CN112322507A - Strain culture medium of Mortierella pseudomorpha and culture method thereof - Google Patents
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Abstract
The invention discloses a culture medium of a strain of Mortierella metamiformis and a culture method thereof. The culture method adopting the culture medium comprises the following steps: s1, separating strains; s2, preparing a culture medium; s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium. The strain is a strain separated from wild cordyceps sinensis, has the characteristics of quick culture and convenient operation, is similar to the active ingredients of cordyceps sinensis, and can be used as a substitute of cordyceps sinensis mycelia.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a strain culture medium of Mortierella metamiformis and a culture method thereof.
Background
The strain is a strain separated from wild cordyceps sinensis, has the characteristics of quick culture and convenient operation, is similar to the active ingredients of cordyceps sinensis, and can be used as a substitute of cordyceps sinensis mycelia. The method of the present invention can produce a large amount of fermentation product having various physiologically active substances in a much shorter production cycle than other methods using insects as hosts. The method of the present invention reproducibly produces high yields of high quality mycelial fermentation products in about 15 days on a fermentation scale. For example, products containing adenosine, guanosine and mannitol, and having a higher arachidonic acid, have been obtained by the process of the invention. At present, the growth conditions of the wild cordyceps sinensis are very harsh and the wild cordyceps sinensis is blindly and destructively harvested and dug by human beings, so that the wild resources are endangered to be extinct. Meanwhile, the growth period is long and the picking is difficult.
Disclosure of Invention
The invention aims to provide a strain culture medium of Mortierella protea and a culture method thereof, which aim to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a culture medium of a strain of Mortierella protea comprises a carbon source, a nitrogen source, salts and a coagulant, wherein the carbon source comprises glucose, sucrose and maltose, the nitrogen source comprises yeast extract powder, peptone and hydrolyzed silkworm pupa protein, the salts adopt sodium citrate, potassium dihydrogen phosphate and magnesium sulfate, and the coagulant adopts agar, wherein the addition of the sodium citrate can obviously improve the fermentation condition of the Mortierella protea and simultaneously improve the adenosine content of fermented mycelia.
Preferably, the mass percentages of the glucose, the sucrose and the maltose in the culture medium are respectively 1% -3%, 1% -2% and 1% -3%.
Preferably, the yeast extract powder, the peptone, the hydrolyzed silkworm pupa protein and the agar account for 1 to 2 percent, 0.3 to 1 percent and 0.8 to 1.2 percent of the culture medium in percentage by mass.
Preferably, the mass percentage of the sodium citrate, the monopotassium phosphate and the magnesium sulfate in the culture medium is 0.1-0.3%, 0.2-0.5% and 0.1-0.3%.
Preferably, the form of the medium is liquid or solid.
Preferably, the pH value of the culture medium is 5.0-6.5.
A culture method of a strain of Mortierella protea, which adopts the culture medium to culture, and is characterized by comprising the following steps:
s1, separating strains;
s2, preparing a culture medium;
s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium.
Preferably, the method for separating the strains in the step S1 includes washing natural and fresh cordyceps sinensis with purified water, washing with sterile water in a sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, removing surface water with sterile paper, cutting off outer skins, avoiding intestinal tracts, slicing the white tissue, inoculating the white tissue on a common separation culture medium, standing and culturing at 5-15 ℃ for 10 days, allowing white hyphae to grow from the surface of the culture medium, and further separating and purifying the white hyphae to obtain the Mortierella morphescens.
Preferably, the preparation method of the culture medium in the step S2 is to weigh the carbon source, the nitrogen source and the salt according to the mass ratio, put the weighed materials into a container, heat and dissolve the materials, mix the materials uniformly, and then sterilize the materials according to a conventional sterilization method.
Compared with the prior art, the invention has the beneficial effects that:
the strain is a strain separated from wild cordyceps sinensis, has the characteristics of quick culture and convenient operation, is similar to the active ingredients of cordyceps sinensis, and can be used as a substitute of cordyceps sinensis mycelia. The method of the present invention can produce a large amount of fermentation product having various physiologically active substances in a much shorter production cycle than other methods using insects as hosts. The method of the present invention reproducibly produces high yields of high quality mycelial fermentation products in about 15 days on a fermentation scale.
