CN110804557A - Culture method and culture medium of irpex cacteus - Google Patents
Culture method and culture medium of irpex cacteus Download PDFInfo
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- CN110804557A CN110804557A CN201911226565.0A CN201911226565A CN110804557A CN 110804557 A CN110804557 A CN 110804557A CN 201911226565 A CN201911226565 A CN 201911226565A CN 110804557 A CN110804557 A CN 110804557A
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- 239000001963 growth medium Substances 0.000 title claims abstract description 65
- 241000222342 Irpex Species 0.000 title claims abstract description 35
- 238000012136 culture method Methods 0.000 title abstract description 8
- 241000222344 Irpex lacteus Species 0.000 claims abstract description 12
- 238000012258 culturing Methods 0.000 claims abstract description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 40
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 36
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 28
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 21
- 229910052799 carbon Inorganic materials 0.000 claims description 21
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 21
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 21
- 239000002609 medium Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 18
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
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- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 13
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 13
- 239000002775 capsule Substances 0.000 description 13
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- 241001619412 Meruliaceae Species 0.000 description 1
- 206010030302 Oliguria Diseases 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- 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
- C12R2001/645—Fungi ; Processes using fungi
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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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
- C12N1/14—Fungi; Culture media therefor
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- 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
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a culture method and a culture medium of irpex cacteus. The invention firstly provides a culture method and a culture medium for Irpex lacteus (PAX-2014-1). The culture obtained by culturing the irpex cacteus PAX-2014-1 can be used for preparing products. The function of the product is as follows: treatment and/or prevention of gout; treatment and/or prevention of hyperuricemia; treatment and/or prevention of chronic glomerulonephritis; treatment and/or prevention of glomerulonephritis; treating and/or preventing nephritis. The invention has great application prospect.
Description
The application is a divisional application with the application number of 201611079486.8, the application date of 2016, 11 and 30, and the name of the invention of a strain of irpex cacteus and a culture method and application thereof.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method and a culture medium of irpex cacteus.
Background
Irpex lacteus (Fr.) Fr. ], also known as Irpex lacteus, belongs to the order polyporales, the family of porpeziaceae (Meruliaceae), the genus Irpex, grows on standing, withered or fallen broad-leaved trees, and can cause white decay of wood, with annual, constant lying down and rolling back, and occasionally lateral growth.
The irpex cacteus is one of medicinal fungi in China, has the efficacy of treating symptoms such as oliguria, edema, lumbago, blood pressure increase and the like, and has anti-inflammatory activity. Moreover, because the harringtonia denticulata contains degradable enzymes such as ligninase, cellulase, laccase and the like and has higher growth speed, the harringtonia denticulata is widely applied to industries such as industrial papermaking, paper pulp modification, papermaking sewage treatment, printing and dyeing sewage treatment, beverage industry, clothing industry, organic synthesis and the like, and is used for treating and repairing the pollution of heavy metals.
Disclosure of Invention
The invention aims to provide a culture method and a culture medium for irpex cacteus.
The invention firstly provides Irpex lacteus (PAX-2014-1). Irpexlateus (Irpexlateus) PAX-2014-1, which has been deposited in China general microbiological culture Collection center (CGMCC, No. 3 of the institute of microbiology, China academy of sciences, North West Lu No.1 of the south Kogyo, Beijing) on 29 th 6 th in 2016, with the collection registration number of CGMCC NO. 12519. Ralstonia alba (Irpex lacteus) PAX-2014-1CGMCC NO.12519, which is called Ralstonia alba PAX-2014-1 for short.
The invention also protects a culture medium for culturing the irpex cacteus PAX-2014-1, which is a culture medium I or a culture medium II. The culture medium I comprises a carbon source, a nitrogen source, calcium carbonate, potassium dihydrogen sulfate, magnesium sulfate and water. The carbon-nitrogen ratio of the culture medium I is 3-15: 1. The carbon-nitrogen ratio of the culture medium I can be specifically 6.67: 1. 12.3: 1 or 9.23: 1. the culture medium II comprises a carbon source, a nitrogen source, a growth factor, potassium dihydrogen sulfate, magnesium sulfate and water. The carbon-nitrogen ratio of the culture medium II is 3-15: 1. The carbon-nitrogen ratio of the culture medium II can be specifically 6.67: 1. 12.3: 1 or 9.23: 1.
the culture medium I can be prepared from the following raw materials per liter: 45-60 g of carbon source, 20-35 g of nitrogen source, 0.5-0.6 g of calcium carbonate, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water. The culture medium I can be prepared from the following raw materials per liter: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of calcium carbonate, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water.
