CN111742785B - Long-term preservation method of cordyceps militaris strain with high cordycepin yield - Google Patents
Long-term preservation method of cordyceps militaris strain with high cordycepin yield Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a long-term preservation method of cordyceps militaris strains with high cordycepin productivity. Inoculating Cordyceps militaris strain to test tube slant culture medium, culturing at 18-25 deg.C in constant temperature incubator until mycelia overgrow the slant to obtain slant strain; cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae; adding protective solution and the hypha-containing fungus blocks into the freezing tube, completely immersing the fungus blocks in the protective solution, preserving the freezing tube by adopting a temperature-changing treatment method for pretreatment, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃. The culture medium containing ginkgo nut powder is adopted, so that the cordycepin production activity of cordyceps militaris strains can be maintained. The invention also adopts a temperature-changing treatment method of reducing the room temperature to-10 ℃, then heating to 4 ℃, and finally reducing to-80 ℃, thereby improving the low-temperature resistance of the strain and keeping the activity of the cordyceps militaris strain.
Description
Technical Field
The invention belongs to the technical field of agricultural microorganisms, and particularly relates to a long-term preservation method of cordyceps militaris strains with high cordycepin productivity.
Background
Cordyceps militaris belongs to the genus Cordyceps of the family Clavicipitaceae of the subdivision Ascomycotina and is an important traditional Chinese medicinal material. Cordycepin contained in Cordyceps militaris is nucleoside antibiotic, has antibacterial, antiinflammatory, antiviral and antitumor activities, and has high nutritive value and medicinal value. The screening and preservation work of cordyceps militaris strains with high cordycepin production capacity is an important prerequisite for separating and extracting cordycepin. The quantity of the cordyceps militaris growing in nature is small, and the yield is low, so that the technology for artificially culturing the high cordycepin cordyceps militaris is gradually increased.
In the prior art, the preservation method of cordyceps militaris strains mainly comprises the steps of test tube inclined plane refrigeration at 4-15 ℃, air-conditioned room preservation at 26 ℃, test tube inclined plane refrigeration at 4-15 ℃, paraffin oil refrigeration at 26 ℃, sterile water air-conditioned room preservation at 26 ℃ and the like, wherein the method for the air-conditioned room preservation at 26 ℃ and the sterile water air-conditioned room preservation at 26 ℃ is suitable for short-term preservation, can effectively ensure the activity of the strains in the current generation, can be taken and used at any time, but is fast in strain propagation at the temperature and is not beneficial to the preservation of the strains in the next generation. The test tube inclined plane refrigeration at the temperature of 4-15 ℃ is an important means for preserving cordyceps militaris strains for a long time due to the fact that the temperature is low and the strains can survive for a long time, but the activity of the strains is reduced after the inclined plane refrigeration for a long time, rejuvenation operation is often needed, rejuvenation is unsuccessful after some strains are refrigerated for a long time, the risk of reducing the cordycepin production activity exists, and the yield of cordycepin is reduced. Therefore, it is necessary to develop a long-term preservation method of cordyceps militaris strains capable of maintaining the production activity of cordycepin.
Disclosure of Invention
The long-term preservation method of the cordyceps militaris strain with high cordycepin production capacity can keep the cordycepin production activity of cordyceps militaris after preservation.
The invention aims to provide a long-term preservation method of cordyceps militaris strains with high cordycepin productivity, which comprises the following steps: inoculating Cordyceps militaris strain to test tube slant culture medium, culturing at 18-25 deg.C in constant temperature incubator until mycelia overgrow the slant to obtain slant strain;
cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae for later use;
adding a protective solution and a fungus block containing hyphae into a freezing tube, completely immersing the fungus block in the protective solution, preserving the freezing tube by adopting a temperature-changing treatment method for pretreatment, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃;
the temperature change range of the temperature change treatment method is as follows: the room temperature is reduced to-10 ℃, then the temperature is increased to 4 ℃, and finally the temperature is reduced to-80 ℃.
