CN114591847A - Cordyceps militaris bacterium culture medium composition for increasing cordycepin content, liquid fermentation method and cordycepin preparation method - Google Patents
Cordyceps militaris bacterium culture medium composition for increasing cordycepin content, liquid fermentation method and cordycepin preparation method Download PDFInfo
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- 241001264174 Cordyceps militaris Species 0.000 title claims abstract description 94
- 238000000855 fermentation Methods 0.000 title claims abstract description 71
- 230000004151 fermentation Effects 0.000 title claims abstract description 71
- 239000007788 liquid Substances 0.000 title claims abstract description 56
- OFEZSBMBBKLLBJ-BAJZRUMYSA-N cordycepin Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)C[C@H]1O OFEZSBMBBKLLBJ-BAJZRUMYSA-N 0.000 title claims abstract description 48
- OFEZSBMBBKLLBJ-UHFFFAOYSA-N cordycepine Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)CC1O OFEZSBMBBKLLBJ-UHFFFAOYSA-N 0.000 title claims abstract description 48
- KQLDDLUWUFBQHP-UHFFFAOYSA-N Cordycepin Natural products C1=NC=2C(N)=NC=NC=2N1C1OCC(CO)C1O KQLDDLUWUFBQHP-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 239000001963 growth medium Substances 0.000 title claims abstract description 28
- 239000000203 mixture Substances 0.000 title claims abstract description 24
- 241000894006 Bacteria Species 0.000 title claims abstract description 16
- 238000000034 method Methods 0.000 title claims abstract description 13
- 238000002360 preparation method Methods 0.000 title abstract description 7
- 239000000843 powder Substances 0.000 claims abstract description 65
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 50
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 25
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 25
- 235000007516 Chrysanthemum Nutrition 0.000 claims abstract description 24
- 229910052602 gypsum Inorganic materials 0.000 claims abstract description 24
- 239000010440 gypsum Substances 0.000 claims abstract description 24
- 235000006533 astragalus Nutrition 0.000 claims abstract description 23
- 244000045232 Canavalia ensiformis Species 0.000 claims abstract description 17
- 235000010520 Canavalia ensiformis Nutrition 0.000 claims abstract description 17
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- 239000006228 supernatant Substances 0.000 claims description 97
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 51
- 238000002156 mixing Methods 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 238000003756 stirring Methods 0.000 claims description 30
- 238000001035 drying Methods 0.000 claims description 29
- 241000723353 Chrysanthemum Species 0.000 claims description 23
- 239000012153 distilled water Substances 0.000 claims description 19
- 238000007796 conventional method Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 238000004587 chromatography analysis Methods 0.000 claims description 14
- 238000000605 extraction Methods 0.000 claims description 14
- 238000010298 pulverizing process Methods 0.000 claims description 14
- 239000011347 resin Substances 0.000 claims description 14
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- 241000007126 Codonopsis pilosula Species 0.000 description 1
- 241000190633 Cordyceps Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
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- 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
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Abstract
The invention provides a cordyceps militaris bacterium culture medium composition for improving cordycepin content, a liquid fermentation method and a cordycepin preparation method, and belongs to the technical field of biology. In a first aspect, the invention provides a cordyceps militaris bacterium culture medium composition for improving cordycepin content, wherein the cordyceps militaris bacterium culture medium composition comprises astragalus mongholicus dregs, green date powder, wall breaking sword bean powder, chrysanthemum flower pollen, gypsum, magnesium sulfate and 2, 4-D; in a second aspect, the invention provides a cordyceps militaris mycelium liquid fermentation method; in a third aspect, the invention also provides a preparation method of cordycepin from cordyceps militaris mycelia. Compared with the prior art, the cordycepin content and the cordycepin purity obtained by performing liquid fermentation on the cordyceps militaris culture medium composition with the cordycepin content increased are both obviously increased.
Description
Technical Field
The invention relates to the field of biotechnology, and particularly relates to a cordyceps militaris bacterium culture medium composition for improving cordycepin content, a liquid fermentation method and a cordycepin preparation method.
Background
Cordyceps militaris is a model species of Ascomycota, Hypocreales, Clavicipitaceae, Cordyceps. The scientific name is Cordycepsmitaris (L.exFr.) Link, Cordyceps militaris, called Cordyceps militaris, or Cordyceps militaris for short, generally the Cordyceps militaris cultured by living pupa is called Cordyceps militaris, the two are the same kind of fungi, but the content difference of the two is large.
