectively, and compared with the control groups, the fermentation yield of echinocandin B was not significantly changed by adding gamma-aminobutyric acid solution at days 0-7 of fermentation period, and the fermentation yield of echinocandin B was significantly improved by more than 10% by adding gamma-aminobutyric acid solution at days 8-11 of fermentation. The addition of gamma-aminobutyric acid at 8-11 days of fermentation is shown to significantly increase the fermentation yield of echinocandin B.
Experimental example 2 Effect of addition of different amounts of gamma-aminobutyric acid on production of echinocandin B in late fermentation (day 8)
Steps (1) - (2) are the same as steps (1) - (2) of example 1.
(3) Fermentation culture: inoculating the seed solution into 9 groups of 3 repeated fermentation media according to 10% of inoculum size, and culturing at 25 ℃ and 250rpm to obtain fermentation liquor 1;
(4) Gamma-aminobutyric acid was added: on day 8, adding 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0mL of gamma-aminobutyric acid solution with the concentration of 100g/L into 9 groups of fermentation liquor 1 respectively, and continuously culturing at the temperature of 25 ℃ and the rotating speed of 250rpm to obtain fermentation liquor;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Step (6) is the same as step (6) of Experimental example 1.
The measurement results are shown in figure 2.
As is clear from FIG. 2, when 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0mL of a 100. 100g/L concentration of gamma-aminobutyric acid solution were added to 9 groups of medium on the 8 th day of the late fermentation period, the fermentation yield of echinocandin B did not significantly change when 6-10mL of the gamma-aminobutyric acid solution was added, i.e., when the concentration was 6-10 g/L, the fermentation yield of echinocandin B was increased when 1-5mL of the gamma-aminobutyric acid solution was added, i.e., when the concentration was 1-5g/L, and the fermentation yield of echinocandin B was increased to the greatest extent when the addition amount of the gamma-aminobutyric acid solution was 3 mL. The addition of gamma-aminobutyric acid can improve the fermentation content of echinocandin B.
Experimental example 3
Steps (1) - (2) are the same as steps (1) - (2) of example 1.
(3) Fermentation culture: respectively inoculating seed solution into 6 control groups and 10 treatment groups according to 10% inoculum size, wherein each group comprises 3 repeated fermentation media, and culturing at 25deg.C and rotation speed of 250rpm for 7 days to obtain fermentation liquor 1;
(4) Gamma-aminobutyric acid was added: clear water was added to each of 3 replicates of the 6 control groups, and 3mL of 100g/L gamma-aminobutyric acid solution was added to each of 3 replicates of the 10 treatment groups. The addition time of gamma-aminobutyric acid in the treatment group is as shown in Table 1, and the addition time of the control group is the same as that of the treatment group. Continuously culturing at 25deg.C and rotation speed of 250rpm to obtain fermentation broth;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
Step (6) is the same as step (6) of Experimental example 1.
The measurement results are shown in FIG. 3.
TABLE 1 time of addition of gamma-aminobutyric acid
Note that: 8-11 are days 8-11 of fermentation culture, respectively, "+" is the addition of gamma-aminobutyric acid solution.
Since the control group was added with clear water, the number of days of addition had no effect on the fermentation yield of echinocandin B, which was the same, 6 control groups were pooled into 1 control group (CK) in fig. 3.
As can be seen from fig. 3, in the latter fermentation period, the fermentation yield of echinocandin B to which γ -aminobutyric acid was added was higher for any two days than for any one day; compared with the fermentation yield of the echinocandin B added with the gamma-aminobutyric acid in any two days, the fermentation yield of the echinocandin B added with the gamma-aminobutyric acid in any three days is high; when the gamma-aminobutyric acid is added for four consecutive days, the fermentation yield of echinocandin B is the highest and is 1581.9mg/L, and compared with a control group, the fermentation yield is improved by about 58.7%. The results show that the addition of gamma-aminobutyric acid in the late fermentation stage can improve the fermentation yield of echinocandin B, and the more days the gamma-aminobutyric acid is added, the more the fermentation yield of echinocandin B is improved.