The culture medium II can be prepared from the following raw materials per liter: 45-60 g of carbon source, 20-35 g of nitrogen source, 0.5-0.6 g of growth factor, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water. The culture medium II can be prepared from the following raw materials per liter: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of growth factor, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water.
The carbon source may specifically be soluble starch and/or glucose.
The nitrogen source may be specifically peptone and/or yeast extract.
The culture medium I can be specifically a culture medium A, a culture medium B or a culture medium C.
The culture medium A per liter can be composed of the following raw materials: 60.0 g of soluble starch, 20.0 g of peptone, 15.0 g of yeast extract, 0.6 g of calcium carbonate, 2.0 g of monopotassium phosphate, 1.5 g of magnesium sulfate and the balance of water. The pH is natural.
The culture medium B per liter can be composed of the following raw materials: 60.0 g of glucose, 15.0 g of peptone, 5.0 g of yeast extract, 0.5 g of calcium carbonate, 1.0 g of monopotassium phosphate, 0.5 g of magnesium sulfate and the balance of water. The pH is natural.
The culture medium per liter can be specifically composed of the following raw materials: 45.0 g of glucose, 15.0 g of peptone, 5.0 g of yeast extract, 0.5 g of calcium carbonate, 1.0 g of monopotassium phosphate, 0.5 g of magnesium sulfate and the balance of water. The pH is natural.
The invention also provides a method for culturing the irpex cacteus PAX-2014-1, which comprises the following steps: the culture medium is adopted to culture the irpex cacteus PAX-2014-1.
The method specifically comprises the following steps:
(1) inoculating single colony of the irpex cacteus PAX-2014-1 on a PDA culture medium plate, and standing and culturing for 5 days at 30 ℃;
(2) the cake of 6 mm in diameter was punched out from the plate on which step (1) was performed with a punch, and 1L of the medium was inoculated with 12 cakes and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
The culture medium provided by the invention has clear components. The culture medium provided by the invention for producing the irpex cacteus mycelium has the advantages of simple process, no heavy metal pollution, high thallus growth speed, high thallus yield, stable product quality and the like.
The invention also provides a preparation method of the extract of the irpex cacteus PAX-2014-1, which is characterized by comprising the following steps: the method comprises the following steps of taking the irpex cacteus PAX-2014-1 as a raw material:
(a1) water extraction;
(a2) precipitating with ethanol;
(a3) and collecting the precipitate.
The water extraction condition in the step (a1) can be specifically 90 ℃ standing extraction, the water extraction condition in the step (a1) can be specifically 90 ℃ standing extraction for 2-6 hours, the proportion of the harpagophytum pectiniferum PAX-2014-1 to water in the step (a1) can be 30-90 ml of water, and the step (a1) specifically comprises the steps of taking ① g of harpagophytum pectiniferum PAX-2014-1 (wet weight), adding 30 ml of water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ② taking the residual thallus in the step ①, adding 30 ml of water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ③ taking the residual thallus in the step ②, adding 30 ml of distilled water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ④, combining the supernatant obtained in the step 4 and the step ②, and concentrating the supernatant into a third of the concentrated supernatant, wherein the concentrated supernatant is obtained in the step ③.
In the step (a2), the ethanol precipitation may be ethanol precipitation. In the step (a2), the alcohol precipitation condition may be specifically 4 ℃. In the step (a2), the alcohol precipitation condition may be specifically that the solution is allowed to stand at 4 ℃ for 24 hours. In the step (a2), the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1: 3. the step (b3) specifically comprises the following steps: 1 part by volume of the concentrate obtained in step (a1) was mixed with 3 parts by volume of absolute ethanol, and the mixture was allowed to stand at 4 ℃ for 24 hours.
