CN103860605A - Preparation method of antrodia-camphorate-fermented full-liquid alcohol extract with avian influenza virus resistance - Google Patents

Preparation method of antrodia-camphorate-fermented full-liquid alcohol extract with avian influenza virus resistance Download PDF

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CN103860605A
CN103860605A CN201310403703.4A CN201310403703A CN103860605A CN 103860605 A CN103860605 A CN 103860605A CN 201310403703 A CN201310403703 A CN 201310403703A CN 103860605 A CN103860605 A CN 103860605A
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antrodia camphorata
fermentation liquid
influenza virus
avian influenza
liquid
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宋爱荣
赵晨
孔超
秦丹
梁大勇
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Qingdao Agricultural University
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Abstract

The invention discloses a preparation method of antrodia-camphorate-fermented full-liquid alcohol extract with avian influenza virus resistance. The preparation method comprises the steps of taking antrodia-camphorate-fermented full liquid or fermented clear liquid or mycelium, extracting by alcohol, filtering, concentrating under reduced pressure, removing pigment, removing low-molecular-weight compounds, removing fat, carrying out alcohol precipitation and eccentric separation, and drying and crushing collected materials and the like. The antrodia-camphorate-fermented full-liquid alcohol extract extracted by the method has obvious inhibiting effect on the avian influenza virus. The preparation method disclosed by the invention has the characteristics of simple process, low cost and good effect.