Drawings
FIG. 1 is a graph comparing components of wild Cordyceps sinensis and Mortierella pseudomorpha.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1, the present invention provides a technical solution: a culture medium of strains of Mortierella metamiformis comprises a carbon source, a nitrogen source, salts and a coagulant, wherein the carbon source comprises glucose, sucrose and maltose, the nitrogen source comprises yeast extract powder, peptone and hydrolyzed silkworm pupa protein, the salts comprise sodium citrate, potassium dihydrogen phosphate and magnesium sulfate, the coagulant comprises agar, the mass percent of the glucose, the sucrose and the maltose in the culture medium is 1%, the mass percent of the yeast extract powder, the peptone, the hydrolyzed silkworm pupa protein and the agar in the culture medium is 1%, 0.3% and 0.8%, the mass percent of the sodium citrate, the potassium dihydrogen phosphate and the magnesium sulfate in the culture medium is 0.1%, 0.2% and 0.1%, the rest components are water, the culture medium is in a liquid state or a solid state, and the pH value of the culture medium is 5.0-6.5.
A culture method of a strain of Mortierella protea adopts the culture medium to culture, and is characterized by comprising the following steps:
s1, separating strains;
s2, preparing a culture medium;
s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium.
Specifically, the method for separating the strains in step S1 includes washing natural fresh cordyceps sinensis with purified water, washing with sterile water in a sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, removing surface water with sterile paper, cutting off outer skin, avoiding intestinal tracts, cutting white tissue into slices, inoculating the slices on a common separation culture medium, standing and culturing at 5-15 ℃ for 10 days, allowing white hyphae to grow from the surface of the culture medium, and further separating and purifying the white hyphae to obtain the Mortierella pseudomorpha.
Specifically, the preparation method of the culture medium in step S2 includes weighing the carbon source, the nitrogen source and the salt according to the mass ratio, placing the weighed materials into a container, heating, dissolving, mixing uniformly, and sterilizing according to a conventional sterilization method.
Example 2
Referring to fig. 1, the present invention provides a technical solution: a culture medium of strains of Mortierella metamiformis comprises a carbon source, a nitrogen source, salts and a coagulant, wherein the carbon source comprises glucose, sucrose and maltose, the nitrogen source comprises yeast extract powder, peptone and hydrolyzed silkworm pupa protein, the salts comprise sodium citrate, potassium dihydrogen phosphate and magnesium sulfate, the coagulant comprises agar, the mass percentages of the glucose, the sucrose and the maltose in the culture medium are respectively 2%, 1.5% and 2%, the mass percentages of the yeast extract powder, the peptone, the hydrolyzed silkworm pupa protein and the agar in the culture medium are respectively 1.5%, 0.6% and 1%, the mass percentages of the sodium citrate, the potassium dihydrogen phosphate and the magnesium sulfate in the culture medium are respectively 0.2%, 0.35% and 0.2%, and the balance of water, the culture medium is in a liquid state or a solid state, and the pH value of the culture medium is 5.0-6.5.
A culture method of a strain of Mortierella protea adopts the culture medium to culture, and is characterized by comprising the following steps:
s1, separating strains;
s2, preparing a culture medium;
s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium.
Specifically, the method for separating the strains in step S1 includes washing natural fresh cordyceps sinensis with purified water, washing with sterile water in a sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, removing surface water with sterile paper, cutting off outer skin, avoiding intestinal tracts, cutting white tissue into slices, inoculating the slices on a common separation culture medium, standing and culturing at 5-15 ℃ for 10 days, allowing white hyphae to grow from the surface of the culture medium, and further separating and purifying the white hyphae to obtain the Mortierella pseudomorpha.
Specifically, the preparation method of the culture medium in step S2 includes weighing the carbon source, the nitrogen source and the salt according to the mass ratio, placing the weighed materials into a container, heating, dissolving, mixing uniformly, and sterilizing according to a conventional sterilization method.