In the (a3), the precipitate was collected by centrifugation. The centrifugation conditions may specifically be: centrifuge at 8000rpm for 30 min.
The method further comprises the steps of: freeze-drying the precipitate obtained in (a3) to obtain a lyophilized powder.
The invention also provides a preparation method of the extract of the irpex cacteus PAX-2014-1, which sequentially comprises the following steps:
(b1) adopting any one of the culture media to culture the irpex cacteus PAX-2014-1, and then collecting thallus precipitates;
(b2) water extraction;
(b3) precipitating with ethanol;
(b4) and collecting the precipitate.
The step (b1) specifically comprises the following steps:
(1) inoculating single colony of the irpex cacteus PAX-2014-1 on a PDA culture medium plate, and standing and culturing for 5 days at 30 ℃;
(2) the cake of 6 mm in diameter was punched out from the plate on which step (1) was performed with a punch, and 1L of the medium was inoculated with 12 cakes and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
(3) After the step (2) is finished, taking the whole culture system, centrifuging at 6000rpm for 20min, and collecting thalli precipitates.
The water extraction condition in the step (b2) can be specifically that the water extraction condition is 90 ℃ standing extraction, the water extraction condition in the step (b2) can be specifically that the water extraction condition is 90 ℃ standing extraction for 2-6 hours, the ratio of the thallus precipitate to water in the step (b2) can be 1 g of thallus precipitate (wet weight) and 30-90 ml of water, the step (b2) specifically comprises the steps of ① taking n g of the thallus precipitate, adding 30 n ml of water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ② taking the thallus remaining in the step ①, adding 30 n ml of water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ③ taking the thallus remaining in the step ②, adding 30 n ml of distilled water, standing extraction for 2 hours at 90 ℃, collecting supernatant, ④ combining the supernatant obtained in the step ①, the supernatant obtained in the step ② and the supernatant obtained in the step ③, then concentrating the mixture under reduced pressure to obtain a third volume of positive concentrated solution.
In the step (b3), the alcohol precipitation may be ethanol precipitation. In the step (b3), the alcohol precipitation condition may be specifically 4 ℃. In the step (b3), the alcohol precipitation condition may be specifically that the solution is allowed to stand at 4 ℃ for 24 hours. In the step (b3), the volume ratio of the concentrated solution to the absolute ethyl alcohol is 1: 3. the step (b3) specifically comprises the following steps: taking 1 volume part of the concentrated solution obtained in the step (b2), mixing with 3 volume parts of absolute ethyl alcohol, and standing at 4 ℃ for 24 hours.
In the (b4), the precipitate was collected by centrifugation. The centrifugation conditions may specifically be: centrifuge at 8000rpm for 30 min.
The method further comprises the steps of: and (c) freeze-drying the precipitate obtained in the step (b4) to obtain freeze-dried powder.
The extract prepared by any method is also in the protection scope of the invention.
The invention also protects a product, the active ingredients of which are as follows (c1) or (c 2):
(c1) harpagophytum procumbens PAX-2014-1;
(c2) any of the above extracts.
The function of the product is as follows (d1) and/or (d2) and/or (d3) and/or (d4) and/or (d 5):
(d1) treatment and/or prevention of gout;
(d2) treatment and/or prevention of hyperuricemia;
(d3) treatment and/or prevention of chronic glomerulonephritis;
(d4) treatment and/or prevention of glomerulonephritis;
(d5) treating and/or preventing nephritis.
The invention also protects the application of the irpex cacteus PAX-2014-1 or any one of the extracts in the preparation of products.
The function of the product is as follows (d1) and/or (d2) and/or (d3) and/or (d4) and/or (d 5):
(d1) treatment and/or prevention of gout;
(d2) treatment and/or prevention of hyperuricemia;
(d3) treatment and/or prevention of chronic glomerulonephritis;
(d4) treatment and/or prevention of glomerulonephritis;
(d5) treating and/or preventing nephritis.
The product is a medicine or a health product.
The invention provides a new strain of irpex cacteus, develops and optimizes a culture medium for culturing the new strain and prepares an extract of the strain. The extract of the irpex cacteus PAX-2014-1 has good curative effect on various diseases, has low side effect and has great application prospect.