Description

A kind of preparation method of Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus
Technical field
The present invention relates to a kind of preparation method of Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus, relate in particular to Antrodia camphorata ( antrodia camphorata ) Ac001, within 21st, be deposited in JIUYUE in 2005 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center "; deposit number is CGMCC No.1460, preservation address is No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, Institute of Microorganism, Academia Sinica.
background technology
About the preparation method of the Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus, have through the Patents retrieving: Chinese Patent Application No. is 99112556.8 process for extracting polyose from Polyporus frondosus fermenting liquor, the production method of 02136517.2 Grifola frondosa fermentation technology and peptidoglycan thereof, the production method of 89105471 1 kinds of polysaccharide-peptide (PSP), 92109345 lentinan and Lentinula edodes mycelium polysaccharide extraction process, 96109095 Chinese glossy ganoderma health beverages, 92111030 glossy ganoderma nurition health care liquids and method for making thereof, the patents such as 93103116 Ganoderma oral liquids, these patent contents are main purpose mainly with extracting polysaccharide greatly, wherein Grifola frondosa, three kinds of extract of active substances of preparing of Coriolous Dersicolor (Fr.) Quel and Ganoderma have antitumor, resisting hypertension, reduce blood glucose, obesity, the drug effects such as anti-hepatitis, but rarely has report for antivirus action, and do not retrieve especially for the report of anti-avian influenza virus.
Summary of the invention
The problem to be solved in the present invention is, for the preparation method of the full liquid fermenting alcohol of the Antrodia camphorata extract of above-mentioned anti-avian influenza virus with and the existing deficiency of pharmacological action of the ethanol extraction of preparation, a kind of preparation method of Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus is provided.The Antrodia camphorata fermentation liquid ethanol extraction that utilizes method of the present invention to extract is inhibited to bird flu virus, and normal cell is had to protective effect, and the method has the advantages that simple process, cost are low, have anti-avian influenza.
The preparation method of the Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus of the present invention, comprises that three kinds are prepared form: one is for directly comprising mycelium and clear liquor and go out active substance with separation of ethanol from Antrodia camphorata bacterium fermentation liquid; It is two for first Antrodia camphorata bacterium fermentation liquid being separated into mycelium and clear liquor, then goes out active substance with separation of ethanol from gained mycelium; It is three for first Antrodia camphorata bacterium fermentation liquid being separated into mycelium and clear liquor, then goes out active substance with separation of ethanol from gained clear liquor.Formed by following steps:
The preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of described anti-avian influenza virus, is characterized in that, described Antrodia camphorata bacterium fermentation liquid ethanol extraction is made by following method:
Figure DEST_PATH_IMAGE001
heating is concentrated: Antrodia camphorata bacterium fermentation liquid or the clear liquor of the liquid fermentation of learning from else's experience are put in container, 50～70 DEG C of temperature, pressure 0.2～0.6Kg/cm2, vacuum is to carry out concentrating under reduced pressure under the condition of 0.02～0.06Mpa, and making fermentation liquid or clear liquor after concentrating under reduced pressure is 1/4～1/2 of original volume.
Figure 54315DEST_PATH_IMAGE002
ethanol extraction: the Antrodia camphorata bacterium fermentation liquid after concentrated or the temperature of clear liquor drop to 30 DEG C once, toward the ethanol that adds 80%-95% in container, make the alcoholic degree of the alcoholic solution of Antrodia camphorata bacterium fermentation liquid after concentrated or clear liquor reach 60～70% left and right, then continue heating, temperature is controlled at 30～70 DEG C, and be 30～240 minutes heat time heating time.
Figure DEST_PATH_IMAGE003
filter: afterwards cooling, with 180～200 object screen filtration Antrodia camphorata bacterium fermentation liquid or clear liquor, collect filtrate, filtering residue is with the 80%-95% ethanol dilution of its 4～8 times of volumes, repeats aforesaid operations 1～2 time, centrifugal collection filtrate;
Figure 150315DEST_PATH_IMAGE004
concentrating under reduced pressure: discard filtering residue after filtration, merge the filtrate of collecting, at 50～70 DEG C of temperature, pressure 0.2～0.6Kg/cm 2, under the condition that vacuum is 0.02～0.06Mpa, carry out concentrating under reduced pressure, reclaim ethanol, make filtrate be concentrated into 1/5～1/10 of original volume;
Figure DEST_PATH_IMAGE005
gleanings is dry: by the filtrate after above-mentioned concentrating, under 50～60 DEG C of temperature conditions, be dried 120～480 hours, unnecessary ethanol volatilizees;
(7) gleanings is pulverized: get the gleanings of above-mentioned oven dry, put under 35 DEG C of conditions and pulverize, after 200 eye mesh screens sieve, be the ethanol extraction of Antrodia camphorata bacterium anti-avian influenza virus activity.
Wherein, step
Figure 157586DEST_PATH_IMAGE001
described Antrodia camphorata bacterium fermentation liquid refer to Antrodia camphorata ( antrodia camphorata ) fermentation liquid and mycelial aggregation.Step
Figure 339037DEST_PATH_IMAGE001
described Antrodia camphorata clear liquor refer to Antrodia camphorata ( antrodia camphorata) the rear clear liquor of fermentation
Described Antrodia camphorata bacterium is fermented by following steps:
(1) liquid seeds is cultivated: adopt 500 milliliters of triangular flasks, every bottled 200 milliliters of liquid culture medium, 10% Antrodia camphorata strain is inoculated into conventional method in the conical flask that fluid medium is housed, adjusting pH is 3-8, cultivation temperature is 20-35 DEG C, shaking flask rotating speed is 80-280r/min, cultivates 7-15 days, can obtain Antrodia camphorata liquid seeds;
(2) ferment tank: first the culture medium in fermentation tank is carried out to sterilizing, sterilizing pressure is 0.1～0.2 kg/cm, and sterilization time is 1-2 hour; In the time that Antrodia camphorata liquid seeds pH value is down to 2.5-4, Antrodia camphorata seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, adjustment pH is 5-8, cultivation temperature 25-35 DEG C, ventilation 0.5-1.1vvm, speed of agitator 100-250r/min, cultivates 7-30 days, when fermentation stops, the color of culture fluid is orange colour.
Described liquid seed culture medium or fermentative medium formula in gram/100 milliliters be:
Corn starch 1～3% sucrose 1～5%
Peptone 0.2～0.6% yeast extract 0.2～0.5%
Magnesium sulfate 0.1～0.3% potassium dihydrogen phosphate 0.05～0.1%
Adopt the preparation method of the Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus of the present invention, have simple process,, the feature of low, the anti-avian influenza virus of cost.The method adopts fermentation liquid and the fermentation liquid thereof of Antrodia Camphorata mycelium to carry out active substance extraction, greatly solved Antrodia camphorata sporophore and mycelium artificial culture difficulty, reduced cost of material and production cost, improved productive rate.
The Antrodia camphorata bacterium ethanol extraction that adopts method of the present invention to extract; the In Vitro Anti bird flu virus drug screening test of doing in China Animal Health and Epidemiology Center's exotic disease research center through Qingdao Agricultural University's medicinal fungi institute; Antrodia camphorata bacterium ethanol extraction has obvious inhibitory action to bird flu virus, and cell is had to protective effect.
The concrete grammar of In Vitro Anti bird flu virus drug screening test is as follows:
Figure 417852DEST_PATH_IMAGE006
, experiment material
1, cell: mdck cell
2, virus: fowl influenza virus strain is H5N1 type
3, culture fluid: containing the DMEM culture fluid of 10% hyclone
Figure DEST_PATH_IMAGE007
, experiment condition
Three grades of laboratorys of bio-safety
Figure 774884DEST_PATH_IMAGE008
, experimental procedure
1, CPE method is measured virus to mdck cell half toxic concentration (TCID50)
Press Reed-Muench method and measure the half cell infection thing infective dose (TCID50) of mdck cell.
2, CPE method is determined the non-toxic concn of medicine to cell in conjunction with mtt assay
Measure OD492 value with enzyme-linked immunosorbent assay instrument, calculate maximal non-toxic concentration (TC0) and the median toxic concentration (TC50) of sample.
3, the evaluation of pesticide effectiveness of the maximal non-toxic concentration of extract to bird flu virus
Adopt mtt assay to measure OD492 value.In the time that the OD492 of application of sample group value is significantly greater than virus control group OD492, show that this sample liquid can significantly suppress viral infection MDCK, has good antiviral activity.
Figure DEST_PATH_IMAGE009
, experimental result
1, the TCID of virus 5obe 10 -4.
2, get 11 concentration of Antrodia camphorata bacterium ethanol extraction 64,32,16,8,4,2,1,0.5,0.25,0.125 and 0.0625mg/ml carry out cytotoxic assay, every concentration is done 10 multiple holes, establishes cell contrast simultaneously.Result shows that maximal non-toxic concentration is 32mg/ml.
3, get 32,16,8,0.8mg/ml medicinal liquid, do 10 multiple holes, observe the inhibitory action to bird flu virus.Virus control and cell contrast are established in test, and result shows that 4 variable concentrations medicines of Antrodia camphorata extract have good inhibitory action to bird flu virus.See the following form.
Below in conjunction with detailed description of the invention, the present invention is further illustrated.
  