Cordyceps militaris is distributed worldwide, and the quantity of natural resources is small. In 1950, German scientist Cunningham observed that insect tissue parasitized by Cordyceps militaris is not easy to rot, and further separated an antibacterial substance, 3' -deoxyadenosine, named as cordycepin. Cordyceps militaris, pupa of a multiple infection lepidoptera insect, is a complex composed of two parts, namely a stroma (i.e. a grass part, also called a sporophore) and a sclerotium (i.e. a corpse part of the insect), and simply speaking, is the combination of an insect body and grass. The traditional Chinese medicine considers that the traditional Chinese medicine can tonify lung yin and kidney yang, is mainly used for treating kidney deficiency, impotence and spermatorrhea, soreness and pain of waist and knees, weakness after illness, chronic cough and weakness, cough with phlegm and blood caused by overexertion, spontaneous perspiration and night sweat and the like, and is a traditional Chinese medicine capable of balancing and regulating yin and yang simultaneously.
Modern researches show that the content of cordycepin in cordyceps militaris mycelia is higher than that of cordyceps militaris fruiting bodies, the cordycepin is extracted from the cordyceps militaris mycelia, the cordyceps militaris mycelia has the advantages of high growth speed of the mycelia, simple fermentation steps, easiness in control and the like, and the production time can be shortened and the production efficiency can be improved by extracting the cordycepin from the mycelia.
In view of the above, the invention provides a fermentation medium composition for producing cordyceps militaris mycelia, a fermentation method thereof and a method for preparing cordycepin.
Disclosure of Invention
The invention aims to provide a cordyceps militaris bacterium culture medium composition for improving cordycepin content, a liquid fermentation method and a cordycepin preparation method.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
in a first aspect, the invention provides a cordyceps militaris bacterium culture medium composition for improving cordycepin content, wherein the cordyceps militaris bacterium culture medium composition comprises astragalus mongholicus dregs, green date powder, wall breaking sword bean powder, chrysanthemum flower pollen, gypsum and magnesium sulfate.
Furthermore, in a preferred embodiment of the invention, the astragalus residue is 5-15 parts by weight, the green date powder is 20-30 parts by weight, the green sword bean powder is 6-16 parts by weight, the chrysanthemum pollen is 0.3-1 part by weight, the gypsum is 0.6-1 part by weight, and the magnesium sulfate is 0.3-1 part by weight.
Further, in a preferred embodiment of the invention, the cordyceps militaris bacterium culture medium composition further comprises 2,4-D in parts by weight.
Further, in a preferred embodiment of the invention, the astragalus residue is 8-12 parts by weight, the green date powder is 23-27 parts by weight, the wall breaking sword bean powder is 9-11 parts by weight, the chrysanthemum pollen is 0.3-1 part by weight, the gypsum is 0.6-1 part by weight, the magnesium sulfate is 0.3-1 part by weight, and the 4-D is 0.0006-0.0010 part by weight.
Furthermore, in a preferred embodiment of the invention, the astragalus residue is 10 parts, the green date powder is 25 parts, the garden pea powder is 10 parts, the chrysanthemum pollen is 0.7 part, the gypsum is 0.8 part, the magnesium sulfate is 0.7 part, and the 2,4-D is 0.0008 part by weight.
In a second aspect, the present invention provides a method for liquid fermentation of cordyceps militaris mycelia, comprising:
5-15 g/L of astragalus residue, 20-30 g/L of green date powder, 6-16 g/L of wall-broken sword bean powder, 0.3-1 g/L of chrysanthemum pollen, 0.6-1 g/L of gypsum and 0.3-1 g/L of magnesium sulfate in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement to a specified amount, adjusting the pH to 6.2-6.8, sterilizing at 121 ℃ for 15-30 min, cooling to obtain a liquid culture medium, inoculating cordyceps militaris strain into the cordyceps militaris strain, performing liquid fermentation, culturing at 22 ℃ in a dark room for 7 days, culturing at 23-25 ℃ under light, ventilating and stirring, performing fermentation for 6-10 days, filtering or centrifuging the cultured fermentation liquid at 3500-8000 rpm, collecting the fermentation supernatant, washing and drying to obtain cordyceps militaris mycelium.
