CN112877219A - High-concentration cholesterol culture medium and preparation method and application thereof - Google Patents
High-concentration cholesterol culture medium and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a high-concentration cholesterol culture medium and a preparation method and application thereof, belonging to the technical field of cholesterol degradation research. The invention provides a high-concentration cholesterol culture medium, the cholesterol content in the culture medium can reach 3g/100mL, the culture medium not only contains nutrient substances required by the growth of Aspergillus oryzae, but also has better dispersity and stability, and is suitable for the research test of the degradation of the cholesterol of microorganisms, particularly Aspergillus oryzae. The invention also provides a preparation method of the high-concentration cholesterol culture medium, which does not involve the use of an organic solvent, has simple and rapid preparation steps, ensures that the prepared culture medium has uniform cholesterol distribution, and does not undergo oxidative degradation when used under normal test conditions. Experiments prove that the culture medium can be used for relevant experimental researches on degrading cholesterol of microorganisms, particularly Aspergillus oryzae.
Description
Technical Field
The invention belongs to the technical field of cholesterol degradation research, and particularly relates to a high-concentration cholesterol culture medium, and a preparation method and application thereof.
Background
Cholesterol is an essential substance of human bodies, accounts for 0.2 percent of the total weight of the human bodies, and when the cholesterol is excessive in the human bodies, hypercholesterolemia can be caused, so that diseases such as arteriosclerosis, coronary heart disease, cerebral apoplexy and the like are caused, and the health of the human bodies is seriously threatened. At present, researchers at home and abroad have more and more researches on the degradation of cholesterol by microorganisms. In the research of exploring the degradation of cholesterol by microorganisms, the preparation of a high-concentration cholesterol culture medium is an important link. However, according to the research on screening and function mechanism of cholesterol-lowering probiotic lactic acid bacteria and the record of phospholipidic and cholestol uptake by mycoplasma cells and membranes, a cholesterol culture medium used for traditional microorganism or cell culture is a lecithin-cholesterol micelle culture medium, the lecithin and cholesterol are required to be dissolved into chloroform, then the chloroform and the cholesterol are dried by nitrogen, the processes of ultrasonic crushing, centrifugation and the like are required to be carried out, the prepared culture medium is required to be stored in a refrigerator at 4 ℃, and the storage time is 2-3 days at most. The preparation process of the culture medium is complex, the preparation can not be completed in a short time, the storage time is short, and the used substances are expensive and only suitable for preparing milligram-level low-concentration cholesterol culture medium. Therefore, the sterile high-concentration cholesterol liquid cell culture medium which is simple, economical, ready-to-use and can be completed by one-man operation is developed, the cost can be greatly reduced, and the experimental efficiency can be improved.
Disclosure of Invention
The invention aims to provide a high-concentration cholesterol culture medium, the content of cholesterol in the culture medium can reach 3g/100mL, the culture medium not only contains nutrient substances required by the growth of Aspergillus oryzae, but also has better dispersity and stability, and the medium is suitable for a cholesterol degradation research test of microorganisms, particularly Aspergillus oryzae.
The second purpose of the invention is to provide a preparation method of the high-concentration cholesterol culture medium, which does not involve the use of organic solvent, has simple and rapid preparation steps, and ensures that the prepared culture medium has uniform cholesterol distribution and can not be subjected to oxidative degradation when used under normal test conditions.
The third purpose of the invention is to propose the application of the culture medium in the research of degrading cholesterol by microorganisms. Experiments prove that the culture medium can be used for relevant experimental researches on degrading cholesterol of microorganisms, particularly Aspergillus oryzae.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
in order to achieve the first object of the present invention, the present invention provides a culture medium for microorganisms containing cholesterol at a high concentration, the culture medium comprising the following raw materials: 0.15-0.25g/100mL of sodium nitrate, 0.08-0.12g/100mL of monopotassium phosphate, 0.03-0.07g/100mL of magnesium sulfate, 0.03-0.07g/100mL of potassium chloride, 0.03-0.07g/100mL of sodium chloride, 0.001-0.003g/100mL of ferrous sulfate, 0.1-3g/100mL of cholesterol and water.
According to a preferred technical scheme, the culture medium comprises the following raw materials: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 0.1-3g/100mL of cholesterol and water.
According to the technical scheme of the preferred embodiment, the culture medium comprises the following raw materials: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 1.5g/100mL of cholesterol and water.
The liquid culture medium adopting the formula can be used for the test of Aspergillus oryzae for degrading cholesterol, the culture medium contains nutrient components required by the growth of Aspergillus oryzae, and in the better culture medium components provided by the invention, the obtained culture medium containing high-concentration cholesterol is in an opaque turbid uniform liquid state, the cholesterol is uniformly and stably dispersed and is not easy to precipitate, so that a good test research basis is provided for the test of degrading cholesterol.
