CN115161355B - Method for preparing high-stability pravastatin by using fermentation process - Google Patents
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- 238000000855 fermentation Methods 0.000 title claims abstract description 87
- 230000004151 fermentation Effects 0.000 title claims abstract description 87
- TUZYXOIXSAXUGO-UHFFFAOYSA-N Pravastatin Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CC(O)C=C21 TUZYXOIXSAXUGO-UHFFFAOYSA-N 0.000 title claims abstract description 39
- TUZYXOIXSAXUGO-PZAWKZKUSA-N pravastatin Chemical compound C1=C[C@H](C)[C@H](CC[C@@H](O)C[C@@H](O)CC(O)=O)[C@H]2[C@@H](OC(=O)[C@@H](C)CC)C[C@H](O)C=C21 TUZYXOIXSAXUGO-PZAWKZKUSA-N 0.000 title claims abstract description 39
- 229960002965 pravastatin Drugs 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 24
- 239000000284 extract Substances 0.000 claims abstract description 93
- 235000019764 Soybean Meal Nutrition 0.000 claims abstract description 37
- 239000004455 soybean meal Substances 0.000 claims abstract description 37
- 241000187362 Actinomadura Species 0.000 claims abstract description 28
- 241000723343 Cichorium Species 0.000 claims abstract description 24
- 235000007542 Cichorium intybus Nutrition 0.000 claims abstract description 23
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 239000002131 composite material Substances 0.000 claims abstract description 6
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 48
- AJLFOPYRIVGYMJ-UHFFFAOYSA-N SJ000287055 Natural products C12C(OC(=O)C(C)CC)CCC=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 AJLFOPYRIVGYMJ-UHFFFAOYSA-N 0.000 claims description 28
- 239000001963 growth medium Substances 0.000 claims description 28
- AJLFOPYRIVGYMJ-INTXDZFKSA-N mevastatin Chemical compound C([C@H]1[C@@H](C)C=CC2=CCC[C@@H]([C@H]12)OC(=O)[C@@H](C)CC)C[C@@H]1C[C@@H](O)CC(=O)O1 AJLFOPYRIVGYMJ-INTXDZFKSA-N 0.000 claims description 28
- BOZILQFLQYBIIY-UHFFFAOYSA-N mevastatin hydroxy acid Natural products C1=CC(C)C(CCC(O)CC(O)CC(O)=O)C2C(OC(=O)C(C)CC)CCC=C21 BOZILQFLQYBIIY-UHFFFAOYSA-N 0.000 claims description 28
- 229950009116 mevastatin Drugs 0.000 claims description 26
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- 239000002609 medium Substances 0.000 claims description 20
- 238000011218 seed culture Methods 0.000 claims description 19
- 244000068988 Glycine max Species 0.000 claims description 15
- 235000010469 Glycine max Nutrition 0.000 claims description 15
- 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 14
- 239000001888 Peptone Substances 0.000 claims description 14
- 108010080698 Peptones Proteins 0.000 claims description 14
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- 239000008103 glucose Substances 0.000 claims description 14
- 235000019319 peptone Nutrition 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 14
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- 239000012138 yeast extract Substances 0.000 claims description 14
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
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- 238000006243 chemical reaction Methods 0.000 abstract description 16
- 238000000605 extraction Methods 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 16
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- 238000010992 reflux Methods 0.000 description 12
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- VGMFHMLQOYWYHN-UHFFFAOYSA-N Compactin Natural products OCC1OC(OC2C(O)C(O)C(CO)OC2Oc3cc(O)c4C(=O)C(=COc4c3)c5ccc(O)c(O)c5)C(O)C(O)C1O VGMFHMLQOYWYHN-UHFFFAOYSA-N 0.000 description 2
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 239000000463 material Substances 0.000 description 2
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- 239000002904 solvent Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000228153 Penicillium citrinum Species 0.000 description 1
- 229940123934 Reductase inhibitor Drugs 0.000 description 1
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- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
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- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
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- -1 trihydroxymethylglutaryl coenzyme A Chemical compound 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/03—Actinomadura
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Abstract
The invention discloses a method for preparing high-stability pravastatin by using a fermentation process, belonging to the technical field of microorganisms. The method comprises the following steps: s01, culturing actinomadura strain seeds; s02, fermenting actinomadura; in the S02 actinomadura fermentation, a fermentation medium contains a composite extract. According to the method for preparing high-stability pravastatin by using the fermentation process, the chicory extract and the soybean meal extract are added into the fermentation medium and in the fermentation process, so that the conversion rate of pravastatin can be remarkably improved. According to the method for preparing the high-stability pravastatin by using the fermentation process, the added extract is common in raw materials, the extraction method is simple, and the cost is low.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a method for preparing high-stability pravastatin by using a fermentation process.