Drawings
FIG. 1 is a photograph of strain PAX-2014-1 after 5 days of culture on potato dextrose agar medium at 25 ℃.
FIG. 2 is a photograph under a microscope of the hyphae of the strain PAX-2014-1.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Control medium: cutting 200g of potato into blocks, boiling, filtering with gauze, collecting filtrate, adding 20g of glucose into the filtrate, and diluting with distilled water to 1000 ml.
PDA culture medium: 200g of potato is cut into blocks and boiled, filtered by gauze and the filtrate is collected, 20g of glucose and 20g of agar are added into the filtrate, and the volume is adjusted to 1000ml by distilled water.
Yishenkang capsule: changchun Yishenkang biopharmaceutical Co., Ltd.
The control irpex cacteus is preserved in China general microbiological culture Collection center with the preservation registration number of CGMCC NO. 1016. The control Rapex cacteus is described in patent application No. 201210461164.5 (the publication number of the patent is CN 102935095B; publication date is 2013-11-27).
Example 1 preparation of Rapex cacteus PAX-2014-1
First, isolation of the Strain
In 2014, a specimen of the irpex cacteus is collected from the Changbai mountain area of China, and then strains are separated through tissue blocks to obtain a plurality of pure cultured strains, wherein one strain is named as the strain PAX-2014-1.
II, identification of the strains
Morphological characteristics of the strain PAX-2014-1: the bacterial colony is white, thin and slightly raised cotton flocculent; the whole colony surface is radially arranged with dense cluster and feather aerial mycelium with interval and transparent mycelium with diameter of 1.2-4 μm and no locking combination. The photograph of the strain PAX-2014-1 after 5 days of culture on potato dextrose agar medium at 25 ℃ is shown in FIG. 1, which is a full plate with a diameter of 9 cm. The microscopic photograph of the hyphae of strain PAX-2014-1 is shown in FIG. 2.
The ITS sequence of the strain PAX-2014-1 is shown in the sequence 1 of the sequence table.
Third, preservation of the Strain
Irpex lacteus (PAX-2014-1) has been deposited in China general microbiological culture Collection center (CGMCC, No. 3 of the institute of microbiology, China academy of sciences) at 29.6.2016 under the China Committee for culture Collection of microorganisms, with the accession number of CGMCC No. 12519. Ralstonia alba (Irpex lacteus) PAX-2014-1CGMCC NO.12519, which is called Ralstonia alba PAX-2014-1 for short.
Example 2 culture of Rapex cacteus PAX-2014-1
Firstly, utilizing the carbon-nitrogen ratio of 6: 1 production of Rapex cacteus powder by Rapex cacteus semisynthetic culture medium
Medium a (natural pH): 60.0 g of soluble starch, 20.0 g of peptone, 15.0 g of yeast extract, 0.6 g of calcium carbonate, 2.0 g of monopotassium phosphate and 1.5 g of magnesium sulfate are taken, dissolved by distilled water and then the volume is fixed to 1000ml by distilled water. The carbon content in the starch is 40% (mass percentage content), the nitrogen content in the peptone is 9% (mass percentage content), and the nitrogen content in the yeast extract is 12% (mass percentage content). The mass ratio of carbon to nitrogen in the medium A is calculated to be 6.67: 1.
1. the single colony of the irpex cacteus PAX-2014-1 is inoculated on a PDA culture medium plate and is statically cultured for 5 days at the temperature of 30 ℃.
2. The 6 mm diameter cake was punched out from the plate for completion of step 1 with a punch, and inoculated into 1L of Medium A (or control medium) for 12 cakes, and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
3. After the step 2 is completed, the whole culture system is taken, centrifuged at 6000rpm for 20min, and thalli precipitates are collected.
4. And (4) freeze-drying the thallus precipitate obtained in the step (3) to obtain freeze-dried powder.
When the culture medium A is adopted, 15.25 g of freeze-dried powder is obtained per liter of culture system (average value of five repeated experiments).
Using the control medium, 3.83 g of lyophilized powder per liter of culture system were obtained (average of five replicates).
