CN103816190A - Preparation method for extracted antradia camphorata fermented whole-liquid active substances for resisting avian influenza virus - Google Patents
Preparation method for extracted antradia camphorata fermented whole-liquid active substances for resisting avian influenza virus Download PDFInfo
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- CN103816190A CN103816190A CN201310404668.8A CN201310404668A CN103816190A CN 103816190 A CN103816190 A CN 103816190A CN 201310404668 A CN201310404668 A CN 201310404668A CN 103816190 A CN103816190 A CN 103816190A
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Abstract
The invention discloses a preparation method for extracted antradia camphorata fermented whole-liquid active substances for resisting avian influenza virus. The method comprises the following step: antradiacomphora fermented whole liquid or fermented clear liquor or mycelium is subjected to heating, filtration, vacuum concentration, alcohol precipitation, centrifugal separation, collection drying, collection smashing and the like. The antradiacomphora mycelium fermented whole-liquid active substances extracted by the method has obvious inhibiting effect on the avian influenza virus; the method has the characteristics of simple process, high yield, low cost and good effect.
Description
(1)technical field
The present invention relates to a kind of anti-avian influenza virus Antrodia camphorata fermentation liquid active substance extract preparation method, 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.
(2)background technology
The preparation method of extracting about the Antrodia camphorata fermentation liquid active substance 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.
(3)summary of the invention
The problem to be solved in the present invention is, the preparation method of extracting for the Antrodia camphorata fermentation liquid active substance of above-mentioned anti-avian influenza virus with and the existing deficiency of pharmacological action of the fermentation broth extract of preparation, the preparation method that provides a kind of Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus to extract.The Antrodia camphorata fermentation liquid active substance that utilizes method of the present invention to extract has obvious inhibitory action to bird flu virus, and normal cell is had to protective effect, and the method has simple process, output is high, cost is low feature.
The preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus of the present invention extracts, comprises that three kinds are prepared form: one is for directly comprising mycelium and clear liquor and isolate active substance from Antrodia camphorata fermentation liquid; It is two for first Antrodia camphorata fermentation liquid being separated into mycelium and clear liquor, then isolates active substance from gained mycelium; It is three for first Antrodia camphorata fermentation liquid being separated into mycelium and clear liquor, then isolates active substance from gained clear liquor.Formed by following steps:
The preparation method that the Antrodia camphorata fermentation liquid active substance of described anti-avian influenza virus extracts, is characterized in that, described Antrodia camphorata mycelium fermented full liquid extract is made by following method:
heating extraction: the Antrodia camphorata fermentation liquid of the liquid fermentation of learning from else's experience or mycelium or clear liquor are put in container, adjusting its pH value is 8～12, put at 60～120 ℃ and heat 100～240 minutes,
alcohol is analysed: in concentrated filtrate, addition is the alcoholic solution of 4～8 times of its volumes and constantly stirs, and mixing speed is 60r/min, after 10～20 minutes, leaves standstill and within 24～48 hours, carries out alcohol and analyse;
(7) gleanings is pulverized: get the gleanings of above-mentioned oven dry, pulverize, after 200 eye mesh screens sieve, be Antrodia camphorata anti-avian influenza virus extract of active substances.Wherein, step
described Antrodia camphorata fermentation liquid refer to Antrodia camphorata (
antrodia camphorata ) fermentation liquid and mycelial aggregation.
Step
described Antrodia camphorata clear liquor refer to Antrodia camphorata (
antrodia camphorata) the rear clear liquor of fermentation.
Step
described Antrodia Camphorata mycelium refer to Antrodia camphorata (
antrodia camphorata) strain fermentation gained mycelium
Described Antrodia camphorata ferments 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 ℃, 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 ℃, ventilation 0.5-1.1vvm, speed of agitator 100-250r/min, cultivates 7-30 days.
Described liquid seed culture medium or fermentative medium formula in gram/100 milliliters be:
Potato starch 1～3% maltose 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%
The preparation method that adopts the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus of the present invention to extract, has simple process, output is high, cost is low feature.The method adopts fermentation liquid, mycelium 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, production cost, increased output, improved productive rate.
The Antrodia camphorata active substance that adopts method of the present invention to extract; the In Vitro Anti bird flu virus drug screening test of doing in Chinese animal health and exotic disease research center, epidemiology center through Qingdao Agricultural University's medicinal fungi institute; Antrodia camphorata fermentation liquid active substance has stronger 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:
1, cell: mdck cell
2, virus: fowl influenza virus strain is H5N1 type
3, culture fluid: containing the DMEM culture fluid of 10% hyclone
Three grades of laboratorys of bio-safety
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 (EC50) 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 antiviral activity.
1, the TCID of virus
5obe 10
-4.
2, remove 5 concentration of Antrodia camphorata extract 16,8,4,2 and 1mg/ml carry out cytotoxic assay, every concentration is done 10 multiple holes, establishes cell contrast simultaneously.Result shows that maximal non-toxic concentration is 16 mg/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.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 the specific embodiment, the present invention is further illustrated.
(4) specific embodiment
embodiment 1:
The preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus extracts
1, fermentative medium formula:
2, potato starch 1%, maltose 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 ℃ 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 ℃, time, the Antrodia camphorata liquid seeds of pH3.5 is transferred in 50L fermentation tank, and temperature is controlled at 28 ℃, and adjusting pH is 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 mycelium fermented full liquid.
