CN107779404A - One plant of white rake teeth bacterium and its cultural method and application - Google Patents
One plant of white rake teeth bacterium and its cultural method and application Download PDFInfo
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- CN107779404A CN107779404A CN201611079486.8A CN201611079486A CN107779404A CN 107779404 A CN107779404 A CN 107779404A CN 201611079486 A CN201611079486 A CN 201611079486A CN 107779404 A CN107779404 A CN 107779404A
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- rake teeth
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- teeth bacterium
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- 241000894006 Bacteria Species 0.000 title claims abstract description 85
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000001556 precipitation Methods 0.000 claims abstract description 21
- 239000000284 extract Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000002994 raw material Substances 0.000 claims abstract description 11
- 241000222344 Irpex lacteus Species 0.000 claims abstract description 10
- 238000003809 water extraction Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims description 56
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 34
- 230000001580 bacterial effect Effects 0.000 claims description 26
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 22
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 19
- 229910052799 carbon Inorganic materials 0.000 claims description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 17
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 17
- 239000001888 Peptone Substances 0.000 claims description 15
- 108010080698 Peptones Proteins 0.000 claims description 15
- 235000019319 peptone Nutrition 0.000 claims description 15
- 239000013049 sediment Substances 0.000 claims description 14
- 239000002609 medium Substances 0.000 claims description 13
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 9
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 9
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 claims description 8
- 239000003102 growth factor Substances 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 4
- 230000036541 health Effects 0.000 claims description 4
- ILRLTAZWFOQHRT-UHFFFAOYSA-N potassium;sulfuric acid Chemical compound [K].OS(O)(=O)=O ILRLTAZWFOQHRT-UHFFFAOYSA-N 0.000 claims description 4
- 201000005569 Gout Diseases 0.000 abstract description 8
- 201000001431 Hyperuricemia Diseases 0.000 abstract description 8
- 206010018364 Glomerulonephritis Diseases 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract 5
- 230000000694 effects Effects 0.000 description 19
- 239000006228 supernatant Substances 0.000 description 17
- 239000000843 powder Substances 0.000 description 16
- 210000002966 serum Anatomy 0.000 description 16
- 239000012153 distilled water Substances 0.000 description 15
- 238000000605 extraction Methods 0.000 description 13
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 12
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 12
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 12
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- 229940116269 uric acid Drugs 0.000 description 12
- 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 description 9
- 239000002775 capsule Substances 0.000 description 9
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 6
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- 235000015895 biscuits Nutrition 0.000 description 6
- 229940109239 creatinine Drugs 0.000 description 6
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- 206010030113 Oedema Diseases 0.000 description 4
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000011591 potassium Substances 0.000 description 4
- 229910052700 potassium Inorganic materials 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
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- 235000019698 starch Nutrition 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 241000233866 Fungi Species 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 206010028813 Nausea Diseases 0.000 description 3
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 3
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- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
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- 238000012113 quantitative test Methods 0.000 description 2
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- 239000010865 sewage Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 206010060891 General symptom Diseases 0.000 description 1
- 244000060234 Gmelina philippensis Species 0.000 description 1
- 241000222342 Irpex Species 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 241001619412 Meruliaceae Species 0.000 description 1
- 206010030302 Oliguria Diseases 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 1
- 241000222383 Polyporales Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
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- 210000003734 kidney Anatomy 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
-
- 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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- C12N1/14—Fungi; Culture media therefor
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- 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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- Natural Medicines & Medicinal Plants (AREA)
- Virology (AREA)
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- General Chemical & Material Sciences (AREA)
- Alternative & Traditional Medicine (AREA)
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Abstract
The invention discloses one plant of white rake teeth bacterium and its cultural method and application.Present invention firstly provides white rake teeth bacterium (Irpex lacteus) PAX 2,014 1, preservation registration number is CGMCC NO.12519, referred to as white rake teeth bacterium PAX 2,014 1.The present invention also protects a kind of preparation method of white rake teeth bacterium PAX 2,014 1 extract, it is characterised in that：It is raw material with white rake teeth bacterium PAX 2,014 1, carries out following steps successively：(a1) water extraction；(a2) alcohol precipitation；(a3) precipitation is collected.The present invention also protects a kind of product, and its active component is following (c1) or (c2)：(c1) white rake teeth bacterium PAX 2,014 1；(c2) extract.The function of product is as follows：Treatment and/or prevention gout；Treatment and/or prevention hyperuricemia；Treatment and/or prevention CGN；Treatment and/or prevention glomerulonephritis；Treatment and/or prevention ephritis.The present invention has great application prospect.