Secondly, utilizing the carbon-nitrogen ratio of 12: 1 production of Rapex cacteus powder by Rapex cacteus semisynthetic culture medium
And (3) culture medium B: 60.0 g of glucose, 15.0 g of peptone, 5.0 g of yeast extract, 0.5 g of calcium carbonate, 1.0 g of monopotassium phosphate and 0.5 g of magnesium sulfate are taken, dissolved by distilled water and then the volume is fixed to 1000ml by distilled water. The carbon content in glucose was 40% (mass percentage content), the nitrogen content in peptone was 9% (mass percentage content), and the nitrogen content in yeast extract was 12% (mass percentage content). The mass ratio of carbon to nitrogen in the culture medium B is calculated to be 12.3: 1.
1. the single colony of the irpex cacteus PAX-2014-1 is inoculated on a PDA culture medium plate and is statically cultured for 5 days at the temperature of 30 ℃.
2. The 6 mm diameter cake was punched out from the plate after completion of step 1 with a punch, and inoculated into 1L of Medium B (or control medium) for 12 cakes, and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
3. After the step 2 is completed, the whole culture system is taken, centrifuged at 6000rpm for 20min, and thalli precipitates are collected.
4. And (4) freeze-drying the thallus precipitate obtained in the step (3) to obtain freeze-dried powder.
When the culture medium B is adopted, 15.36 g of freeze-dried powder is obtained per liter of culture system (average value of five repeated experiments).
Using the control medium, 3.83 g of lyophilized powder per liter of culture system were obtained (average of five replicates).
Thirdly, utilizing the carbon-nitrogen ratio of 9: 1 producing the irpex cacteus powder by the irpex cacteus semisynthetic culture medium
Culture medium C: taking 45.0 g of glucose, 15.0 g of peptone, 5.0 g of yeast extract, 0.5 g of calcium carbonate, 1.0 g of monopotassium phosphate and 0.5 g of magnesium sulfate, dissolving with distilled water, and then fixing the volume to 1000ml with distilled water. The carbon content in glucose was 40% (mass percentage content), the nitrogen content in peptone was 9% (mass percentage content), and the nitrogen content in yeast extract was 12% (mass percentage content). The mass ratio of carbon to nitrogen in the culture medium C is calculated to be 9.23: 1.
1. the single colony of the irpex cacteus PAX-2014-1 is inoculated on a PDA culture medium plate and is statically cultured for 5 days at the temperature of 30 ℃.
2. The 6 mm diameter cake was punched out from the plate after completion of step 1 using a punch, and inoculated into 1L of medium C (or control medium) for 12 cakes, and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
3. After the step 2 is completed, the whole culture system is taken, centrifuged at 6000rpm for 20min, and thalli precipitates are collected.
4. And (4) freeze-drying the thallus precipitate obtained in the step (3) to obtain freeze-dried powder.
Using medium C, 16.27 g of lyophilized powder per liter of culture system were obtained (average of five replicates).
Using the control medium, 3.83 g of lyophilized powder per liter of culture system were obtained (average of five replicates).
Example 3 preparation of capsules from Rapex cacteus PAX-2014-1
Culture medium C: taking 45.0 g of glucose, 15.0 g of peptone, 5.0 g of yeast extract, 0.5 g of calcium carbonate, 1.0 g of monopotassium phosphate and 0.5 g of magnesium sulfate, dissolving with distilled water, and then fixing the volume to 1000ml with distilled water.
1. The single colony of the irpex cacteus PAX-2014-1 is inoculated on a PDA culture medium plate and is statically cultured for 5 days at the temperature of 30 ℃.
2. The 6 mm diameter cake was punched out from the plate for completion of step 1 with a punch, and inoculated to 1L of medium C for 12 cakes, and cultured at 30 ℃ for 72 hours with shaking at 150 rpm.
3. After the step 2 is completed, the whole culture system is taken, centrifuged at 6000rpm for 20min, and thalli precipitates are collected.
4. The bacterial pellet obtained in step 3 (wet weight: n g, n is positive number) was taken, 30 × n ml of distilled water was added, and the mixture was allowed to stand at 90 ℃ for 2 hours to extract, and the supernatant was collected.