According to the needs of the actual situation, can also be through adding the appropriate concentration of agar to make into solid medium, preferably, the culture medium also includes the concentration of 1-2g/100ml agar. After agar is added to the medium, cholesterol can be maintained in a uniformly distributed state in the medium for a long period of time after the medium is coagulated.
In order to achieve the second object of the present invention, the present invention provides a method for preparing the above-mentioned microbial culture medium containing high concentration of cholesterol, comprising the steps of:
s1: dissolving sodium nitrate, monopotassium phosphate, magnesium sulfate, potassium chloride, sodium chloride and ferrous sulfate in water to obtain a mixed solution; when the culture medium contains agar, heating and melting the agar, and mixing the agar with the mixed solution to obtain mixed gel;
s2: sterilizing the mixed solution or the mixed gel, and standing to obtain a sterile culture medium;
s3: adding cholesterol into the sterile culture medium, and uniformly mixing to obtain the product.
According to a better technical scheme, the sterilization of S2 is high-temperature high-pressure sterilization; preferably, the high-temperature and high-pressure sterilization is sterilization in an autoclave, the sterilization temperature is 115-130 ℃, and the sterilization time is 15-30 min. The mixed solution or mixed gel contains various components including sodium nitrate, potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, sodium chloride, ferrous sulfate, etc., and various nutritional components are not lost after being sterilized in a sterilizer at 121 ℃ for 15 min.
According to a preferred technical scheme, the standing temperature of S2 is 4-25 ℃. The sterilized culture medium can be stored at room temperature for later use, the optimal storage condition is a refrigerator at 4 ℃, the culture medium can be stored for a long time, and the culture medium can be stored for more than one month under the condition of no mixed bacteria. When it is necessary to use, the sterilized culture medium may be taken out from the low-temperature environment, and cholesterol and the microorganism may be added and mixed in the same manner as in S3.
According to a preferred technical scheme, the purity of the cholesterol S3 is more than 90%.
Because the microorganism culture process is in a sterile state, after the CD culture medium without the cholesterol is sterilized, sterilized cholesterol and the microorganism to be cultured are added into a hundred-grade super clean bench. According to a preferred technical scheme, before the mixing in S3, ultraviolet sterilization is carried out on the cholesterol in a hundred-grade ultra-clean bench for 30-40 min. High-purity cholesterol is hardly subjected to oxidative degradation in an ultra-clean bench by adopting ultraviolet sterilization, and has good stability.
According to a preferred embodiment, the step S3 is performed by a conventional mixing method, and preferably, the step S is performed by shaking the mixture up and down by hand at least 20 times to uniformly disperse the cholesterol in the mixed solution or gel.
According to a preferred technical scheme, when the culture medium contains agar, the step of heating the sterile culture medium in a microwave oven to be melted before the step of mixing S3 is further included.
In order to achieve the third object of the present invention, the present invention proposes the use of the microorganism culture medium containing high concentration of cholesterol or the microorganism culture medium containing high concentration of cholesterol prepared by the above method in the research of degrading cholesterol by microorganisms; preferably, the microorganism is aspergillus oryzae.
The Aspergillus oryzae is cultured under the liquid medium components provided by the invention, and the result shows that the Aspergillus oryzae has almost complete degradation effect on cholesterol in the medium with the cholesterol concentration of 1.5g/100ml after 48 hours, and has the advantage of high degradation efficiency.
The technical scheme of the invention has the beneficial effects that: the cholesterol sterile culture medium provided by the invention is prepared by preparing a common sterile culture medium and adding sterilized high-purity cholesterol before use, so that the prepared high-concentration cholesterol culture medium is relatively stable, and the cholesterol cannot be oxidized and degraded. The prepared sterile culture medium can be stored at room temperature for a long time. The invention can be immediately prepared into the high-concentration cholesterol culture medium which can be stored for a long time and is economic and effective only by simple processes of weighing, dissolving, sterilizing and the like without complicated processes of dissolving cholesterol into an organic solvent, carrying out ultrasonic crushing, centrifuging and the like, and the culture medium has uniform and stable distribution of cholesterol, is not easy to precipitate, is economic and convenient, improves the experimental efficiency, has low price of prepared raw materials and reduces the experimental cost.
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FIG. 1 shows the growth of Aspergillus oryzae in a high-concentration cholesterol liquid medium over time;
wherein a is 0 h; b is 48 h; c is 72 h; d is 96 h.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 described in further detail below with reference to examples.