Description
Technical field
The invention belongs to biological technical field, and in particular to one plant of white rake teeth bacterium and its cultural method and application.
Background technology
White rake teeth bacterium [Irpex lacteus (Fr.) Fr.], alias Irpex lacteus, belong to Aphyllophorales, wrinkle pore fungi section
(Meruliaceae), rake teeth Pseudomonas (Irpex), the live standing tree of broad-leaved or coniferous tree, dry wood is grown on or is fallen on wood, 1 year
Give birth to, Chang Pingfu warps, side is given birth to once in a while, can cause the white rot of timber.
White rake teeth bacterium is one kind of China's medicinal fungus, has the work(of the symptoms such as treatment oliguria, edema, pain in the back, blood pressure rise
Effect, while there is anti-inflammatory activity.Also, because its contain the degradability such as lignoenzyme, cellulase and laccase enzyme and compared with
The fast speed of growth, white rake teeth bacterium be widely used in commercial paper, pulp modifying, Sewage, dyeing and printing sewage processing,
The industries such as beverage industry, apparel industry, organic synthesis, and for the pollution control and reparation of heavy metal.
The content of the invention
It is an object of the invention to provide one plant of white rake teeth bacterium and its cultural method and application.
Present invention firstly provides white rake teeth bacterium (Irpex lacteus) PAX-2014-1.White rake teeth bacterium (Irpex
Lacteus) PAX-2014-1, it is commonly micro- China Committee for Culture Collection of Microorganisms has been preserved on June 29th, 2016
(abbreviation CGMCC, address are Bio-Centers：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute), preservation registration number is CGMCC NO.12519.White rake teeth bacterium (Irpex lacteus) PAX-2014-1CGMCC
NO.12519, referred to as white rake teeth bacterium PAX-2014-1.
The present invention also protects the culture medium for cultivating white rake teeth bacterium PAX-2014-1, is culture medium I or culture medium II.Institute
Stating culture medium I includes carbon source, 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 concretely 6.67：1、12.3：1 or 9.23：1.The culture medium II includes carbon source, nitrogen
Source, growth factor, potassium dihydrogen sulfate, magnesium sulfate and water.The carbon-nitrogen ratio of the culture medium II is 3-15:1.The culture medium II
Carbon-nitrogen ratio concretely 6.67：1、12.3：1 or 9.23：1.
Every liter of culture medium I can be made up of the following raw material：45-60 grams of carbon source, 20-35 grams of nitrogen source, calcium carbonate 0.5-0.6
Gram, 1.0-2.0 grams of potassium dihydrogen phosphate, 0.5-1.5 grams of magnesium sulfate, surplus is water.Every liter of culture medium I can be by the following raw material group
Into：45-60 grams of carbon source, 15-20 grams of peptone, 5-15 grams of yeast extract, 0.5-0.6 grams of calcium carbonate, potassium dihydrogen phosphate 1.0-
2.0 grams, 0.5-1.5 grams of magnesium sulfate, surplus are water.
Every liter of culture medium II can be made up of the following raw material：45-60 grams of carbon source, 20-35 grams of nitrogen source, growth factor 0.5-
0.6 gram, 1.0-2.0 grams of potassium dihydrogen phosphate, 0.5-1.5 grams of magnesium sulfate, surplus is water.Every liter of culture medium II can be by following former
Material composition：45-60 grams of carbon source, 15-20 grams of peptone, 5-15 grams of yeast extract, 0.5-0.6 grams of growth factor, biphosphate
1.0-2.0 grams of potassium, 0.5-1.5 grams of magnesium sulfate, surplus are water.
The carbon source concretely soluble starch and/or glucose.
The nitrogen source concretely peptone and/or yeast extract.
The culture medium I concretely culture medium first, culture medium second or culture medium third.
Every liter of culture medium first can be specifically made up of the following raw material：60.0 grams of soluble starch, 20.0 grams of peptone, ferment
Female 15.0 grams of extract, 0.6 gram of calcium carbonate, 2.0 grams of potassium dihydrogen phosphate, 1.5 grams of magnesium sulfate, surplus is water.Natural pH.
Every liter of culture medium second can be specifically made up of the following raw material：60.0 grams of glucose, 15.0 grams of peptone, yeast carry
5.0 grams of thing, 0.5 gram of calcium carbonate, 1.0 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate are taken, surplus is water.Natural pH.