Background
Pravastatin (Pravastatin) is a trihydroxymethylglutaryl coenzyme A reductase inhibitor, which can reduce VLDL (very low density lipoprotein) and LDLC (low density lipoprotein), especially reduce total cholesterol obviously, but has weak effect on phospholipid but not triglyceride.
At present, the fermentation method for producing pravastatin is a two-step fermentation process, firstly, a secondary metabolite Mevastatin, also called Compactin, is obtained by fermentation production of microorganisms such as penicillium citrinum and the like, and then the Mevastatin is converted into the pravastatin through the conversion action of microbial enzymes.
The culture medium of actinomadura significantly affects the efficiency of pravastatin conversion. CN108070623A discloses a method for improving the fermentation yield of pravastatin, wherein bran extract is added into a fermentation medium. CN102757986A discloses a fermentation process for producing pravastatin by converting compactin with actinomadura, adding a trace element solution into a fermentation medium. CN108977469A discloses an application of rice bran meal extract in producing pravastatin, and the rice bran meal extract is added into a culture medium.
Disclosure of Invention
Researchers of the invention widely screen common plant fruits, vegetables and food crops, and discover the influence of chicory extract and soybean meal extract on the conversion of mevastatin into pravastatin by actinomadura, and the patent is based on the discovery.
The invention discloses a method for preparing high-stability pravastatin by using a fermentation process, which comprises the following steps:
s01, culturing actinomadura strain seeds;
s02, fermenting actinomadura;
in the S02 actinomadura fermentation, a fermentation medium contains a compound extract, and the compound extract comprises a chicory extract and a soybean meal extract.
In some preferred embodiments of the present invention, the method further comprises the step of supplementing the complex extract in the fermentation of S02 actinomadura.
In some preferred embodiments of the invention, the chicory extract is a chicory root extract or a chicory stem extract.
In some preferred embodiments of the present invention, the complex extract comprises chicory root extract and soybean meal extract.
In some preferred embodiments of the present invention, the chicory extract is prepared by extraction with aqueous ethanol. Preferably, chicory root or stem is ground, soaked in 10 times (mL/g) of 95% ethanol for 24h, then extracted under reflux in a water bath kettle at 80 ℃ for 2h, repeated for 2 times, the filtrates are combined, concentrated under reduced pressure in vacuum and then freeze-dried to obtain the chicory root extract.
In some preferred embodiments of the present invention, the soybean meal extract is prepared by first extracting with an aqueous ethanol solution and then extracting with ethyl acetate. Preferably, the low-temperature soybean meal is extracted by refluxing 10 times (mL/g) of 60-80% ethanol water solution in a water bath kettle at 55-70 ℃ for 1.5-2.5h, the extraction is carried out for 1-3 times, the filtrate is combined, the vacuum reduced pressure concentration is carried out, the ethanol is recovered, 1.5-2.5 times (mL/g) of ethyl acetate is added, the ethyl acetate phase is subjected to vacuum reduced pressure concentration and then freeze drying is carried out, and the soybean meal extract is obtained.
In some preferred embodiments of the present invention, the weight ratio of the chicory extract to the soybean meal extract in the composite extract is (1-5): (1-5), preferably (2-4): 1.
in some preferred embodiments of the present invention, the complex extract is present in the fermentation medium in an amount of 0.3-2g/L, preferably 1-1.5g/L.
In some preferred embodiments of the invention, the supplemental complex extract is added after fermentation for 50-80h in an amount of 0.3-2g/L culture medium, preferably 1-1.5g/L culture medium.