Example 3
Referring to fig. 1, the present invention provides a technical solution: a culture medium of strains of Mortierella metamiformis comprises a carbon source, a nitrogen source, salts and a coagulant, wherein the carbon source comprises glucose, sucrose and maltose, the nitrogen source comprises yeast extract powder, peptone and hydrolyzed silkworm pupa protein, the salts comprise sodium citrate, potassium dihydrogen phosphate and magnesium sulfate, the coagulant comprises agar, the glucose, the sucrose and the maltose account for 3%, 2% and 3% of the culture medium respectively, the yeast extract powder, the peptone, the hydrolyzed silkworm pupa protein and the agar account for 2%, 1% and 1.2% of the culture medium respectively, the rest components are water, the sodium citrate, the potassium dihydrogen phosphate and the magnesium sulfate account for 0.3%, 0.5% and 0.3% of the culture medium respectively, the culture medium is in a liquid state or a solid state, and the pH value of the culture medium is 5.0-6.5.
A culture method of a strain of Mortierella protea adopts the culture medium to culture, and is characterized by comprising the following steps:
s1, separating strains;
s2, preparing a culture medium;
s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium.
Specifically, the method for separating the strains in step S1 includes washing natural fresh cordyceps sinensis with purified water, washing with sterile water in a sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, removing surface water with sterile paper, cutting off outer skin, avoiding intestinal tracts, cutting white tissue into slices, inoculating the slices on a common separation culture medium, standing and culturing at 5-15 ℃ for 10 days, allowing white hyphae to grow from the surface of the culture medium, and further separating and purifying the white hyphae to obtain the Mortierella pseudomorpha.
Specifically, the preparation method of the culture medium in step S2 includes weighing the carbon source, the nitrogen source and the salt according to the mass ratio, placing the weighed materials into a container, heating, dissolving, mixing uniformly, and sterilizing according to a conventional sterilization method.
Comparative example 1
Cleaning natural fresh Cordyceps with purified water, washing with sterile water in sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, removing surface water with sterile paper, cutting off outer skin, avoiding intestinal tract, cutting white tissue into slices, inoculating to separation culture medium surface, standing at 5-15 deg.C for 10 days to allow white hypha to grow out from the culture medium surface, and further separating and purifying the white hypha to obtain Mortierella morphescens.
Comparative example 2
The invention provides a technical scheme that: a method for fermenting Mortierella morphescens mycelium is provided.
Preparing a fermentation liquid culture medium, wherein the concentration of glucose is 20g/L, the concentration of sucrose is 10g/L, the concentration of maltose is 20g/L, the concentration of yeast extract powder is 10g/L, the concentration of peptone is 10g/L, the concentration of potassium dihydrogen phosphate is 3g/L, the concentration of hydrolyzed silkworm pupa protein is 5g/L, and the concentration of magnesium sulfate is 1.5 g/L; placing into a container, heating, dissolving with 1L pure water, mixing, and sterilizing by conventional sterilization method.
And step two, inoculating the Mortierella pseudomorpha separated in the comparative example 1 into the liquid culture medium obtained in the step one, and standing and fermenting for 14 days at the culture temperature of 25 ℃ in an aerobic environment to obtain fermentation hyphae.
Comparative example 3
The invention provides a technical scheme that: a method for fermenting Mortierella morphescens mycelium is provided. The difference from the practical example 2 is that 1g/L sodium citrate is added in the first step.
The content comparison of adenosine in the Mortierella morphescens mycelium prepared in comparative example 2 of the invention is detected by using natural Cordyceps sinensis mycelium as a control group according to the following method, and is determined by liquid chromatography under the chromatographic condition that a Shim-packGISTC18 chromatographic column (5um,4.6x250mm) is selected; phosphate buffer (pH6.5) and methanol (85: 15) are used as mobile phases, the flow rate is 1.0ml/min, the detection wavelength is 260nm, and the sample injection amount is 5 uL; column temperature 25 deg.C
Respectively sampling 0.5g of wild cordyceps sinensis, the samples of comparative example 2 and comparative example 3, placing the samples into a conical flask with a plug, adding 10mL of methanol with the volume fraction of 90% into the conical flask, plugging the plug of the conical flask tightly, shaking up uniformly, weighing, heating and refluxing for 30min, cooling, weighing again, then complementing the weight loss amount with the methanol with the volume fraction of 90%, shaking up uniformly, filtering with a 0.45 mu m microporous membrane, taking the filtrate as a sample solution, taking the sample solution, and testing the adenosine content (mu g/g) in the samples according to the chromatographic conditions.