Preferably, in the above preservation method, the formula of the test tube slant culture medium is as follows: 180g of potato, 20-30g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural.
Preferably, in the above preservation method, the formula of the test tube slant culture medium is as follows: 180g of potato, 20g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural.
Preferably, in the above preservation method, the tube slant medium is prepared by the following method:
weighing potatoes, ginkgo nut powder, glucose, monopotassium phosphate, dipotassium phosphate, magnesium sulfate and agar according to the formula;
slicing potatoes, adding the sliced potatoes into 800mL of water with the volume of less than 500-;
adding glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar into the potato filtrate, supplementing 1000mL of distilled water, heating and stirring until all materials are dissolved, adding ginkgo nut powder, uniformly mixing, sterilizing, and placing an inclined plane.
Preferably, in the preservation method, the temperature-changing treatment method carries out preservation pretreatment according to the temperature sequence of room temperature, 4 ℃, 10 ℃, 4 ℃,20 ℃ and 80 ℃.
Preferably, in the preservation method, the temperature-changing treatment method specifically includes: placing the freezing tube filled with the fungus blocks at room temperature for standing for 0.5-1 hour, preserving at 4 ℃ for 12-12.5 hours, preserving at-10 ℃ for 12-12.5 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day, and preserving at-80 ℃ for one day.
Preferably, in the preservation method, the protective solution is liquid paraffin or a sterile glycerol solution with a volume fraction of 30-35%.
Preferably, in the above preservation method, when the cryopreservation tube is taken for inspection, the cryopreservation tube is taken out from the environment of-80 ℃, then is placed at-20 ℃ for 12 hours and at 4 ℃ for 12 hours in sequence, and finally is naturally restored to room temperature and then is used for inspection.
Compared with the prior art, the long-term preservation method of the cordyceps militaris strain with high cordycepin production capacity provided by the invention has the following beneficial effects:
the culture medium containing the ginkgo nut powder is adopted, and the ginkgo nut powder provides nutrition for the growth of cordyceps militaris on one hand, and can keep the cordycepin production activity of cordyceps militaris strains on the other hand.
The invention also adopts a temperature change treatment method of reducing the room temperature to-10 ℃, then heating to 4 ℃, and finally reducing to-80 ℃, thereby improving the low temperature resistance of the strain. The mycelia grow fast when the strains are activated after ultralow temperature preservation at minus 80 ℃, the degradation degree of the mycelia is small, the dry weight of the mycelia is high, and the cordycepin production capacity in the fermentation process of the cordyceps militaris liquid is high.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods in the following examples, which are not specified in specific conditions, are generally conducted under conventional conditions, and the steps thereof will not be described in detail since they do not relate to the invention. The "room temperature" in the examples and experiments described below is 20-30 ℃. In the following examples and experiments, 30% volume fraction sterile glycerol was prepared as follows: weighing 30mL of glycerol, adding distilled water, diluting to 100mL, and autoclaving at 121 deg.C for 30 min.
Example 1
A long-term preservation method of Cordyceps militaris strains with high cordycepin productivity comprises the following steps:
inoculating Cordyceps militaris strains to a test tube slant culture medium, wrapping the outer surface of a test tube with newspaper (namely, culturing in a dark light environment), and culturing in a constant-temperature incubator at 20 +/-1 ℃ until hyphae grow over the slant to obtain slant strains;
cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae for later use;
adding 30% of aseptic glycerol and hypha-containing fungus blocks into a freezing tube, completely immersing the fungus blocks in the 30% of aseptic glycerol, preserving the freezing tube by adopting a temperature-variable treatment method, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃;
the temperature-changing treatment method comprises the following specific steps: and placing the freezing tube filled with the fungus blocks at room temperature for standing for 0.5 hour, preserving at 4 ℃ for 12 hours, preserving at-10 ℃ for 12 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day and preserving at-80 ℃ for one day.