In some preferred embodiments of the present invention, in S01, the seed culture medium is inoculated with the slant-seeded actinomadura strain, and cultured in a triangular flask shaker at 30-34 ℃ for 55-65h to obtain a seed solution;
preferably, each liter of the seed culture medium comprises 25-35g of glucose, 15-25g of yeast extract, 3-8g of soybean peptone, K 2 HPO 4 ·3H 2 O1-3g，MgSO 4 ·7H 2 0.5-1g of O and 0.5-1.5g of soybean oil.
In some preferred embodiments of the invention, in S02, the fermentation medium is inoculated with the seed solution in a volume ratio of 5-15%, after culturing at 31-34 ℃ for 35-45h in a biological fermentation tank, the temperature is adjusted to 28-30 ℃, mevastatin is continuously added for 170-185h, the concentration of mevastatin in the medium is maintained at 0.3-0.8g/L, and the fermentation is ended within 190-200 h;
preferably, the fermentation medium per liter comprises 45-55g of glucose, 20-30g of yeast extract, 5-10g of soybean peptone and K 2 HPO 4 ·3H 2 O2-5g，MgSO 4 ·7H 2 0.5-1.5g of O, 0.3-2g of compound extract and 0.5-1.5g of soybean oil.
In some preferred embodiments of the present invention, the fermentation tank is supplemented with the complex extract in an amount of 0.5-1.5g/L fermentation broth in S01 at the time of cultivation for 90-120 h.
In some preferred embodiments of the present invention, in order to examine the growth rate of actinomadura and the conversion rate of pravastatin at different concentrations of mevastatin, the following formula was used for estimation:
wherein,
as maximum specific growth rate, h -1 ；
Is mevastatin concentration, g.L -1 ；
Is the concentration of actinomadura, g.L -1 ；
And
is a constant number of times, and is,
is pravastatin concentration, g.L -1 ；
Wherein,
the yield coefficient of Madura actinomycetes to mevastatin is g.g -1 ；
The yield coefficient of the actinomadura to pravastatin is g.g -1 ；
Through the regression analysis of the Monod model,
the content of the acid was 0.26,
the content of the acid was 0.95,
the content of the acid was 0.08,
is 0.06.
The parameters can be used for well estimating the growth of actinomadura and the transformation of pravastatin, and can be used for adjusting the concentration of proper mevastatin in fermentation liquor.
In the invention, the chicory is a plant in the cichorium of the compositaeCichoriumintybusThe low-temperature soybean meal is a commercial product and meets the standard of low-temperature edible soybean meal (GB/T21494-2008).
The beneficial technical effects of the invention are as follows:
according to the method for preparing high-stability pravastatin by using the fermentation process, the chicory extract and the soybean meal extract are added into the fermentation medium and in the fermentation process, so that the conversion rate of pravastatin can be remarkably improved.
According to the method for preparing the high-stability pravastatin by using the fermentation process, the added extract is common in raw materials, the extraction method is simple, and the cost is low.
The method for preparing high-stability pravastatin by utilizing the fermentation process can well estimate the growth of actinomadura and the transformation of pravastatin, and is used for adjusting the concentration of proper mevastatin in fermentation liquor.
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.
The experimental procedures used in the following examples and comparative examples are conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples and comparative examples are commercially available unless otherwise specified.
In the following examples and/or comparative examples, 2 volumes of ethyl acetate were added at an initial low temperature soybean meal dose, i.e., 200mL of ethyl acetate was added per 100g of low temperature soybean aqueous ethanol meal. Detecting the concentration of mevastatin in the fermentation broth every 10h during the fermentation process, and adding mevastatin to 0.5g/L if the concentration is lower than 0.4 g/L. For ease of feeding, it may be dissolved with a small amount of solvent.
Unless otherwise specified, the examples and comparative examples are parallel tests with identical components, component contents, preparation steps, preparation parameters.
Example 1
A method for preparing high-stability pravastatin by utilizing a fermentation process comprises the following steps:
(1) Preparation of chicory root extract
100g of dried chicory root is taken, crushed, soaked in 1000mL of 95% ethanol for 24h, extracted under reflux in a water bath kettle at 80 ℃ for 2h, and repeated for 2 times. Mixing filtrates, vacuum concentrating under reduced pressure, and freeze drying to obtain the extract.