Comparing the content of adenosine in wild Cordyceps. The content of adenosine in the Mortierella pseudomorpha mycelia is greatly increased. The content of adenosine in mycelium produced by the culture medium added with sodium citrate is also greatly improved compared with the common culture medium.
| Sample name | Natural wild cordyceps | Comparative example 2 | Comparative example 3 |
| Adenosine content mg/g | 0.388 | 2.9 | 4.4 |
Comparison of the biotransformation efficiency of comparative example 2 and comparative example 3
Comparing comparative example 2 and comparative example 3, culturing for 14 days at the same time, weighing dry weight, and calculating the biotransformation rate to obtain that the biotransformation rate of mycelium produced by the culture medium added with sodium citrate is higher.
| Comparative example 2 | Comparative example 33 | |
| Biological conversion rate | 32％ | 45.23％ |
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A strain culture medium of Mortierella metamiformis is characterized in that: the culture medium comprises a carbon source, a nitrogen source, salts and a coagulant, wherein the carbon source comprises glucose, sucrose and maltose, the nitrogen source comprises yeast extract powder, peptone and hydrolyzed silkworm pupa protein, the salts adopt sodium citrate, potassium dihydrogen phosphate and magnesium sulfate, and the coagulant adopts agar.
2. The culture medium for Mortierella proteus strain according to claim 1, wherein: the glucose, the sucrose and the maltose account for 1 to 3 percent, 1 to 2 percent and 1 to 3 percent of the culture medium respectively by mass percent.
3. The culture medium for Mortierella proteus strain according to claim 1, wherein: the yeast extract powder, the peptone, the hydrolyzed silkworm pupa protein and the agar account for 1 to 2 percent, 0.3 to 1 percent and 0.8 to 1.2 percent of the culture medium in percentage by mass.
4. The culture medium for Mortierella proteus strain according to claim 1, wherein: the sodium citrate, the monopotassium phosphate and the magnesium sulfate account for 0.1-0.3 percent, 0.2-0.5 percent and 0.1-0.3 percent of the culture medium in percentage by mass.
5. The culture medium for Mortierella proteus strain according to claim 1, wherein: the form of the culture medium is liquid or solid.
6. The culture medium for Mortierella proteus strain according to claim 1, wherein: the pH value of the culture medium is 5.0-6.5.
7. A method for culturing a Mortierella pseudomorpha strain by using the culture medium of claim 1, comprising the steps of:
s1, separating strains;
s2, preparing a culture medium;
s3, inoculating the strain separated in the step S1 to the surface of a fermentation medium, and performing static culture for 14 days at the temperature of 20-30 ℃ in an aerobic environment to obtain the fermentation mycelium.
8. The method for culturing Mortierella proteus strain according to claim 7, wherein: the separation method of the strains in the step S1 comprises the steps of cleaning natural fresh cordyceps sinensis with purified water, washing with sterile water in a sterile environment, sterilizing with 75% ethanol, washing with sterile water for multiple times, absorbing surface water with sterile paper, cutting off outer skins, avoiding intestinal tracts, cutting white tissues into thin slices, inoculating the thin slices on a common separation culture medium, standing and culturing for 10 days at the temperature of 5-15 ℃, allowing white hyphae to grow out of the surface of the culture medium, and further separating and purifying the white hyphae to obtain the Mortierella pseudomorpha.
9. The method for culturing Mortierella proteus strain according to claim 7, wherein: the preparation method of the culture medium in the step S2 comprises the steps of weighing the carbon source, the nitrogen source and the salt according to the mass ratio, putting the weighed materials into a container, heating, dissolving, uniformly mixing, and then sterilizing according to a conventional sterilization method.
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