5. And (4) adding 30 x n milliliters of distilled water into the residual thallus in the step (4), standing and extracting for 2 hours at 90 ℃, and collecting supernatant.
6. And (3) adding 30 x n milliliters of distilled water into the residual thallus in the step (5), standing and extracting for 2 hours at 90 ℃, and collecting supernatant.
7. And (3) combining the supernatant obtained in the step (4), the supernatant obtained in the step (5) and the supernatant obtained in the step (6), and then concentrating under reduced pressure to one third of the volume to obtain a concentrated solution.
8. And (3) mixing 1 volume part of the concentrated solution obtained in the step (7) with 3 volume parts of absolute ethyl alcohol, standing at 4 ℃ for 24 hours, centrifuging at 8000rpm for 30min, collecting precipitates, and freeze-drying to obtain freeze-dried powder.
9. And (3) filling 0.3g of the freeze-dried powder obtained in the step (8) into each capsule shell to obtain the PAX-2014-1 capsule.
And (3) replacing the irpex cacteus PAX-2014-1 with the control irpex cacteus to carry out the steps to obtain the control capsule.
Example 4 application of Rapex cacteus PAX-2014-1 in treating gout and hyperuricemia
24 patients with hyperuricemia complicated with gout (male 16 cases, female 8 cases), 4 patients with gout without hyperuricemia (male 2 cases, female 2 cases), and 4 patients with hyperuricemia without gout (all males). All the patients were informed consenting volunteers diagnosed in the third hospital. Of the patients, 22 men and 10 women had an age of 33-78 years and a course of disease of 5 months-6 years. The treatment process has eliminated: 1) pregnant women; 2) those allergic to the drug and any component thereof; 3) patients with serious primary diseases of cardiovascular, liver, hematopoietic system, etc.; 4) HIV patients and other severely immunocompromised persons; 5) psychotic patients; 6) it is not adhered to taking medicine.
The patients were treated as follows: the PAX-2014-1 capsule prepared in example 3 is taken 3 times a day for 3 times each time, and the capsule is continuously taken for 60 days.
After completion of the treatment, evaluation of the treatment effect and evaluation of side effects were carried out.
The criteria for the evaluation of the therapeutic effect were as follows:
the healing process comprises the steps of completely eliminating the pain, red swelling and other symptoms of ① diseased parts, enabling the uric acid content in ② serum to reach the health standard, and judging the healing process to be complete when the uric acid content in the 3526 serum meets the ① and ②;
① has obvious effects that compared with the prior treatment, the symptoms such as pain, red swelling and the like of the diseased part are obviously improved, the uric acid content in ② serum does not reach the health standard, but the reduced value of the uric acid content in the serum is more than or equal to 20 mu mol/L compared with the prior treatment, the pain of the ③ diseased part completely disappears, the uric acid content in the serum is not increased compared with the prior treatment, and the condition that ③ is met or ① and ② are met simultaneously is judged to be the obvious effect;
① has obviously improved symptoms such as pain, red swelling and the like of the diseased part compared with before treatment, ② has no obvious reduction and no increase of the content of uric acid in the serum compared with before treatment, and the results of ① and ② are met to judge that the traditional Chinese medicine is effective;
①, compared with before treatment, the symptoms of pain, red swelling and the like of the diseased part are not obviously improved or aggravated, ② compared with before treatment, the content of uric acid in blood serum is not obviously reduced or increased, and the conditions of ① and ② are judged to be ineffective;
health standards for uric acid content in serum: the content of uric acid in the blood serum of the male is 150-440 mu mol/L; the content of uric acid in the serum of a female is 95-360 mu mol/L.
The criteria for the evaluation of side effects were as follows: dry mouth and/or stomach discomfort and/or nausea.
The mean values of uric acid content, creatinine content and urea nitrogen content in the patient's serum before treatment (day before treatment started), after treatment (day after treatment ended) are shown in table 1.