In example 1, the formulation of the tube slant medium is as follows: 180g of potato, 20g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural. The ginkgo nut powder is 80-mesh powder formed by crushing ginkgo nuts.
The test tube slant culture medium is prepared according to the following method:
the formula comprises potato, ginkgo nut powder, glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar;
slicing potato, adding 500mL (the amount of water is based on the following standard: submerging potato slices, and submerging potato slices after boiling for 20 min), boiling for 20min, filtering with four layers of gauze, and collecting potato filtrate;
adding glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar into the potato filtrate, supplementing 1000mL of distilled water, heating and stirring until all materials are dissolved, adding ginkgo nut powder, uniformly mixing, sterilizing, and placing an inclined plane (since the ginkgo nut powder is possibly precipitated after sterilization, uniformly mixing by vortex before placing the inclined plane so that the ginkgo nut powder is uniformly dispersed in an inclined plane culture medium as far as possible).
Example 2
A long-term preservation method of Cordyceps militaris strains with high cordycepin productivity comprises the following steps:
inoculating Cordyceps militaris strains to a test tube slant culture medium, wrapping the outer surface of a test tube with newspaper, and culturing in a constant temperature incubator at 20 +/-1 ℃ until hyphae grow over the slant to obtain slant strains;
cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae for later use;
adding liquid paraffin and a fungus block containing hyphae into a freezing tube, completely immersing the fungus block in the liquid paraffin, preserving the freezing tube by adopting a temperature-changing treatment method for pretreatment, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃;
the temperature-changing treatment method comprises the following specific steps: and placing the freezing tube filled with the fungus blocks at room temperature for standing for 1 hour, preserving at 4 ℃ for 12.5 hours, preserving at-10 ℃ for 12.5 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day and preserving at-80 ℃ for one day.
In example 2, the formulation and preparation method of the test tube slant medium are the same as those of example 1.
Example 3
A long-term preservation method of Cordyceps militaris strains with high cordycepin productivity comprises the following steps:
inoculating Cordyceps militaris strains to a test tube slant culture medium, wrapping the outer surface of a test tube with newspaper, and culturing in a constant temperature incubator at 20 +/-1 ℃ until hyphae grow over the slant to obtain slant strains;
cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae for later use;
adding 30% of aseptic glycerol and hypha-containing fungus blocks into a freezing tube, completely immersing the fungus blocks in the 30% of aseptic glycerol, preserving the freezing tube by adopting a temperature-variable treatment method, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃;
the temperature-changing treatment method comprises the following specific steps: and placing the freezing tube filled with the fungus blocks at room temperature for standing for 0.5 hour, preserving at 4 ℃ for 12 hours, preserving at-10 ℃ for 12 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day and preserving at-80 ℃ for one day.
In example 3, the formulation of the tube slant medium is as follows: 180g of potato, 30g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural. The ginkgo nut powder is 60-mesh powder formed by crushing ginkgo nuts.
The test tube slant culture medium is prepared according to the following method:
the formula comprises potato, ginkgo nut powder, glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar;
slicing potato, adding 800mL of water (the amount of water is based on the following standard: submerging potato slices, and submerging potato slices after boiling for 20 min), boiling for 20min, filtering with six layers of gauze, and collecting potato filtrate;
adding glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar into the potato filtrate, supplementing 1000mL of distilled water, heating and stirring until all materials are dissolved, adding ginkgo nut powder, uniformly mixing, sterilizing, and placing an inclined plane (since the ginkgo nut powder is possibly precipitated after sterilization, uniformly mixing by vortex before placing the inclined plane so that the ginkgo nut powder is uniformly dispersed in an inclined plane culture medium as far as possible).
To investigate the effects of the present invention, we performed the following experiments.
1. Design of experiments
Test strains: "Haizhou No. one" cordyceps militaris and CICC No.14014 cordyceps militaris.