(4) detailed description of the invention
embodiment 1:
The preparation method of the Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus:
1 fermentative medium formula:
Corn starch 1%, sucrose 1%, peptone 0.2%, yeast extract 0.2%, magnesium sulfate 0.1% potassium dihydrogen phosphate 0.05%.
2 liquid seeds are cultivated:
Adopt 500 milliliters of triangular flasks, every bottled 200 milliliters of liquid culture medium.Fermentation culture method is, 10% Antrodia camphorata strain is inoculated into conventional method in the conical flask that fluid medium is housed, and adjusts pH 6, and to cultivate under 28 DEG C of conditions, shaking flask rotating speed is 150r/min, and vibrations are cultivated 15 days.Can obtain Antrodia camphorata liquid seeds.
3. ferment tank:
Adopt 50L fermentation tank to carry out Antrodia camphorata liquid fermentation, fermentation tank sterilizing pressure is 0.1～0.2 kg/cm, and sterilization time is 1.5 hours, when tank body temperature drops to below 30 DEG C, time, the Antrodia camphorata liquid seeds of pH3.5 is transferred in 50L fermentation tank, and temperature is controlled at 28 DEG C, pH 6, ventilation 0.5～1.1vvm in tank, mixing speed is controlled at the condition of 200 revs/min, cultivates 15 days, can obtain Antrodia camphorata bacterium fermentation liquid.
The preparation of 4 fermentation liquid ethanol extractions:
heating is concentrated: the Antrodia camphorata of the liquid fermentation of learning from else's experience ( antrodia camphorata) fermentation liquid 2000ml, be put in 3000ml container, at temperature 50 C, pressure 0.6Kg/cm 2, under the condition that vacuum is 0.04Mpa, carry out concentrating under reduced pressure, making fermentation liquid or clear liquor after concentrating under reduced pressure is 1/4 of original volume.
Figure 887382DEST_PATH_IMAGE002
ethanol extraction: the Antrodia camphorata fermentation liquid after concentrated or the temperature of clear liquor drop to below 30 DEG C, toward the ethanol that adds 85% in container, make the alcoholic degree of the alcoholic solution of Antrodia camphorata fermentation liquid after concentrated or clear liquor reach 68%, then continue heating, temperature is controlled at 50 DEG C, and be 60 minutes heat time heating time.
filter: afterwards cooling, with 180 object screen filtration Antrodia camphorata ethanol extracts, collect filtrate, filtering residue is with 80% ethanol dilution of its 8 times of volumes, repeats aforesaid operations 2 times, centrifugal collection filtrate;
Figure 794475DEST_PATH_IMAGE004
filtrate decompression is concentrated: after centrifugal, discards filtering residue, merges the filtrate of collecting, and at temperature 50 C, pressure 0.6Kg/cm 2, under the condition that vacuum is 0.04～0.06Mpa, carry out concentrating under reduced pressure, reclaim ethanol, make filtrate be concentrated into 1/5 of original volume;
Figure 189553DEST_PATH_IMAGE005
gleanings is dry: by the filtrate after above-mentioned concentrating, and vacuum drying 320 hours under 50 DEG C of temperature conditions, unnecessary ethanol and moisture volatilize;
(7) gleanings is pulverized: get the gleanings of above-mentioned oven dry, put under 35 DEG C of conditions and pulverize, after 200 eye mesh screens sieve, obtain 1.65 grams of the ethanol extractions of Antrodia camphorata anti-avian influenza virus activity.
  