Every liter of culture medium third can be specifically made up of the following raw material：45.0 grams of glucose, 15.0 grams of peptone, yeast carry
5.0 grams of thing, 0.5 gram of calcium carbonate, 1.0 grams of potassium dihydrogen phosphate, 0.5 gram of magnesium sulfate are taken, surplus is water.Natural pH.
The present invention also protects a kind of method for cultivating white rake teeth bacterium PAX-2014-1, comprises the following steps：Use to take up an official post
The one white rake teeth bacterium PAX-2014-1 of medium culture.
Methods described specifically comprises the following steps：
(1) white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days；
(2) beaten from the flat board for completing step (1) with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations
To culture medium described in 1L, 30 DEG C, 150rpm shaken cultivations 72 hours.
Culture medium provided by the invention, constituent are clear and definite.White rake teeth bacterium bacterium is produced using culture medium provided by the invention
Filament have technique it is simple, in the absence of heavy metal pollution, that thalli growth speed is fast, thalline yield is high, product quality is stable etc. is excellent
Point.
The present invention also protects a kind of preparation method of white rake teeth bacterium PAX-2014-1 extract, it is characterised in that：With white
Rake teeth bacterium PAX-2014-1 is raw material, carries out following steps successively：
(a1) water extraction；
(a2) alcohol precipitation；
(a3) precipitation is collected.
In (a1), the condition of water extraction is concretely：90 DEG C stand extraction.In (a1), the condition of water extraction is specific
Can be：90 DEG C stand extraction 2-6 hours.In (a1), white rake teeth bacterium PAX-2014-1 and water proportioning can be：1 gram of white rake
Hedgehog fungus PAX-2014-1 (weight in wet base)：30-90 milliliter water.(a1) specifically comprises the following steps：1. take n grams of white rake teeth bacterium PAX-
2014-1 (weight in wet base), 30*n milliliter water is added, 90 DEG C stand extraction 2 hours, collect supernatant；2. take step 1. remaining bacterium
Body, 30*n milliliter water is added, 90 DEG C stand extraction 2 hours, collect supernatant；3. taking step 2. remaining thalline, 30*n is added
Milliliter distilled water, 90 DEG C stand extraction 2 hours, collect supernatant；4. 2. 1. supernatant that step is obtained, step obtain upper
3. supernatant that clear liquid and step obtain merges, and is then concentrated under reduced pressure into 1/3rd volumes, obtains concentrate；N is positive number.
In (a2), alcohol precipitation concretely ethanol precipitation.In (a2), the condition of alcohol precipitation is concretely stood for 4 DEG C.
In (a2), the condition of alcohol precipitation concretely stands 24 hours for 4 DEG C.In (a2), the concentrate and absolute ethyl alcohol
Volume proportion is 1：3.(b3) specifically comprises the following steps：The concentrate for taking 1 parts by volume step (a1) to obtain, with 3 volumes
Part absolute ethyl alcohol mixing, 4 DEG C stand 24 hours.
In (a3), by the way that precipitation is collected by centrifugation.The condition of the centrifugation is concretely：8000rpm centrifuges 30min.
Methods described also comprises the following steps：(a3) obtained precipitation is freeze-dried, obtains freeze-dried powder.
The present invention also protects a kind of preparation method of white rake teeth bacterium PAX-2014-1 extract, includes following step successively
Suddenly：
(b1) the white rake teeth bacterium PAX-2014-1 of medium culture described in any of the above is used, then collects bacterial sediment；
(b2) water extraction；
(b3) alcohol precipitation；
(b4) precipitation is collected.
(b1) specifically comprises the following steps：
(1) white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days；
(2) beaten from the flat board for completing step (1) with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations
To culture medium described in 1L, 30 DEG C, 150rpm shaken cultivations 72 hours.
(3) after completing step (2), a cultivating system is rounded, 6000rpm centrifugation 20min, collects bacterial sediment.
In (b2), the condition of water extraction is concretely：90 DEG C stand extraction.In (b2), the condition of water extraction is specific
Can be：90 DEG C stand extraction 2-6 hours.In (b2), the proportioning of the bacterial sediment and water can be：1 gram of bacterial sediment is (wet
Weight)：30-90 milliliter water.(b2) specifically comprises the following steps：1. taking the n grams of bacterial sediment, 30*n milliliter water is added,
90 DEG C stand extraction 2 hours, collect supernatant；2. taking step 1. remaining thalline, 30*n milliliter water is added, 90 DEG C stand extraction
2 hours, collect supernatant；3. taking step 2. remaining thalline, 30*n milliliter distilled water is added, 90 DEG C stand extraction 2 hours, receive
Collect supernatant；4. 3. supernatant that 2. supernatant and step that 1. supernatant that step is obtained, step obtain obtain merges, so
After be concentrated under reduced pressure into 1/3rd volumes, obtain concentrate；N is positive number.