(2) Preparation of a Bean pulp extract
Collecting 100g low temperature soybean meal, adding 250ml purified water, adding 750ml 95% ethanol, reflux extracting in 60 deg.C water bath for 2 hr, and repeating for 2 times. Mixing the filtrates, vacuum concentrating under reduced pressure, recovering ethanol, adding 2 times volume of ethyl acetate, vacuum concentrating the ethyl acetate phase under reduced pressure, and freeze drying to obtain the soybean meal extract.
(3) Seed culture:
inoculating the slant seeds of actinomadura in the seed culture medium, and culturing for 60h at 32 ℃ in a triangular flask shaker to obtain a seed solution.
The seed culture medium per liter comprises 30g of glucose, 20g of yeast extract, 5g of soybean peptone 2 HPO 4 ·3H 2 O2g，MgSO 4 ·7H 2 O0.5g, soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
(4) Fermentation culture
Inoculating the seed solution into the fermentation culture medium according to the volume ratio of 10%, culturing at 32 ℃ for 40h in a biological fermentation tank, adjusting the temperature to 30 ℃, continuously adding mevastatin for 180h, keeping the concentration of mevastatin in the culture medium at 0.5g/L, and ending the fermentation for 192 h. The fermentor was supplemented with 1g/L of the complex extract at 110h of incubation.
The fermentation medium per liter comprises 50g of glucose, 25g of yeast extract, 7g of soybean peptone, K 2 HPO 4 ·3H 2 O3g，MgSO 4 ·7H 2 O1g, compound extract 1g and soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
The 1g of the composite extract comprises 0.75g of chicory root extract and 0.25g of soybean meal extract.
Example 2
A method for preparing high-stability pravastatin by utilizing a fermentation process comprises the following steps:
(1) Preparation of chicory root extract
100g of dried chicory root is taken, crushed, soaked in 1000mL of 95% ethanol for 24h, extracted under reflux in a water bath kettle at 80 ℃ for 2h, and repeated for 2 times. Mixing filtrates, vacuum concentrating under reduced pressure, and freeze drying to obtain the extract.
(2) Preparation of a Bean pulp extract
Collecting 100g low temperature soybean meal, adding 250ml purified water, adding 750ml 95% ethanol, reflux extracting in 60 deg.C water bath for 2 hr, and repeating for 2 times. Mixing the filtrates, vacuum concentrating under reduced pressure, recovering ethanol, adding 2 times volume of ethyl acetate, vacuum concentrating the ethyl acetate phase under reduced pressure, and freeze drying to obtain the soybean meal extract.
(3) Seed culture:
inoculating the slant seeds of actinomadura in the seed culture medium, and culturing for 60h at 32 ℃ in a triangular flask shaker to obtain a seed solution.
The seed culture medium per liter comprises 30g of glucose, 20g of yeast extract, 5g of soybean peptone 2 HPO 4 ·3H 2 O2g，MgSO 4 ·7H 2 O0.5g, soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
(4) Fermentation culture
Inoculating the seed solution into the fermentation culture medium according to the volume ratio of 10%, culturing at 32 deg.C for 40h in a biological fermentation tank, adjusting the temperature to 30 deg.C, continuously adding mevastatin for 180h, keeping the concentration of mevastatin in the culture medium at 0.5g/L, and finishing the fermentation within 192 h. The fermentor was supplemented with 1g/L of the complex extract at 110h of incubation.
The fermentation medium per liter comprises 50g of glucose, 25g of yeast extract, 7g of soybean peptone, K 2 HPO 4 ·3H 2 O3g，MgSO 4 ·7H 2 O1g, compound extract 1g and soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
The 1g of the compound extract comprises 0.50g of chicory root extract and 0.50g of soybean meal extract.
Example 3
A method for preparing high-stability pravastatin by utilizing a fermentation process comprises the following steps:
(1) Preparation of chicory root extract
100g of dried chicory root is taken, crushed, soaked in 1000mL of 95% ethanol for 24h, extracted under reflux in a water bath kettle at 80 ℃ for 2h, and repeated for 2 times. Mixing filtrates, vacuum concentrating under reduced pressure, and freeze drying to obtain the extract.