Blank control group: the strain before preservation.
The preservation control group: inoculating Cordyceps militaris strain to PDA (adding 0.01g of microorganism B per liter of culture medium)1) On slant culture medium, newspaper wrapping the outer surface of test tube, culturing in 20 + -1 deg.C constant temperature incubatorGrowing the filaments on the inclined plane to obtain inclined plane strains; cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae; adding 30% volume fraction of aseptic glycerol and mycelia containing bacteria block into the freezing tube, completely immersing the bacteria block in 30% volume fraction of aseptic glycerol, preserving the freezing tube at-80 deg.C for one day, taking the freezing tube for inspection, and selecting the surviving bacteria for long-term preservation at-80 deg.C.
Group A: the bacterial species were preserved as described in example 1.
Group B: the bacterial species were preserved as described in example 2.
Group C: the bacterial species were preserved as described in example 3.
Group D: the method is basically the same as that of the preserved control group, except that:
the formula of the test tube slant culture medium is as follows: potato 200g, glucose 20g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 0.5g, magnesium sulfate 0.5g, agar 20g, distilled water make up to 1000mL, natural pH.
The test tube slant culture medium is prepared according to the following method:
the formula comprises potato, glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar; slicing potatoes, adding the sliced potatoes into 500mL of water, boiling for 20min, filtering with four layers of gauze, and collecting potato filtrate; adding glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar into the potato filtrate, supplementing 1000mL of distilled water, heating and stirring until all materials are dissolved, sterilizing after uniformly mixing, and placing on an inclined plane.
Group E: the method is basically the same as that of the preserved control group, except that:
after 30% of sterile glycerol completely submerges the bacterium blocks, preserving the freezing tube by adopting a temperature-changing treatment method, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at the temperature of minus 80 ℃. The temperature-changing treatment method comprises the following specific steps: and placing the freezing tube filled with the fungus blocks at room temperature for standing for 0.5 hour, preserving at 4 ℃ for 12 hours, preserving at-10 ℃ for 12 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day and preserving at-80 ℃ for one day.
The strains were preserved for 60 days and 90 days, respectively, according to the experimental design described above. The blank control group was not preserved and the relevant experiments were performed directly.
2. Strain revival
The preserved strain is inoculated on the test tube slant culture medium prepared in the test tube slant culture medium in the embodiment 1, the culture is carried out at the temperature of 20 +/-1 ℃, and a strain block with the diameter of 5mm is taken as a revival strain for standby after hyphae grow fully.
The blank control group was run directly without rejuvenation.
3. Measurement of growth rate of mycelia after preservation
Inoculating a 5 mm-sized block of revived strain in the center of a PDA (personal digital assistant) plate with the diameter of 9cm, culturing at 20 +/-1 ℃, and observing the growth speed of hyphae, wherein the growth speed of the strain is the diameter/time of a colony. Each experiment was set up in 3 replicates and averaged. The results in Table 1 show that the hypha growth rate of two Cordyceps militaris strains is degraded after being preserved by the common method (preservation control group), but the hypha growth rate is not degraded after being preserved by the method (A-C) of the embodiment 1-3.
TABLE 1 hyphal growth rates of different experimental groups
4. Measurement of the Dry weight of the mycelia after preservation
Inoculating 5mm size fungus block of revived strain in 100ml triangular flask containing 50ml liquid PDA culture medium, culturing at 20 + -1 deg.C for 15d, filtering to remove liquid culture medium, washing mycelium with distilled water twice, oven drying mycelium at 60 deg.C to constant weight, and weighing. Each experiment was set up in 3 replicates and averaged. The results in Table 2 show that the hypha dry weight of two Cordyceps militaris strains is degraded after being preserved by the common method (preservation control group), but the hypha dry weight is not degraded after being preserved for 60 days by the method (A-C groups) of the embodiments 1-3; after 90 days of storage in groups A-C, the dry weight of hyphae remained consistent with that of the blank control.