In (b3), alcohol precipitation concretely ethanol precipitation.In (b3), the condition of alcohol precipitation is concretely stood for 4 DEG C.
In (b3), the condition of alcohol precipitation concretely stands 24 hours for 4 DEG C.In (b3), the concentrate and absolute ethyl alcohol
Volume proportion is 1：3.(b3) specifically comprises the following steps：The concentrate for taking 1 parts by volume step (b2) to obtain, with 3 volumes
Part absolute ethyl alcohol mixing, 4 DEG C stand 24 hours.
In (b4), by the way that precipitation is collected by centrifugation.The condition of the centrifugation is concretely：8000rpm centrifuges 30min.
Methods described also comprises the following steps：(b4) obtained precipitation is freeze-dried, obtains freeze-dried powder.
The extract that any of the above methods described is prepared falls within protection scope of the present invention.
The present invention also protects a kind of product, and its active component is following (c1) or (c2)：
(c1) white rake teeth bacterium PAX-2014-1；
(c2) extract described in any of the above.
The function of the product is following (d1) and/or (d2) and/or (d3) and/or (d4) and/or (d5)：
(d1) treat and/or prevent gout；
(d2) treat and/or prevent hyperuricemia；
(d3) treat and/or prevent CGN；
(d4) treat and/or prevent glomerulonephritis；
(d5) treat and/or prevent ephritis.
The present invention also protects application of any extracts of white rake teeth bacterium PAX-2014-1 or more in product is prepared.
The function of the product is following (d1) and/or (d2) and/or (d3) and/or (d4) and/or (d5)：
(d1) treat and/or prevent gout；
(d2) treat and/or prevent hyperuricemia；
(d3) treat and/or prevent CGN；
(d4) treat and/or prevent glomerulonephritis；
(d5) treat and/or prevent ephritis.
The product is medicine or health products.
The invention provides one plant of white rake teeth bacterium new strains and research and develop optimize for cultivate the culture medium of the new strains with
And it is prepared for the extract of the bacterial strain.White rake teeth bacterium PAX-2014-1 extract is respectively provided with the effect of good for a variety of diseases
And side effect is low, there is great application prospect.
Brief description of the drawings
Fig. 1 be bacterial strain PAX-2014-1 on potato dextrose agar 25 DEG C culture 5 days after photo.
Fig. 2 is the photo of bacterial strain PAX-2014-1 mycelia under the microscope.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with and repeats to test three times, as a result make even
Average.
Control medium：200g potato stripping and slicing liquors, with filtered through gauze and collect filtrate, and 20g grapes are added in filtrate
Sugar, 1000ml is settled to distilled water.
PDA culture medium：200g potato stripping and slicing liquors, with filtered through gauze and collect filtrate, and 20g glucose is added in filtrate
With 20 grams of agar, 1000ml is settled to distilled water.
Kidney-nourishing recovering capsule：Changchun Yi-Shen-Kang Biology Pharmacy Co., Ltd.
White rake teeth bacterium is compareed, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation
Registration number is CGMCC NO.1016.Compare white rake teeth bacterium and be recorded in the patent of Application No. 201210461164.5 (the patent
Authorization Notice No. be：CN 102935095B；Authorized announcement date is：2013-11-27).
The acquisition of embodiment 1, white rake teeth bacterium PAX-2014-1
First, the separation of bacterial strain
2014, the sample of white rake teeth bacterium is gathered from Chinese Changbaishan area, then by tissue block isolated strains, is obtained
The bacterial strain of some pure cultures, will wherein one plant be named as bacterial strain PAX-2014-1.
2nd, the identification of bacterial strain
Bacterial strain PAX-2014-1 morphological feature：Bacterium colony is white, thin, and the cotton slightly risen is cotton-shaped；In whole bacterium colony
Surface radial arrangement some intensive tufted and penniform aerial mycelium, and mycelia has every, transparent, 1.2-4 μm of diameter,
Without clamp connection.Bacterial strain PAX-2014-1 on potato dextrose agar 25 DEG C culture 5 days after photo see Fig. 1,
Cover with diameter 9cm flat board.The photo of bacterial strain PAX-2014-1 mycelia under the microscope is shown in Fig. 2.