(2) Preparation of a Bean pulp extract
Collecting 100g low temperature soybean meal, adding 250ml purified water, adding 750ml 95% ethanol, reflux extracting in 60 deg.C water bath for 2 hr, and repeating for 2 times. Mixing the filtrates, vacuum concentrating, recovering ethanol, adding 2 times volume of ethyl acetate, vacuum concentrating, and freeze drying to obtain the soybean meal extract.
(3) Seed culture:
inoculating the slant seeds of actinomadura in the seed culture medium, and culturing for 60h at 32 ℃ in a triangular flask shaker to obtain a seed solution.
The seed culture medium per liter comprises 30g of glucose, 20g of yeast extract, 5g of soybean peptone 2 HPO 4 ·3H 2 O2g，MgSO 4 ·7H 2 O0.5g, soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
(4) Fermentation culture
Inoculating the seed solution into the fermentation culture medium according to the volume ratio of 10%, culturing at 32 deg.C for 40h in a biological fermentation tank, adjusting the temperature to 30 deg.C, continuously adding mevastatin for 180h, keeping the concentration of mevastatin in the culture medium at 0.5g/L, and finishing the fermentation within 192 h. The fermentor was supplemented with 1g/L of the complex extract at 110h of incubation.
The fermentation medium per liter comprises 50g of glucose, 25g of yeast extract, 7g of soybean peptone, K 2 HPO 4 ·3H 2 O3g，MgSO 4 ·7H 2 O1g, compound extract 1g and soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
The 1g of the composite extract comprises 0.25g of chicory root extract and 0.75g of soybean meal extract.
Example 4
A method for preparing high-stability pravastatin by utilizing a fermentation process comprises the following steps:
(1) Preparation of chicory root extract
100g of dried chicory root is taken, crushed, soaked in 1000mL of 95% ethanol for 24h, extracted under reflux in a water bath kettle at 80 ℃ for 2h, and repeated for 2 times. Mixing filtrates, vacuum concentrating under reduced pressure, and freeze drying to obtain the extract.
(2) Preparation of a Bean pulp extract
Collecting 100g low temperature soybean meal, adding 250ml purified water, adding 750ml 95% ethanol, reflux extracting in 60 deg.C water bath for 2 hr, and repeating for 2 times. Mixing the filtrates, vacuum concentrating under reduced pressure, recovering ethanol, adding 2 times volume of ethyl acetate, vacuum concentrating the ethyl acetate phase under reduced pressure, and freeze drying to obtain the soybean meal extract.
(3) Seed culture:
inoculating the slant seeds of actinomadura in the seed culture medium, and culturing for 60h at 32 ℃ in a triangular flask shaker to obtain a seed solution.
The seed culture medium per liter comprises 30g of glucose, 20g of yeast extract, 5g of soybean peptone 2 HPO 4 ·3H 2 O2g，MgSO 4 ·7H 2 O0.5g, soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
(4) Fermentation culture
Inoculating the seed solution into the fermentation culture medium according to the volume ratio of 10%, culturing at 32 deg.C for 40h in a biological fermentation tank, adjusting the temperature to 30 deg.C, continuously adding mevastatin for 180h, keeping the concentration of mevastatin in the culture medium at 0.5g/L, and finishing the fermentation within 192 h. The fermentor was supplemented with 1g/L of the complex extract at 110h of incubation.
The fermentation medium per liter comprises 50g of glucose, 25g of yeast extract, 7g of soybean peptone, K 2 HPO 4 ·3H 2 O3g，MgSO 4 ·7H 2 O1g, compound extract 0.8g and soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
The 1g of the composite extract comprises 0.75g of chicory root extract and 0.25g of soybean meal extract.
Example 5
A method for preparing high-stability pravastatin by utilizing a fermentation process comprises the following steps:
(1) Preparation of chicory root extract
100g of dried chicory root is taken, crushed, soaked in 1000mL of 95% ethanol for 24h, extracted under reflux in a water bath kettle at 80 ℃ for 2h, and repeated for 2 times. Mixing filtrates, vacuum concentrating under reduced pressure, and freeze drying to obtain the extract.