embodiment 2:
The preparation method of the Antrodia camphorata fermentation liquid ethanol extraction of anti-avian influenza virus:
1 fermentative medium formula:
Corn starch 1%, sucrose 1%,, peptone 0.2%, yeast extract 0.2%, magnesium sulfate 0.1% potassium dihydrogen phosphate 0.05%.
2 liquid seeds are cultivated:
Adopt 500 milliliters of triangular flasks, every bottled 200 milliliters of liquid culture medium.Fermentation culture method is, 10% Antrodia camphorata strain is inoculated into conventional method in the conical flask that fluid medium is housed, and pH7, to cultivate under 30 DEG C of conditions, shaking flask rotating speed is 200r/min, vibrations are cultivated 10 days.Can obtain Antrodia camphorata liquid seeds.
3. ferment tank:
Adopt 50L fermentation tank to carry out Antrodia camphorata liquid fermentation, fermentation tank sterilizing pressure is 0.1～0.2 kg/cm, and sterilization time is 2 hours, when tank body temperature drops to below 30 DEG C, time, the Antrodia camphorata liquid seeds of pH3 is transferred in 50L fermentation tank, and temperature is controlled at 30 DEG C, pH 6, ventilation 0.5～1.1vvm in tank, mixing speed is controlled at the condition of 180 revs/min, cultivates 10 days, can obtain Antrodia camphorata bacterium fermentation liquid.
The preparation of 4 fermentation liquid ethanol extractions:
By the Antrodia camphorata of 50L liquid fermentation (Antrodia camphorata) fermentation liquid, move on in extraction pot by pipet road, at temperature 70 C, pressure 0.6Kg/cm 2, under the condition that vacuum is 0.04～0.06Mpa, carry out concentrating under reduced pressure, making fermentation liquid or clear liquor after concentrating under reduced pressure is 1/2 of original volume; In chuck, squeeze into circulating water cooling, the fermentation liquid temperature after concentrated drops to below 30 DEG C, toward the ethanol that adds 95% in container, make the alcoholic degree of the alcoholic solution of Antrodia camphorata fermentation liquid after concentrated or clear liquor reach 70%, then continue heating, temperature is controlled at 70 DEG C, and be 120 minutes heat time heating time.Afterwards cooling, with 180 object screen filtrations, collect filtrate, filtering residue is with 95% ethanol dilution of its 4 times of volumes, repeats aforesaid operations 2 times, centrifugal collection filtrate; Merge the filtrate of collecting, concentrating under reduced pressure, temperature is controlled at 70 DEG C of left and right, pressure 0.4Kg/cm 2, vacuum is 0.06Mpa, reclaims ethanol, filtrate is concentrated into 1/10 of original volume; By above-mentioned concentrated solution, vacuum drying 480 hours under 55 DEG C of temperature conditions, unnecessary ethanol and moisture volatilize; Get oven dry gleanings, put under 35 DEG C of conditions and pulverize, after 200 eye mesh screens sieve, it is 90.6 grams that ethanol extraction weighs.

Claims (10)