Bacterial strain PAX-2014-1 ITS sequence is shown in the sequence 1 of sequence table.
3rd, the preservation of bacterial strain
White rake teeth bacterium (Irpex lacteus) PAX-2014-1, is preserved in China Microbiological bacterium on June 29th, 2016
(abbreviation CGMCC, address are kind preservation administration committee common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica), preservation registration number is CGMCC NO.12519.White rake teeth bacterium (Irpex lacteus)
PAX-2014-1CGMCC NO.12519, referred to as white rake teeth bacterium PAX-2014-1.
The culture of embodiment 2, white rake teeth bacterium PAX-2014-1
First, it is 6 using carbon-nitrogen ratio：1 white rake teeth bacterium semisynthetic medium produces white rake teeth bacterium bacterium powder
Culture medium first (natural pH)：Take 60.0 grams of soluble starch, 20.0 grams of peptone, 15.0 grams of yeast extract, carbon
Sour 0.6 gram of calcium, 2.0 grams of potassium dihydrogen phosphate, 1.5 grams of magnesium sulfate, are dissolved with distilled water, are then settled to 1000 millis with distilled water
Rise.Carbon content in starch is 40% (weight/mass percentage composition), and the nitrogen content in peptone is 9% (weight/mass percentage composition), ferment
Nitrogen content in female extract is 12% (weight/mass percentage composition).It is computed, the mass ratio of carbon and nitrogen is 6.67 in culture medium first：
1。
1st, white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days.
2nd, beaten from the flat board for completing step 1 with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations to 1L
Culture medium first (or control medium), 30 DEG C, 150rpm shaken cultivations 72 hours.
3rd, after completing step 2, a cultivating system is rounded, 6000rpm centrifugation 20min, collects bacterial sediment.
4th, the bacterial sediment for obtaining step 3 is freeze-dried, and obtains freeze-dried powder.
During using culture medium first, every liter of cultivating system obtains 15.25 grams of freeze-dried powder (average value of five repetition experiments).
During using control medium, every liter of cultivating system obtains 3.83 grams of freeze-dried powder (average value of five repetition experiments).
2nd, it is 12 using carbon-nitrogen ratio：1 white rake teeth bacterium semisynthetic medium produces white rake teeth bacterium bacterium powder
Culture medium second：Take 60.0 grams of glucose, 15.0 grams of peptone, 5.0 grams of yeast extract, 0.5 gram of calcium carbonate, phosphoric acid
1.0 grams of potassium dihydrogen, 0.5 gram of magnesium sulfate, are dissolved with distilled water, are then settled to 1000 milliliters with distilled water.Carbon in glucose
Content is 40% (weight/mass percentage composition), and the nitrogen content in peptone is 9% (weight/mass percentage composition), the nitrogen in yeast extract
Content is 12% (weight/mass percentage composition).It is computed, the mass ratio of carbon and nitrogen is 12.3 in culture medium second：1.
1st, white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days.
2nd, beaten from the flat board for completing step 1 with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations to 1L
Culture medium second (or control medium), 30 DEG C, 150rpm shaken cultivations 72 hours.
3rd, after completing step 2, a cultivating system is rounded, 6000rpm centrifugation 20min, collects bacterial sediment.
4th, the bacterial sediment for obtaining step 3 is freeze-dried, and obtains freeze-dried powder.
During using culture medium second, every liter of cultivating system obtains 15.36 grams of freeze-dried powder (average value of five repetition experiments).
During using control medium, every liter of cultivating system obtains 3.83 grams of freeze-dried powder (average value of five repetition experiments).
3rd, it is 9 using carbon-nitrogen ratio：1 white rake teeth bacterium semisynthetic medium produces white rake teeth bacterium bacterium powder
Culture medium third：Take 45.0 grams of glucose, 15.0 grams of peptone, 5.0 grams of yeast extract, 0.5 gram of calcium carbonate, phosphoric acid
1.0 grams of potassium dihydrogen, 0.5 gram of magnesium sulfate, are dissolved with distilled water, are then settled to 1000 milliliters with distilled water.Carbon in glucose
Content is 40% (weight/mass percentage composition), and the nitrogen content in peptone is 9% (weight/mass percentage composition), the nitrogen in yeast extract
Content is 12% (weight/mass percentage composition).It is computed, the mass ratio of carbon and nitrogen is 9.23 in culture medium third：1.
1st, white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days.