(2) Preparation of a Bean pulp extract
Collecting 100g low temperature soybean meal, adding 250ml purified water, adding 750ml 95% ethanol, reflux extracting in 60 deg.C water bath for 2 hr, and repeating for 2 times. Mixing the filtrates, vacuum concentrating under reduced pressure, recovering ethanol, adding 2 times volume of ethyl acetate, vacuum concentrating the ethyl acetate phase under reduced pressure, and freeze drying to obtain the soybean meal extract.
(3) Seed culture:
inoculating the slant seeds of actinomadura in the seed culture medium, and culturing for 60h at 32 ℃ in a triangular flask shaker to obtain a seed solution.
The seed culture medium per liter comprises 30g of glucose, 20g of yeast extract, 5g of soybean peptone 2 HPO 4 ·3H 2 O2g，MgSO 4 ·7H 2 O0.5g, soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
(4) Fermentation culture
Inoculating the seed solution into the fermentation culture medium according to the volume ratio of 10%, culturing at 32 ℃ for 40h in a biological fermentation tank, adjusting the temperature to 30 ℃, continuously adding mevastatin for 180h, keeping the concentration of mevastatin in the culture medium at 0.5g/L, and ending the fermentation for 192 h. The fermentor was supplemented with 1g/L of the complex extract at 110h of incubation.
The fermentation medium per liter comprises glucose 50g, yeast extract 25g, soybean peptone 7g, K 2 HPO 4 ·3H 2 O3g，MgSO 4 ·7H 2 O1g, compound extract 1.5g and soybean oil 1.0g. Sterilizing at 121 deg.C for 20min.
The 1g of the compound extract comprises 0.75g of chicory root extract and 0.25g of soybean meal extract.
Comparative example 1
The difference from example 1 is that the chicory root extract of the fermentation medium is replaced by a chicory stem extract, wherein the chicory stem extract is extracted in the same way as the chicory root extract.
Comparative example 2
The difference from example 1 is that the material of the ethanol phase (i.e., remaining after separating the ethyl acetate phase) was concentrated under reduced pressure in vacuo and then freeze-dried to obtain the soybean meal extract.
Comparative example 3
The difference from example 1 is that the fermentation medium does not contain complex extracts.
Examples of the experiments
The fermentation-terminated culture liquids of examples and comparative examples were taken, and the contents of mevastatin and pravastatin therein were measured to calculate the conversion rate. Conversion = amount of pravastatin/amount of mevastatin × 100%. The results are shown in Table 1.
TABLE 1 Effect of Complex extracts on conversion
In the same column of data, different lower case letters represent significant differences, and P is less than 0.05
As can be seen from Table 1, the conversion rates of examples 1-5, in which the chicory root extract and the soybean meal extract were added, were significantly better than those of comparative example 3, in which no chicory root extract and soybean meal extract were added, indicating the effect of the chicory root extract and the soybean meal extract on the conversion rates. Of examples 1-3, example 1 was optimal, showing the significant effect of different ratios of chicory root extract and soybean meal extract on conversion. Compared with the comparative example 3, the conversion rate of the comparative example 2 is not significantly different, and compared with the example 1, the conversion rate of the comparative example 2 is significantly different, which shows the important influence of the soybean meal extraction solvent. Compared with example 1 and comparative example 3, the conversion rate of comparative example 1 is significantly different, which shows that the chicory root extract and the chicory stem extract are not influenced by the source.
Taking the actinomadura actinomyces hyphae which are finished to ferment in the examples and the comparative examples, washing with distilled water, centrifuging to obtain wet thalli, and counting the biomass, wherein the results show that the biomass of the examples 1-5 is not statistically significantly different from that of the comparative example 1, which indicates that the effect of the chicory root extract and the soybean meal extract on the conversion rate is not based on the actinomadura actinomyces biomass.
While the preferred embodiments and examples of the present invention have been described in detail, the present invention is not limited to the embodiments and examples, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.