TABLE 2 Dry weight of hyphae of different experimental groups
5. Content determination of cordycepin produced by preserved strain
According to 'Tangjiapeng, Wangjiaxiong' and plant growth regulator, cordyceps militaris liquid surface culture is promoted to produce cordycepin [ J ]. food industry science and technology, 2018 and v.39; no.402(10) 128-. The results in Table 3 show that the cordycepin yield of two Cordyceps militaris strains is reduced after the two Cordyceps militaris strains are preserved by a common method (preservation control group), while the cordycepin yield of Cordyceps militaris strains is not degraded after the two Cordyceps militaris strains are preserved for 60 days and 90 days by the method (A-C groups) of the embodiment 1-3.
TABLE 3 cordycepin yields for different experimental groups
In the above experiments, the parameter tests of all the deposited strains were measured after activation, and these data are effective for evaluating the growth rate of the mycelia after reactivation of the strains after long-term storage.
It should be noted that, when the present invention relates to a numerical range, it should be understood that two endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those in the embodiment, in order to prevent redundancy, the present invention describes a preferred embodiment. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. A long-term preservation method of cordyceps militaris strains with high cordycepin productivity is characterized by comprising the following steps:
inoculating Cordyceps militaris strain to test tube slant culture medium, culturing at 18-25 deg.C in constant temperature incubator until mycelia overgrow the slant to obtain slant strain;
the formula of the test tube slant culture medium is as follows: 180g of potato, 20-30g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural;
cutting the slant strains into pieces in a sterile environment to obtain fungus pieces containing hyphae;
adding a protective solution and a fungus block containing hyphae into a freezing tube, completely immersing the fungus block in the protective solution, preserving the freezing tube by adopting a temperature-changing treatment method for pretreatment, then taking the freezing tube for inspection, and selecting the surviving strains for long-term preservation at-80 ℃;
the temperature-changing treatment method comprises the following steps: placing the freezing tube filled with the fungus blocks at room temperature for standing for 0.5-1 hour, preserving at 4 ℃ for 12-12.5 hours, preserving at-10 ℃ for 12-12.5 hours, preserving at 4 ℃ for one day, preserving at-20 ℃ for one day, and preserving at-80 ℃ for one day.
2. The long-term storage method of cordyceps militaris strains with high cordycepin production capacity according to claim 1, wherein the formula of the test tube slant culture medium is as follows: 180g of potato, 20g of ginkgo nut powder, 20g of glucose, 0.5g of monopotassium phosphate, 0.5g of dipotassium phosphate, 0.5g of magnesium sulfate, 20g of agar and distilled water, wherein the total volume is 1000mL, and the pH value is natural.
3. The long-term storage method of cordyceps militaris strains with high cordycepin production capacity according to claim 1, wherein the test tube slant culture medium is prepared by the following method:
weighing potato, ginkgo nut powder, glucose, monopotassium phosphate, dipotassium phosphate, magnesium sulfate and agar according to the formula of claim 1;
slicing potatoes, adding the sliced potatoes into 800mL of water with the volume of less than 500-;
adding glucose, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate and agar into the potato filtrate, supplementing 1000mL of distilled water, heating and stirring until all materials are dissolved, adding ginkgo nut powder, uniformly mixing, sterilizing, and placing an inclined plane.
4. The long-term storage method of cordyceps militaris strains with high cordycepin production capacity according to claim 1, wherein the protective solution is liquid paraffin or a sterile glycerol solution with a volume fraction of 30-35%.
5. The long-term storage method of Cordyceps militaris strains with high cordycepin production capacity according to claim 1, wherein the frozen tube is taken out from the environment of-80 ℃ for inspection, and then placed at-20 ℃ for 12 hours, then at 4 ℃ for 12 hours, and then naturally returned to room temperature for inspection.
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