TABLE 1
| Before treatment | After treatment | |
| Uric acid (mu mol/L) | 569,+/-182 | 512,+/-105 |
| Creatinine (mu mol/L) | 327.6,+/-114.6 | 238.5,+/-79.2 |
| Urea nitrogen (mmol/L) | 9.79,+/-5.81 | 7.75,+/-3.88 |
Of the 32 patients: 3 cases are cured, 14 cases are obviously effective, 11 cases are effective, 4 cases are ineffective, and the total effective rate is 87.5 percent; 17 cases in which pain completely disappeared at the affected site, and 28 cases in which the overall symptoms were significantly improved; 5 cases with dry mouth side effects, 4 cases with stomach upset side effects, 0 case with nausea side effects.
The results show that the irpex cacteus PAX-2014-1 extract can obviously improve the pain and red swelling symptoms of patients with hyperuricemia and/or gout, reduce the uric acid content, the creatinine content and the urea nitrogen content in blood serum and has low side effect.
Example 5 application of Rapex cacteus PAX-2014-1 in treatment of chronic glomerulonephritis
A first group: 129 patients with chronic glomerulonephritis (69 men and 60 women). Second group: 132 patients with chronic glomerulonephritis (64 men and 68 women). Third group: 132 patients with chronic glomerulonephritis (62 men and 70 women). All the patients were informed consenting volunteers diagnosed in the third hospital. The treatment process has eliminated: 1) pregnant women; 2) those allergic to the drug and any component thereof; 3) patients with serious primary diseases of cardiovascular, liver, hematopoietic system, etc.; 4) HIV patients and other severely immunocompromised persons; 5) psychotic patients; 6) it is not adhered to taking medicine.
Two groups of patients were treated as follows:
a first group: the PAX-2014-1 capsule prepared in example 3 is taken 2 capsules each time, 3 times a day and continuously for 30 days.
Second group: the control capsules prepared in example 3 were administered 2 capsules at a time, 3 times a day, for 30 consecutive days.
Third group: the capsule is administered 2 capsules each time, 3 times a day for 30 days.
The criteria for the evaluation of the therapeutic effect were as follows:
the recovery comprises that ① edema and other symptoms and signs completely disappear, ② urine protein examination is continuously negative and/or 24 hours urine protein quantification is continuously less than 200mg, ③ high power micturition erythrocyte disappears, ④ urine sediment count is normal, ⑤ renal function is recovered to be normal (the recovery of renal function is represented by normal creatinine content in serum and normal urea nitrogen content in serum, 62-115 mu mol/L of creatinine in male is normal, 53-97 mu mol/L of creatinine in female is normal, 3.2-7.1 mmol/L of urea nitrogen content in serum is normal, and the requirements are also met from ① to ⑤;
the obvious effect is that ① edema and other symptoms and signs basically disappear, the ② urine protein examination is continuously reduced by more than 50 percent, ③ high power urine red blood cells under the microscope are less than 3, the ④ urine sediment count is close to normal, the difference between ⑤ renal function and the normal value is not more than 15 percent, and the requirements of ① to ⑤ are met;
the clinical application has the advantages that ① edema and other symptoms and signs are obviously improved, ② urine protein examination is continuously reduced by 1 + or 24 hours, the quantitative urine protein is continuously reduced by more than 25 percent, ③ high-power urine red blood cells under a microscope are less than 5, ④ renal function is improved, and the requirements of ① to ④ are met;
and (4) invalidation: the clinical manifestations and the above examination items were not improved or aggravated significantly.
Of the 129 patients in the first group: 18 cases are cured, 45 cases are obviously effective, 65 cases are effective, 1 case is ineffective, and the total effective rate is 99.2%; there were 2 cases of dry mouth side effects and 1 case of stomach discomfort side effects.
In the second group of 132 patients, 16 patients are cured, 44 patients with obvious effect, 66 patients with effect and 6 patients with no effect, and the total effective rate is 95.4%; 6 cases with dry mouth side effects, 3 cases with stomach discomfort side effects, and 2 cases with anorexia side effects.
In 132 patients in the third group, 14 patients are cured, 40 patients with obvious effect, 69 patients with effect and 9 patients with no effect, and the total effective rate is 93.1%; 32 cases with dry mouth side effect, 40 cases with stomach discomfort side effect, 20 cases with anorexia side effect, and 12 cases with nausea side effect.