2nd, beaten from the flat board for completing step 1 with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations to 1L
Culture medium third (or control medium), 30 DEG C, 150rpm shaken cultivations 72 hours.
3rd, after completing step 2, a cultivating system is rounded, 6000rpm centrifugation 20min, collects bacterial sediment.
4th, the bacterial sediment for obtaining step 3 is freeze-dried, and obtains freeze-dried powder.
During using culture medium the third, every liter of cultivating system obtains 16.27 grams of freeze-dried powder (average value of five repetition experiments).
During using control medium, every liter of cultivating system obtains 3.83 grams of freeze-dried powder (average value of five repetition experiments).
Embodiment 3, with white rake teeth bacterium PAX-2014-1 prepare capsule
Culture medium third：Take 45.0 grams of glucose, 15.0 grams of peptone, 5.0 grams of yeast extract, 0.5 gram of calcium carbonate, phosphoric acid
1.0 grams of potassium dihydrogen, 0.5 gram of magnesium sulfate, are dissolved with distilled water, are then settled to 1000 milliliters with distilled water.
1st, white rake teeth bacterium PAX-2014-1 single bacterium colonies are inoculated on PDA culture medium flat board, 30 DEG C of quiescent cultures 5 days.
2nd, beaten from the flat board for completing step 1 with card punch and take a diameter of 6 millimeters of bacteria cake, every 12 pure culture biscuits involvng inoculations to 1L
Culture medium third, 30 DEG C, 150rpm shaken cultivations 72 hours.
3rd, after completing step 2, a cultivating system is rounded, 6000rpm centrifugation 20min, collects bacterial sediment.
4th, the bacterial sediment (weight in wet base=n grams, n are positive number) that step 3 obtains is taken, adds 30*n milliliter distilled water, 90 DEG C quiet
Extraction 2 hours is put, collects supernatant.
5th, the remaining thalline of step 4 is taken, adds 30*n milliliter distilled water, 90 DEG C stand extraction 2 hours, collect supernatant.
6th, the remaining thalline of step 5 is taken, adds 30*n milliliter distilled water, 90 DEG C stand extraction 2 hours, collect supernatant.
7th, the supernatant that the supernatant and step 6 that the supernatant that obtains step 4, step 5 obtain obtain merges, and then subtracts
Pressure is concentrated into 1/3rd volumes, obtains concentrate.
8th, the concentrate for taking 1 parts by volume step 7 to obtain, mixed with 3 parts by volume absolute ethyl alcohols, 4 DEG C stand 24 hours, then
8000rpm centrifuges 30min, collects precipitation and is freeze-dried, obtains freeze-dried powder.
9th, each capsule shells load the freeze-dried powder that 0.3g steps 8 obtain, and obtain PAX-2014-1 capsules.
Replace white rake teeth bacterium PAX-2014-1 to carry out above-mentioned steps with white rake teeth bacterium is compareed, obtain compareing capsule.
The application of embodiment 4, white rake teeth bacterium PAX-2014-1 in treatment gout and hyperuricemia
Hyperuricemia merges the patient 24 (man 16, female 8) of gout, the patient with gout of hyperuricemia does not occur
4 (man 2, female 2), the Patients with Hyperuricemia 4 (whole males) of gout does not occur.Above patient is to be cured through front three
The volunteer for the informed consent that institute makes a definite diagnosis.In patient, male 22, women 10, between age 33-78 year, the course of disease 5 months -6
Between year.Excluded in therapeutic process：1) gravid woman；2) to this medicine and any of which composition allergy sufferers；3) cardiovascular, liver
Severe primary disease patient be present in dirty, hemopoietic system etc.；4) HIV patient and other immune system persons of being badly damaged；5) mental disease
Patient；6) pill taker is not adhered to.
Patient is treated by following scheme：Take the PAX-2014-1 capsules of the preparation of embodiment 3,3 tablets each time, daily 3
It is secondary, continuously take 60 days.
After completing treatment, carry out therapeutic effect judge and side effect is judged.