Claims (10)
1. A method for improving fermentation yield of echinocandin B, which is characterized by comprising the following steps: the method comprises the following steps:
(1) Slant culture: inoculating Aspergillus nidulans to PDA slant culture medium, and culturing at 25-30deg.C for 10-15 days to obtain slant thallus;
(2) Seed culture: inoculating the bevel thalli into a seed culture medium, and culturing for 2-3 days at the temperature of 25-30 ℃ and the rotating speed of 250rpm to obtain seed liquid;
(3) Fermentation culture: inoculating the seed liquid into a fermentation culture medium, and performing shaking culture at constant temperature to obtain a fermentation liquid 1;
(4) Gamma-aminobutyric acid was added: adding gamma-aminobutyric acid solution into the fermentation broth 1, and performing shaking culture at constant temperature to obtain a fermentation broth;
(5) And (3) separating and purifying: separating and purifying the fermentation liquor to obtain echinocandin B.
2. The method for improving fermentation yield of echinocandin B according to claim 1, wherein: in the step (1), the PDA slant culture medium is as follows: 200g/L of potato, 20g/L of glucose and 20g/L of agar, wherein the solvent is water, and the pH is natural.
3. The method for improving fermentation yield of echinocandin B according to claim 1, wherein: in the step (2), the seed culture medium is: 20g/L of soybean cake powder, 10g/L of yeast powder and 10g/L of peptone, wherein the solvent is water, and the pH is natural.
4. The method for improving fermentation yield of echinocandin B according to claim 1, wherein: in the step (3), the inoculation amount of the seed liquid is 5-10% of that of the fermentation medium according to the volume percentage.
5. The method for increasing fermentation yield of echinocandin B according to any one of claims 1-4, characterized by: in the step (3), the fermentation medium comprises the following components in percentage by weight: 80g/L of methyl oleate, 10g/L of glucose, 8g/L of peptone, 30g/L of soybean cake powder and K 2 HPO 4 ·3H 2 O 5.0g/L，MgSO 4 ·7H 2 O 0.8g/L，MnSO 4 ·H 2 O 0.3g/L，FeSO 4 ·7H 2 O 0.1g/L，CaCl 2 0.5g/L。
6. The method for improving fermentation yield of echinocandin B according to claim 5, wherein: in step (3), the pH of the fermentation medium is 6.0-6.8.
7. The method for improving fermentation yield of echinocandin B according to claim 1, wherein: in the step (4), the concentration of the gamma-aminobutyric acid solution is 50-100g/L.
8. The method for improving fermentation yield of echinocandin B according to claim 7, wherein: in the step (4), the addition amount of the gamma-aminobutyric acid solution is 1.0 to 5.0% by mass of the fermentation broth 1.
9. The method for increasing fermentation production of echinocandin B according to any one of claims 1-4 or 6-8, characterized by: in the step (4), the addition time of the gamma-aminobutyric acid solution is 8-11 days of the fermentation period, and the gamma-aminobutyric acid solution is added into the fermentation liquid 1.
10. The method for improving fermentation yield of echinocandin B according to claim 1, wherein: the conditions of the shaking table constant temperature culture are that the temperature is 25 ℃ and the rotating speed is 220-280rpm.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238867A1 (en) * | 2007-12-13 | 2009-09-24 | Scott Jenkins | Nanoparticulate Anidulafungin Compositions and Methods for Making the Same |
| CN103509840A (en) * | 2013-10-12 | 2014-01-15 | 浙江工业大学 | Method for increasing yield of anidulafungin precursor compound Echinocandin B |
| CN108342437A (en) * | 2018-02-27 | 2018-07-31 | 浙江工业大学 | A method of utilizing aspergillus nidulans fermentation high yield echinocandin B |
| CN115747283A (en) * | 2021-09-03 | 2023-03-07 | 杭州中美华东制药有限公司 | Method for preparing pneumocandin B0 through fermentation |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090238867A1 (en) * | 2007-12-13 | 2009-09-24 | Scott Jenkins | Nanoparticulate Anidulafungin Compositions and Methods for Making the Same |
| CN103509840A (en) * | 2013-10-12 | 2014-01-15 | 浙江工业大学 | Method for increasing yield of anidulafungin precursor compound Echinocandin B |
| CN108342437A (en) * | 2018-02-27 | 2018-07-31 | 浙江工业大学 | A method of utilizing aspergillus nidulans fermentation high yield echinocandin B |
| CN115747283A (en) * | 2021-09-03 | 2023-03-07 | 杭州中美华东制药有限公司 | Method for preparing pneumocandin B0 through fermentation |
| WO2023030496A1 (en) * | 2021-09-03 | 2023-03-09 | 杭州中美华东制药有限公司 | Method for preparing pneumocandin b0 by fermentation |
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