SEQUENCE LISTING
<110> Guo Qing Zi
<120> culture method and culture medium for irpex cacteus
<130>GNCYX192710
<170>PatentIn version 3.5
<160>1
<210>1
<211>634
<212>DNA
<213>Irpex lacteus
<400>1
cattatcgag ttttgaacgg gttgtagctg gcctctcacg aggcatgtgc acgcctggct 60
catccactct taacctctgt gcactttatg taagagaaaa aaatggtgga agcttccagg 120
atctcgcgag aggtcttcgg ttgaacaagc cgtttttctt tcttatgttt tactacaaac 180
gcttcagtta tagaatgtca actgtgtata acacatttat atacaacttt cagcaacgga 240
tctcttggct ctcgcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca 300
gaattcagtg aatcatcgaa tctttgaacg caccttgcac tccttggtat tccgaggagt 360
atgcctgttt gagtctcatg gtattctcaa cccctaaatt tttgtaatga aggtttagcg 420
ggcttggact tggaggttgt gtcggccctt gtcggtcgac tcctctgaaa tgcattagcg 480
tgaatcttac ggatcgcctt cagtgtgata attatctgcg ctgtggtgtt gaagtattta 540
tggtgttcat gcttcgaacc gtctccttgc cgagacaatc atttgacaat ctgagctcaa 600
atcaggtagg actacccgct gaacttaagc atat 634
Claims (4)
1. The culture medium for Irpex lacteus (PAX-2014-1 is culture medium I or culture medium II;
the culture medium I comprises a carbon source, a nitrogen source, calcium carbonate, potassium dihydrogen sulfate, magnesium sulfate and water; the carbon-nitrogen ratio of the culture medium I is 3-15: 1;
the culture medium II comprises a carbon source, a nitrogen source, a growth factor, potassium dihydrogen sulfate, magnesium sulfate and water; the carbon-nitrogen ratio of the culture medium II is 3-15: 1;
rapex lacteus (Irpex lacteus) PAX-2014-1, and the preservation registration number is CGMCC NO. 12519.
2. The culture medium of claim 1, wherein:
the culture medium I per liter is composed of the following raw materials: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of calcium carbonate, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water;
the culture medium II per liter is composed of the following raw materials: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of growth factor, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water.
3. A method for culturing Irpex lacteus (PAX-2014-1) comprises the following steps: culturing the irpex cacteus of claim 1 with medium i or medium ii;
the culture medium I comprises a carbon source, a nitrogen source, calcium carbonate, potassium dihydrogen sulfate, magnesium sulfate and water; the carbon-nitrogen ratio of the culture medium I is 3-15: 1;
the culture medium II comprises a carbon source, a nitrogen source, a growth factor, potassium dihydrogen sulfate, magnesium sulfate and water; the carbon-nitrogen ratio of the culture medium II is 3-15: 1;
rapex lacteus (Irpex lacteus) PAX-2014-1, and the preservation registration number is CGMCC NO. 12519.
4. The method of claim 3, wherein:
the culture medium I per liter is composed of the following raw materials: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of calcium carbonate, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water;
the culture medium II per liter is composed of the following raw materials: 45-60 g of carbon source, 15-20 g of peptone, 5-15 g of yeast extract, 0.5-0.6 g of growth factor, 1.0-2.0 g of monopotassium phosphate, 0.5-1.5 g of magnesium sulfate and the balance of water.
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| US8460897B1 (en) * | 2009-12-17 | 2013-06-11 | Eclipse Bioproducts, LLC | Methods of culturing fungi and producing cellulases and chitin |
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| WO2017132523A1 (en) * | 2016-01-28 | 2017-08-03 | University of Alaska Anchorage | Thermal insulation material from mycelium and forestry byproducts |
| CN106635821B (en) * | 2016-08-13 | 2019-10-25 | 辽宁省农业科学院微生物工程中心 | A kind of mycorrhizal fungi and the preparation method and application thereof for promoting blueberry nutrient to absorb |
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| US8460897B1 (en) * | 2009-12-17 | 2013-06-11 | Eclipse Bioproducts, LLC | Methods of culturing fungi and producing cellulases and chitin |
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