Therapeutic effect judgment criteria is as follows：
Recovery from illness：1. the symptoms such as site of pathological change pain, redness are wholly absent；2. the uric acid content in serum reaches healthy mark
It is accurate；Meet 1. and 2. the above is judged as fully recovering simultaneously；
It is effective：1. compared with pre-treatment, the symptom such as site of pathological change pain, redness takes an evident turn for the better；2. the uric acid in serum
Content is not reaching to health standards, but the uric acid content in serum reduces numerical value >=20 μm ol/L compared with pre-treatment；3. fall ill
Tract pain is wholly absent, and uric acid in serum content does not raise compared with pre-treatment；1. and 2. 3. or simultaneously satisfaction meets
It is judged as effective；
Effectively：1. compared with pre-treatment, the symptom such as site of pathological change pain, redness takes an evident turn for the better；2. compared with pre-treatment,
Uric acid content in serum, which does not significantly reduce, also not to be raised；Meet 1. and 2. the above is judged as effectively simultaneously；
It is invalid：1. compared with pre-treatment, the symptom such as site of pathological change pain, redness is not improved or aggravated；2. with controlling
Compared before treatment, the uric acid content in serum is not significantly reduced or raised；It is invalid to meet 1. and 2. the above is judged as simultaneously；
The health standards of uric acid content in serum：Uric acid content in the serum of male is 150~440 μm of ol/L；Female
Uric acid content in the serum of property is 95~360 μm of ol/L.
Side effect judgment criteria is as follows：Dry and/or have a stomach upset and/or nausea.
Before treatment (the previous day that treatment starts), after treatment (second day for the treatment of end), the uric acid in the serum of patient
The average value of content, creatinine content and urea nitrogen content is shown in Table 1.
Table 1
| Before treatment | After treatment | |
| Uric acid (μm ol/L) | 569,+/-182 | 512,+/-105 |
| Creatinine (μm ol/L) | 327.6,+/-114.6 | 238.5,+/-79.2 |
| Urea nitrogen (mmol/L) | 9.79,+/-5.81 | 7.75,+/-3.88 |
In 32 patients：Recovery from illness 3, effective 14, effective 11, invalid 4, total effective rate 87.5%；Site of pathological change
Be wholly absent 17 of pain, be obviously improved 28 of general symptom；There is 5 of dry side effect, side effect of having a stomach upset
4,0 of nauseous side effect.
Result above shows that white rake teeth bacterium PAX-2014-1 extracts can significantly improve hyperuricemia and/or gout
The pain of patient and red and swollen symptom, uric acid content, creatinine content and urea nitrogen content in serum are reduced, and side effect is very low.
The application of embodiment 5, white rake teeth bacterium PAX-2014-1 in CGN is treated
First group：Chronic glomerulonephritis patients 129 (man 69, female 60).Second group：CGN is suffered from
Person 132 (man 64, female 68).3rd group：Chronic glomerulonephritis patients 132 (man 62, female 70).Above patient
It is the volunteer for the informed consent made a definite diagnosis through Grade A hospital.Excluded in therapeutic process：1) gravid woman；2) to this medicine and its
In any composition allergy sufferers；3) there is severe primary disease patient in angiocarpy, liver, hemopoietic system etc.；4) HIV patient and other
The immune system person of being badly damaged；5) mental patient；6) pill taker is not adhered to.
Two groups of patients are treated by following scheme respectively：
First group：The PAX-2014-1 capsules of the preparation of embodiment 3 are taken, 2 tablets each time, 3 times a day, are continuously taken 30 days.
Second group：The control capsule of the preparation of embodiment 3 is taken, 2 tablets each time, 3 times a day, is continuously taken 30 days.
3rd group：Kidney-nourishing recovering capsule is taken, 2 tablets each time, 3 times a day, is continuously taken 30 days.
Therapeutic effect judgment criteria is as follows：
Recovery from illness：1. the symptoms such as oedema and sign are wholly absent；2. urine protein examination continues negative and/or twenty-four-hour urine albumen
Quantitative continuous is less than 200mg；3. urine erythrocyte disappears under high power lens；4. arena counts normal；5. normal (the kidney of graft function
Functional rehabilitation Normal appearances are that creatinine in serum content is normal and urea in serum nitrogen content is normal；Male's creatinine in serum content
62~115 μm of ol/L are normal, and 53~97 μm of ol/L of women creatinine in serum content are normal, urea in serum nitrogen content 3.2
~7.1mmol/L is normal；Meet the above 1. to 5. simultaneously；
It is effective：1. the symptoms such as oedema and sign disappear substantially；2. urine protein examination persistently reduces more than 50%；3. high power lens
Lower urine erythrocyte is less than 3；4. arena is counted close to normal；5. renal function is differed with normal value no more than 15%；Simultaneously
1. to 5. more than meeting；
Effectively：1. the symptoms such as oedema and sign are clearly better；2. urine protein examination persistently reduces by 1+or twenty-four-hour urine egg
White quantitative continuous reduces more than 25%；3. urine erythrocyte is less than 5 under high power lens；4. renal function has improvement；Simultaneously meet with
On 1. to 4.；
It is invalid：Clinical manifestation and above inspection project are not improved or aggravated.