Further, in the cordyceps militaris mycelium liquid fermentation medium, the astragalus residue is 8-12 g/L, the green date powder is 23-27 g/L, the broken sword bean powder is 9-11 g/L, the chrysanthemum pollen is 0.3-1 g/L, the gypsum is 0.6-1 g/L, the magnesium sulfate is 0.3-1 g/L and the 2,4-D is 0.0006-0.0010 g/L, distilled water is added to supplement to a specified amount, the pH value is 6.2-6.8, sterilization is carried out at 121 ℃ for 15-30 min, then the liquid culture medium is prepared, the cordyceps militaris mother strain is inoculated conventionally and then subjected to liquid fermentation, after culturing at 22 ℃ in a dark room for 7D, culturing under light at 24 ℃, stirring, ventilating for 8D, the cultured fermentation time is 4000rpm, the fermented supernatant is reserved, mycelia are collected, and are washed and dried to obtain the cordyceps militaris mycelium.
Further, in a preferred embodiment of the invention, in the cordyceps militaris mycelium liquid fermentation medium, 10g/L of astragalus residue, 25g/L of green date powder, 10g/L of wall breaking sword bean powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate and 0.0008g/L of 2,4-D are added, distilled water is supplemented to a specified amount, the pH value is 6.2-6.8, the mixture is sterilized at 121 ℃ for 15-30 min and then cooled to prepare a liquid culture medium, the cordyceps militaris mother strain is inoculated conventionally and then subjected to liquid fermentation, after being cultured at 22 ℃ in a dark room for 7D, the culture medium is cultured under light, the temperature is 24 ℃, ventilation and stirring are carried out, the fermentation time is 8D, the cultured fermentation broth is centrifuged at 4000rpm, the fermentation supernatant is reserved, mycelia are collected, and the cordyceps militaris mycelium is obtained through washing and drying.
In a third aspect, the invention also provides a preparation method of cordycepin from cordyceps militaris mycelium, which comprises the following steps: after crushing by a conventional method, mixing the mycelium powder with water in a ratio of 1: 13-1: 23, adjusting the pH value to 7-9, carrying out water bath at 85-98 ℃, stirring and extracting for 3-5 h, and centrifuging at 4000rpm to obtain a supernatant; concentrating the obtained supernatant to 10-20% of the original volume, adding 30-35% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residues for 1-2 times according to the extraction steps, and merging supernate; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Further, in a preferred embodiment of the invention, the mycelium powder is mixed with water at a ratio of 1: 18 in a 90 ℃ water bath at 33% ethanol after conventional comminution.
The cordyceps militaris mother strain used for liquid fermentation in the invention is prepared by a conventional method.
Compared with the prior art, the invention has the beneficial effects that:
compared with the prior art, the dry weight of the mycelia, the cordycepin amount and the cordycepin purity of the cordyceps militaris are all obviously improved.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are further described in detail below with reference to examples:
example 1
5g/L of astragalus medicine residues, 30g/L of green date powder, 6g/L of wall-broken sword bean powder, 1g/L of chrysanthemum pollen, 0.8g/L of gypsum and 0.3g/L of magnesium sulfate in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the components to a specified amount, keeping the pH value at 6.8, sterilizing at 121 ℃ for 30min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 25 deg.C under ventilation and stirring for 6d, centrifuging the cultured fermentation liquid at 6000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 13, adjusting pH to 9, extracting in water bath at 85 deg.C under stirring for 5 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 20% of the original volume, adding 30% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 2 times according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Example 2
15g/L of astragalus medicine residues, 20g/L of green date powder, 16g/L of wall-breaking sword bean powder, 0.3g/L of chrysanthemum pollen, 0.8g/L of gypsum and 1g/L of magnesium sulfate in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the components to a specified amount, adjusting the pH value to 6.2, sterilizing at 121 ℃ for 15min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 23 deg.C under ventilation and stirring for 10d, press-filtering the cultured fermentation liquid with 60kg pressure, collecting mycelia, washing, and drying to obtain Cordyceps militaris mycelia.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 23, adjusting pH to 7, extracting in water bath at 98 deg.C under stirring for 3 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 10% of the original volume, adding 35% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Example 3
8g/L of astragalus residue, 27g/L of green jujube powder, 9g/L of broken sword bean powder, 1g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.3g/L of magnesium sulfate, 2, and 0.0006g/L of 4-D in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the distilled water to a specified amount, adjusting the pH value to 6.8, sterilizing at 121 ℃ for 20min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 23 deg.C under ventilation and stirring for 10d, centrifuging the cultured fermentation liquid at 2800rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 13, adjusting pH to 9, extracting in water bath at 85 deg.C under stirring for 5 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 20% of the original volume, adding 30% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 2 times according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Example 4
12g/L of astragalus medicine residues, 23g/L of green date powder, 11g/L of wall-breaking sword bean powder, 0.3g/L of chrysanthemum pollen, 0.8g/L of gypsum, 1g/L of magnesium sulfate, 2, and 0.0010g/L of 4-D in a cordyceps militaris mycelium liquid fermentation culture medium, adding distilled water to supplement the distilled water to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 20min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 3000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 23, adjusting pH to 7, extracting in water bath at 98 deg.C under stirring for 3 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 10% of the original volume, adding 35% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Example 5
In the cordyceps militaris mycelium liquid fermentation medium, astragalus dregs are 10g/L, green date powder is 25g/L, wall-broken sword bean powder is 10g/L, chrysanthemum pollen is 0.7g/L, gypsum is 0.8g/L, magnesium sulfate is 0.7g/L and 2,4-D is 0.0008g/L, distilled water is added to supplement the mixture to a specified amount, the pH value is 6.5, and the mixture is cooled after being sterilized for 25min at 121 ℃.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and (4) mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain the cordycepin.