Claims (6)
1. A method for preparing high-stability pravastatin by utilizing a fermentation process is characterized by comprising the following steps:
s01, culturing actinomadura strain seeds;
s02, fermenting actinomadura;
in the S02 actinomadura fermentation, a fermentation medium contains a compound extract, and the compound extract comprises a chicory extract and a soybean meal extract;
the chicory extract is a chicory root extract or a chicory stem extract;
the preparation method of the chicory extract comprises the steps of extracting by using ethanol water;
the preparation method of the soybean meal extract comprises the steps of firstly extracting by using an ethanol water solution, and then extracting by using ethyl acetate;
the weight ratio of the chicory extract to the soybean meal extract in the composite extract is (1-5): (1-5);
in S01, each liter of seed culture medium comprises 25-35g of glucose, 15-25g of yeast extract, 3-8g of soybean peptone 2 HPO 4 ·3H 2 O1-3g，MgSO 4 ·7H 2 0.5-1g of O and 0.5-1.5g of soybean oil;
in S02, each liter of fermentation medium comprises 45-55g of glucose, 20-30g of yeast extract, 5-10g of soybean peptone, and K 2 HPO 4 ·3H 2 O2-5g，MgSO 4 ·7H 2 0.5-1.5g of O, 0.3-2g of compound extract and 0.5-1.5g of soybean oil.
2. The method of preparing pravastatin with high stability through fermentation according to claim 1, further comprising the step of supplementing the complex extract during the fermentation of S02 actinomadura.
3. The method for preparing high-stability pravastatin by fermentation process according to claim 1 or 2, wherein the content of the complex extract in the fermentation medium in S02 is 0.3-2g/L;
and/or, in S02, adding the compound extract after fermentation is carried out for 50-80h, wherein the adding amount is 0.3-2g/L of culture solution.
4. The method for preparing pravastatin with high stability by fermentation process according to claim 1 or 2, wherein in S01, slant seeds of actinomadura are inoculated to the seed culture medium, and the mixture is cultured for 55-65h at 30-34 ℃ in a triangular flask shaker to obtain a seed solution.
5. The method for preparing high-stability pravastatin by a fermentation process as claimed in claim 1 or 2, wherein the fermentation medium is inoculated with the seed solution at a volume ratio of 5-15% in S02, the mixture is cultured at 31-34 ℃ for 35-45 hours in a biological fermentation tank, the temperature is adjusted to 28-30 ℃, mevastatin is continuously added to 170-185 hours, the concentration of mevastatin in the medium is maintained at 0.3-0.8g/L, and the fermentation is terminated within 190-200 hours.
6. The method for preparing pravastatin with high stability through fermentation according to claim 1 or 2, wherein the fermentation tank is supplemented with the complex extract in an amount of 0.5 to 1.5g/L of the fermentation broth in S01 during the cultivation for 90 to 120 hours.
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| US5942423A (en) * | 1995-06-07 | 1999-08-24 | Massachusetts Institute Of Technology | Conversion of compactin to pravastatin by actinomadura |
| EP1452602A1 (en) * | 2003-02-25 | 2004-09-01 | Antibiotic Co., | Method for production of pravastatin by fermentation |
| TWI307360B (en) * | 2004-12-03 | 2009-03-11 | Teva Gyogyszergyar Zartkoruen Mukodo Reszvenytarsasag | Process for constructing strain having compactin hydroxylation ability |
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| CN101524107A (en) * | 2008-03-07 | 2009-09-09 | 马俊 | Chicory coffee beverage and method for producing chicory extract |
| CN102757986B (en) * | 2011-04-27 | 2014-09-03 | 广东蓝宝制药有限公司 | Fermentation process for producing pravastatin by transforming compactin by using actinomadura yumaense |
| CN105821086B (en) * | 2016-05-05 | 2017-03-22 | 广东蓝宝制药有限公司 | Process for producing pravastatin on large scale |
| CN108070623A (en) * | 2016-11-10 | 2018-05-25 | 北大方正集团有限公司 | A kind of method for improving Pravastatin fermentation yield |
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| US20220049200A1 (en) * | 2018-12-10 | 2022-02-17 | Sung Min Lee | Apparatus for producing fermented soybean meal |
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