In first group of 129 patients：Recovery from illness 18, effective 45, effective 65, invalid 1, total effective rate
99.2%；There are 2,1 of side effect of having a stomach upset of dry side effect.
In second group of 132 patients, fully recover 16, effective 44, effective 66, invalid 6, total effective rate
95.4%；Occur 6 of dry side effect, 3 of side effect of having a stomach upset, receive 2 of poor side effect.
In 3rd group of 132 patients, fully recover 14, effective 40, effective 69, no effect 9, total effective rate
93.1%；Occur 32 of dry side effect, 40 of side effect of having a stomach upset, receive poor side effect 20, nauseous side effect 12
Example.
Sequence table
<110>The sub- Guo Shang prints of Guo Qing
<120>One plant of white rake teeth bacterium and its cultural method and application
<130> GNCYX161649
<160> 1
<210> 1
<211> 634
<212> DNA
<213>White rake teeth bacterium（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 (10)
1. white rake teeth bacterium (Irpex lacteus) PAX-2014-1, its preservation registration number is CGMCC NO.12519.
It is culture medium I or culture medium II 2. for the culture medium of white rake teeth bacterium described in claim 1；
The culture medium I includes carbon source, nitrogen source, calcium carbonate, potassium dihydrogen sulfate, magnesium sulfate and water；The carbon-nitrogen ratio of the culture medium I
For 3-15:1；
The culture medium II includes carbon source, nitrogen source, growth factor, potassium dihydrogen sulfate, magnesium sulfate and water；The carbon of the culture medium II
Nitrogen ratio is 3-15:1.
3. culture medium as claimed in claim 2, it is characterised in that：
Every liter of culture medium I can be made up of the following raw material：45-60 grams of carbon source, 15-20 grams of peptone, yeast extract 5-15
Gram, 0.5-0.6 grams of calcium carbonate, 1.0-2.0 grams of potassium dihydrogen phosphate, 0.5-1.5 grams of magnesium sulfate, surplus is water；
Every liter of culture medium II can be made up of the following raw material：45-60 grams of carbon source, 15-20 grams of peptone, yeast extract 5-15
Gram, 0.5-0.6 grams of growth factor, 1.0-2.0 grams of potassium dihydrogen phosphate, 0.5-1.5 grams of magnesium sulfate, surplus is water.
4. a kind of method for cultivating white rake teeth bacterium described in claim 1, comprises the following steps：Using training described in Claims 2 or 3
Support white rake teeth bacterium described in base culture claim 1.
A kind of 5. preparation method of the extract of white rake teeth bacterium described in claim 1, it is characterised in that：With described in claim 1
White rake teeth bacterium PAX-2014-1 is raw material, carries out following steps successively：
(a1) water extraction；
(a2) alcohol precipitation；
(a3) precipitation is collected.
6. the preparation method of the extract of white rake teeth bacterium, in turn includes the following steps described in a kind of claim 1：
(b1) using white rake teeth bacterium PAX-2014-1, Ran Houshou described in medium culture claim 1 described in Claims 2 or 3
Collect bacterial sediment；
(b2) water extraction；
(b3) alcohol precipitation；
(b4) precipitation is collected.
7. the extract that the methods described of claim 5 or 6 is prepared.
8. a kind of product, its active component is following (c1) or (c2)：
(c1) white rake teeth bacterium described in claim 1；(c2) extract described in claim 7.
9. application of the extract in product is prepared described in white rake teeth bacterium described in claim 1 or claim 7.
10. product as claimed in claim 8 or application as claimed in claim 9, it is characterised in that：The product is medicine
Or health products.
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| CN102690147B (en) * | 2012-06-06 | 2013-12-25 | 吉林大学 | Liquid nutrient medium for Irpex lacteus fermentation and preparation method thereof |
| CN102935095B (en) * | 2012-11-16 | 2013-11-27 | 长春益肾康生物制药有限公司 | Bidirectional solid fermentation irpex cacteus mycoplasm extractive and preparation method and application thereof |
| CN104138395B (en) * | 2014-07-11 | 2017-10-24 | 吉林大学 | Purposes of the Irpex lacteus in anti-hypoxia medicament is prepared |
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| CN110791438A (en) | 2020-02-14 |
| CN110804557A (en) | 2020-02-18 |
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