1. a preparation method for the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus, is characterized in that, described Antrodia camphorata bacterium fermentation liquid ethanol extraction is made by following method:
(1) heating is concentrated: the Antrodia camphorata bacterium fermentation liquid of the liquid fermentation of learning from else's experience and mycelium or clear liquor are put in container, at 50～70 DEG C of temperature, pressure 0.2～0.6Kg/cm 2, under the condition that vacuum is 0.02～0.06Mpa, carry out concentrating under reduced pressure, making fermentation liquid or clear liquor after concentrating under reduced pressure is 1/4～1/2 of original volume;
(2) ethanol extraction: the Antrodia camphorata bacterium fermentation liquid after concentrated or the temperature of clear liquor drop to below 30 DEG C, toward the ethanol that adds 80%-95% in container, make the alcoholic degree of the alcoholic solution of Antrodia camphorata bacterium fermentation liquid after concentrated or clear liquor reach 60～70% left and right, then continue heating, temperature is controlled at 30～70 DEG C, and be 30～120 minutes heat time heating time;
(3) filter: afterwards cooling, with 180～200 object screen filtration Antrodia camphorata bacterium fermentation liquid or clear liquor, collect filtrate, filtering residue is with the 80%-95% ethanol dilution of its 4～8 times of volumes, repeats aforesaid operations 1～2 time, centrifugal collection filtrate;
(4) concentrating under reduced pressure: discard filtering residue after centrifugal, merge the filtrate of collecting, at 50～70 DEG C of temperature, pressure 0.2～0.6Kg/cm 2, under the condition that vacuum is 0.02～0.06Mpa, carry out concentrating under reduced pressure, reclaim ethanol, make filtrate be concentrated into 1/5～1/10 of original volume;
(5) gleanings is dry: by above-mentioned concentrated solution, under 50～60 DEG C of temperature conditions, be dried 120～480 hours, unnecessary ethanol and moisture volatilize;
(6) gleanings is pulverized: get the gleanings of above-mentioned oven dry, put under 35 DEG C of conditions and pulverize, after 200 eye mesh screens sieve, be the ethanol extraction of Antrodia camphorata bacterium anti-avian influenza virus activity;
Wherein, the described Antrodia camphorata bacterium fermentation liquid of step (1) refer to Antrodia camphorata ( antrodia camphorata) fermentation liquid and mycelial aggregation,
The described Antrodia camphorata clear liquor of step (1) refer to Antrodia camphorata ( antrodia camphorata) the rear clear liquor of fermentation.
2. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1, is characterized in that described Antrodia camphorata fermentation liquid obtains Antrodia camphorata fermentation mycelium and Antrodia camphorata fermented clear liquid through separating.
3. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1 or 2, is characterized in that, described Antrodia camphorata fermentation liquid obtains by following steps:
(1) liquid seeds is cultivated: adopt 500 milliliters of triangular flasks, every bottled 200 milliliters of liquid culture medium, 10% Antrodia camphorata strain is inoculated into conventional method in the conical flask that fluid medium is housed, adjusting pH is 3-8, cultivation temperature is 20-35 DEG C, shaking flask rotating speed is 80-280r/min, cultivates 7-15 days, can obtain Antrodia camphorata liquid seeds;
(2) ferment tank: first the culture medium in fermentation tank is carried out to sterilizing, sterilizing pressure is 0.1～0.2 kg/cm, and sterilization time is 1-2 hour; In the time that Antrodia camphorata liquid seeds pH value is down to 2.5-4, the Antrodia camphorata seed in shaking flask is inoculated in the culture fluid of 50L fermentation tank, adjustment pH is 5-8, cultivation temperature 25-35 DEG C, ventilation 0.5-1.1vvm, speed of agitator 100-250r/min, cultivate 7-30 days, can obtain Antrodia camphorata fermentation liquid.
4. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 2, is characterized in that, described liquid seed culture medium or fermentative medium formula in gram/100 milliliters be:
Corn starch 1～3% sucrose 1～5%
Peptone 0.2～0.6% yeast extract 0.2～0.5%
Magnesium sulfate 0.1～0.3% potassium dihydrogen phosphate 0.05～0.1%.
5. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1, is characterized in that, described Antrodia camphorata bacterial strain be called Antrodia camphorata ( antrodia camphorata) Ac001.
6. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1, is characterized in that described in step (4), filtrate is ethanol water, and concentration is 60-95%.
7. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1, it is characterized in that gleanings Dryly use vacuum drying described in step (6), vacuum 0.02-0.06Mpa, 50～60 DEG C of temperature, dry 120～480 hours.
8. the preparation method of the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1, while it is characterized in that gleanings described in step (7) is pulverized, put under 35 DEG C of conditions and pulverize, adopt 200 object Chinese medicine grinder to pulverize and sieve to gleanings.
9. the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 1 is in the purposes of preparing in Anti-avian influenza virus drugs.
10. the Antrodia camphorata bacterium fermentation liquid ethanol extraction of anti-avian influenza virus as claimed in claim 9, in the purposes of preparing in Anti-avian influenza virus drugs, is characterized in that the content of described Antrodia camphorata fermentation liquid ethanol extraction in medicine is 10%-20%.
CN201310403703.4A 2013-09-09 2013-09-09 Preparation method of antrodia-camphorate-fermented full-liquid alcohol extract with avian influenza virus resistance Pending CN103860605A (en)

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WO2021218974A1 (en) * 2020-04-28 2021-11-04 Alps Biotech Co., Ltd. Uses of antrodia cinnamomea extract in manufacturing products for reducing expression and treating associated diseases of angiotensin converting enzyme 2
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Application publication date: 20140618