Comparative example 1
10g/L of glucose, 10g/L of astragalus dregs, 25g/L of green date powder, 10g/L of semen trichosanthis powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate, 2,4-D of 2 and 0.0008g/L in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 25min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 2
10g/L of peptone, 10g/L of astragalus dregs, 25g/L of green date powder, 10g/L of semen trichosanthis powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate, 2,4-D of 2 and 0.0008g/L in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 25min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 3
10g/L of codonopsis pilosula dregs, 10g/L of astragalus dregs, 25g/L of green date powder, 10g/L of semen trichosanthis powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate, 2,4-D of magnesium sulfate and 0.0008g/L of distilled water are added to supplement the total amount, the pH value is 6.5, and the cordyceps militaris mycelium liquid fermentation culture medium is cooled after being sterilized at the temperature of 121 ℃ for 25 min.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 4
10g/L of glucose, 25g/L of green date powder, 10g/L of semen phaseoli radiati powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate, 2, 0.0008g/L of 4-D in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the mixture to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 25min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 5
10g/L of peptone, 10g/L of astragalus dregs, 25g/L of green date powder, 0.7g/L of chrysanthemum pollen, 0.8g/L of gypsum, 0.7g/L of magnesium sulfate and 2, 0.0008g/L of 4-D in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the components to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 25min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 6
In the cordyceps militaris mycelium liquid fermentation medium, astragalus dregs are 10g/L, green date powder is 25g/L, sword bean powder is 10g/L, chrysanthemum pollen is 0.7g/L, gypsum is 0.8g/L, magnesium sulfate is 0.7g/L and 2,4-D is 0.0008g/L, distilled water is added to supplement the materials to a specified amount, the pH value is 6.5, and the materials are sterilized at 121 ℃ for 25min and then cooled.
Inoculating Cordyceps militaris strain stock seed conventionally, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C, ventilating and stirring, fermenting for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at a ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
Comparative example 7
12g/L of astragalus medicine residues, 30g/L of green date powder, 12g/L of wall-breaking sword bean powder, 0.84g/L of chrysanthemum pollen, 0.96g/L of gypsum, 0.84g/L of magnesium sulfate, 2, and 0.00096g/L of 4-D in a cordyceps militaris mycelium liquid fermentation medium, adding distilled water to supplement the distilled water to a specified amount, adjusting the pH to 6.5, sterilizing at 121 ℃ for 25min, and cooling.
Inoculating Cordyceps militaris strain, performing liquid fermentation, culturing at 22 deg.C in dark room for 7d, culturing under light at 24 deg.C under ventilation and stirring for 8d, centrifuging the cultured fermentation liquid at 4000rpm, collecting the supernatant, washing, and drying to obtain Cordyceps militaris mycelium.
Pulverizing by conventional method, mixing mycelium powder with water at ratio of 1: 18, adjusting pH to 8, extracting in water bath at 90 deg.C under stirring for 4 hr, centrifuging at 4000rpm, and collecting supernatant; concentrating the obtained supernatant to 15% of the original volume, adding 33% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residue for 1 time according to the above extraction steps, and mixing the supernatants; and (4) mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain the cordycepin.
The beneficial effects of the invention are as follows:
| cordycepin amount (g/L) | Cordycepin purity (%) | |
| Example 1 | 12.8 | 86.6 |
| Example 2 | 13.7 | 87.9 |
| Example 3 | 13.5 | 89.5 |
| Example 4 | 15.2 | 90.8 |
| Example 5 | 16.8 | 92.5 |
| Comparative example 1 | 9.5 | 80.0 |
| Comparative example 2 | 8.6 | 76.1 |
| Comparative example 3 | 9.0 | 79.6 |
| Comparative example 4 | 9.2 | 81.2 |
| Comparative example 5 | 8.9 | 78.5 |
| Comparative example 6 | 8.6 | 79.9 |
| Comparative example 7 | 10.5 | 82.4 |
As can be seen from the above table, in the embodiments 1 to 5 of the invention, the cordycepin content and the cordycepin purity obtained by performing liquid fermentation on the cordyceps militaris culture medium composition with the cordycepin content increased are both significantly increased compared with the comparative examples 1 to 7.