The preparation of 4 fermentation liquid extracts:
(2) afterwards cooling, with 200 object screen filtration Antrodia camphorata fermentation liquids, collect filtrate, filtering residue is with the distilled water diluting of its 4 times of volumes, repeats aforesaid operations 1 time, regathers filtrate;
(3) concentrating under reduced pressure: discard filtering residue after centrifugal, merge the filtrate of collecting, 55 ℃ of temperature, pressure 0.5Kg/cm2, carries out concentrating under reduced pressure under the condition that vacuum is 0.04Mpa, make filtrate be concentrated into 1/10～1/20 of original volume;
(4) alcohol is analysed: in concentrated filtrate, addition is 95% the ethanol of 4 times of its volumes and constantly stirs, and mixing speed is 60r/min, and after 10 minutes, standing 24h carries out alcohol and analyses;
(5) centrifugalize: alcohol is abandoned supernatant after analysing, precipitate moves in centrifuge tube, and take rotating speed as the centrifugal 15min of 5000r/min, centrifugal rear abandoning supernatant, collects centrifugal sediment;
(6) gleanings is dry: by the centrifugal sediment of above-mentioned collection, under 55 ℃ of temperature conditions, put air dry oven inner drying 48 hours;
(7) gleanings is pulverized: get the gleanings of above-mentioned oven dry, pulverize, after 200 eye mesh screens sieve, obtain 20.57 grams of the extracts of Antrodia camphorata fermentation liquid anti-avian influenza virus activity.
embodiment 2:
The preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus extracts,
1 fermentative medium formula:
Potato starch 2%, maltose 3%, peptone 0.1%, yeast extract 0.3%, 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.Does is fermentation culture method 5% Antrodia camphorata strain to be inoculated in the conical flask that fluid medium is housed to pH with conventional method?, to cultivate under 30 ℃ 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, sterilization time is 2 hours, in the time that tank body temperature drops to below 30 ℃, the Antrodia camphorata liquid seeds of pH3 is transferred in 50L fermentation tank, temperature is controlled at 30 ℃, regulating pH is 6, ventilation 0.5～1.1vvm in tank, and mixing speed is controlled at the condition of 180 revs/min, cultivate 10 days, can obtain Antrodia camphorata mycelium fermented full liquid.
The preparation of 4 fermentation liquid extracts:
By the Antrodia camphorata of 50L liquid fermentation (
antrodia camphorata) fermentation liquid, move on in extraction pot by pipet road, adjust fermentation liquid pH to 9 left and right, heat up to the logical steam of extraction pot chuck, in the time that temperature rises to 100 ℃, timing, stops heating up after maintaining 2 hours, in chuck, squeezes into circulating water cooling.When temperature drops to 50 ℃ of left and right, extracting solution is moved on in concentration tank after by secondary 200 object screen filtrations, carry out concentrating under reduced pressure, thickening temperature is controlled at 50 ℃, and vacuum is 0.06Mpa, and tank pressure is controlled at 0.2Kg/cm
2, when concentrated, add 0.1 jin/50L of defoamer (Oleum Glycines), in the time that extracting solution is concentrated to 5L left and right, moved on in alcohol extraction tank, and add 95% ethanol 20L, stir gently, mixing speed is 60r/min, after 15 minutes, standing alcohol is analysed, after 36 hours, supernatant is moved to seasoning in waste liquid tank by pipeline, precipitate carries out centrifugal after emitting, centrifugal employing low speed large capacity centrifuge, rotating speed is 5000r/min, time is controlled at 20 minutes, after centrifugal, centrifugal sediment is placed on and in stainless steel disc, carries out vacuum drying, when dry, vacuum is 0.05Mpa, temperature is controlled at 60 ℃, after 36 hours, extract is taken out, after pulverizing with 200 object Chinese medicine grinder, weigh is 560 grams.
Claims (10)
1. the preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus extracts, is characterized in that, described Antrodia camphorata mycelium fermented full liquid active substance is made by following method:
heating: the Antrodia camphorata fermentation liquid of the liquid fermentation of learning from else's experience or mycelium or clear liquor are put in container, and adjusting its pH value is 8～12, put at 60～120 ℃ and heat 100～240 minutes;
concentrating under reduced pressure: discard filtering residue after centrifugal, merge the filtrate of collecting, at 50～70 ℃ 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, make filtrate be concentrated into 1/10～1/20 of original volume;
(7) gleanings is pulverized: get the gleanings of above-mentioned oven dry, pulverize, after 200 eye mesh screens sieve, be Antrodia camphorata anti-avian influenza virus extract of active substances.
2. the preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus as claimed in claim 1 extracts, 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 mycelium fermented full liquid extract 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 ℃, 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 ℃, 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 that the Antrodia camphorata mycelium fermented full liquid active substance of anti-avian influenza virus as claimed in claim 3 extracts, is characterized in that, described liquid seed culture medium or fermentative medium formula in gram/100 milliliters be:
Potato starch 1～3% maltose 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%.
The Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus as claimed in claim 1 extract preparation method, it is characterized in that, described Antrodia camphorata bacterial strain be called Antrodia camphorata (
antrodia camphorata).
6. the preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus as claimed in claim 1 extracts, is characterized in that described in step (4), alcoholic solution is methanol or ethanol water, and concentration is 60-95%.
The Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus as claimed in claim 1 extract preparation method, it is characterized in that gleanings Dryly use vacuum drying described in step (6), vacuum 0.02-0.06Mpa, drying time 12-36 hour.
8. the preparation method that the Antrodia camphorata fermentation liquid active substance of anti-avian influenza virus as claimed in claim 1 extracts, is characterized in that when gleanings described in step (7) is pulverized adopting 200 object Chinese medicine grinder to pulverize and sieve to gleanings.
9. the Antrodia camphorata fermentation liquid active substance 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 fermentation liquid active substance 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 active substance in medicine is 10%-20%.
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