The details which are not described in the present invention are the common general knowledge which can be selected by the ordinary skilled person in the art. Although the invention has been described in detail hereinabove with respect to specific embodiments thereof, it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A cordyceps militaris bacterium culture medium composition for increasing cordycepin content is characterized by comprising astragalus mongholicus dregs, green date powder, wall breaking sword bean powder, chrysanthemum flower pollen, gypsum and magnesium sulfate.
2. The cordyceps militaris bacterium culture medium composition according to claim 1, wherein the cordyceps militaris bacterium culture medium composition further comprises 2, 4-D.
3. The cordyceps militaris bacterium culture medium composition according to claim 1, wherein the astragalus mongholicus dregs are 5-15 parts by weight, the green date powder is 20-30 parts by weight, the garcinia esculenta powder is 6-16 parts by weight, the chrysanthemum pollen is 0.3-1 part by weight, the gypsum is 0.6-1 part by weight, and the magnesium sulfate is 0.3-1 part by weight.
4. The cordyceps militaris bacterium culture medium composition according to claim 2, wherein the astragalus mongholicus dregs are 8-12 parts by weight, the green date powder is 23-27 parts by weight, the wall breaking sword bean powder is 9-11 parts by weight, the chrysanthemum flower pollen is 0.3-1 part by weight, the gypsum is 0.6-1 part by weight, the magnesium sulfate is 0.3-1 part by weight, and the 2,4-D is 0.0006-0.0010 part by weight.
5. The cordyceps militaris bacterium culture medium composition according to claim 2, wherein the astragalus mongholicus dregs are 10 parts, the green date powder is 25 parts, the wall breaking sword bean powder is 10 parts, the chrysanthemum pollen is 0.7 part, the gypsum is 0.8 part, the magnesium sulfate is 0.7 part, and the 2,4-D is 0.0008 part by weight.
6. A cordyceps militaris mycelium liquid fermentation method is characterized in that the cordyceps militaris mycelium culture medium composition disclosed in claims 1-5 is supplemented with distilled water to a specified amount, the pH value is 6.2-6.8, the cordyceps militaris mycelium culture medium composition is sterilized at 121 ℃ for 15-30 min and then cooled to prepare a liquid culture medium, a cordyceps militaris strain mother strain is inoculated into the liquid culture medium for fermentation, the cordyceps militaris strain mother strain is cultured at 22 ℃ in a dark room for 7 days and then is cultured under light at 23-25 ℃, ventilation and stirring are carried out, the fermentation time is 6-10 days, the cultured fermentation liquid is filtered or centrifuged at 3500-8000 rpm, the fermentation supernatant is reserved, mycelia are collected, and the cordyceps militaris mycelium is obtained after washing and drying.
7. The fermentation process according to claim 6, wherein the liquid fermentation temperature is 24 ℃, the fermentation time is 8d, and the fermentation broth centrifugation speed is 4000 rpm.
8. A method for preparing cordycepin from Cordyceps militaris is characterized by pulverizing the Cordyceps militaris mycelium of claim 6 to obtain mycelium powder; after the mycelium powder is crushed by a conventional method, mixing the mycelium powder with water in a ratio of 1: 13-1: 23, adjusting the pH value to 7-9, carrying out water bath at 85-98 ℃, stirring and extracting for 3-5 h, and centrifuging at 4000rpm to obtain a supernatant; concentrating the obtained supernatant to 10-20% of the original volume, adding 30-35% ethanol, centrifuging at 4000rpm, and collecting the supernatant; extracting the centrifuged mycelium residues for 1-2 times according to the extraction steps, and merging supernate; and mixing the supernatant with the fermented supernatant, performing D101 macroporous resin chromatography, and concentrating and drying the analytic solution to obtain cordycepin.
9. The method for preparing cordycepin from Cordyceps militaris as claimed in claim 8, wherein the mycelium powder is mixed with water at a ratio of 1: 18 after the conventional pulverization.
10. The method for preparing cordycepin from Cordyceps militaris as claimed in claim 8, wherein said water bath is 90 deg.C and said ethanol concentration is 33%.
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