Example 1:
a cholesterol sterile CD liquid culture medium comprises effective component contents: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 1.5g/100mL of cholesterol and water.
The preparation method comprises the following specific steps:
s1, accurately weighing sodium nitrate, monopotassium phosphate, magnesium sulfate, potassium chloride, sodium chloride and ferrous sulfate by using an electronic balance, adding deionized water to dissolve, subpackaging in a triangular flask, and sealing;
s2, sterilizing the sealed triangular flask filled with the culture medium in an autoclave at 121 ℃ for 20min, and standing and storing at 4 ℃ to obtain a sterile CD liquid culture medium for later use;
s3, spreading the weighed cholesterol out and placing the cholesterol on a hundred-grade super clean bench, closing a glass window of the super clean bench, and carrying out ultraviolet sterilization for 35 min.
The high purity cholesterol was 95% pure.
And S4, after the sterilization is finished, adding the sterilized cholesterol into a liquid sterile CD liquid culture medium in a hundred-grade super clean bench, and fully shaking up the liquid sterile CD liquid culture medium to obtain a final culture medium which is an opaque, turbid and uniform liquid high-concentration cholesterol culture medium.
Cholesterol sterile CD liquid media with cholesterol concentrations of 0.1g/100ml, 0.5g/100ml, 1.0g/100ml, 2.0g/100ml, 2.5g/100ml, 3.0g/100ml and 3.5g/100ml were prepared in the same manner as described above, and the above-mentioned flasks containing cholesterol of different solubilities were placed in an environment of 10-20 ℃ to observe the occurrence of cholesterol precipitation in the liquid media. Precipitation was observed every 6 hours, and as a result, it was found that only cholesterol medium with a concentration of 3.5g/100ml produced precipitates after standing for 6 hours. No precipitate is generated after other test groups are placed for 48 hours, and therefore, the culture medium system has good stability and is not easy to generate precipitate when the concentration of cholesterol is 0.1-3g/100 ml.
Example 2
A cholesterol sterile CD solid culture medium comprises effective component contents: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 1.5g/100mL of agar, 1.5g/100mL of cholesterol and water.
The preparation method comprises the following specific steps:
s1, accurately weighing sodium nitrate, monopotassium phosphate, magnesium sulfate, potassium chloride, sodium chloride, ferrous sulfate, agar and deionized water by using an electronic balance, dissolving, subpackaging in a triangular flask, and sealing;
s2, sterilizing the sealed triangular flask filled with the culture medium in an autoclave at 121 ℃ for 20min, and standing and storing at 4 ℃ to obtain a sterile CD solid culture medium for later use;
s3, spreading the weighed cholesterol out and placing the cholesterol on a hundred-grade super clean bench, closing a glass window of the super clean bench, and carrying out ultraviolet sterilization for 35 min.
The high purity cholesterol was 95% pure.
And S4, after the sterilization is finished, adding the sterilized cholesterol into the liquid sterile CD solid culture medium in a hundred-grade super clean bench, and fully shaking up the mixture until the final culture medium state is an opaque, turbid, uniform and high-concentration cholesterol solid culture medium.
Wherein the liquid sterile CD solid culture medium is obtained by heating and melting the sterile CD solid culture medium and cooling to 50 ℃. The results show that cholesterol can be maintained in a uniform distribution state in the medium for a long period of time when the medium is coagulated.
Example 3
After 100. mu.L of Aspergillus oryzae spore suspension was added to the high-concentration cholesterol CD liquid medium (cholesterol concentration of 1.5g/100mL, and loading of 100mL) of example 1 for growth, the cholesterol CD medium to which the Aspergillus oryzae spore suspension had been added was placed in a shaker at 30 ℃ and fermented for 96 hours, and the appearance photographs of the growth at different times are shown in FIG. 1, in which a is 0 hours; b is 48 h; c is 72 h; d is 96 h. As can be seen from FIG. 1, the medium changed from turbid to clear after 48h with time, since cholesterol was insoluble in water, thus indicating that cholesterol in the high-concentration cholesterol medium was decreasing with time, and that Aspergillus oryzae gradually utilized and metabolized cholesterol. The high-concentration cholesterol CD liquid culture medium provided by the invention can be used for research in the field of cholesterol, and in the culture medium, Aspergillus oryzae has higher metabolic efficiency on cholesterol, so that most of cholesterol in the culture medium can be consumed after 48 hours.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. 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.
Claims (10)
1. A microbial culture medium containing high cholesterol concentration, which is characterized by comprising the following raw materials: 0.15-0.25g/100mL of sodium nitrate, 0.08-0.12g/100mL of monopotassium phosphate, 0.03-0.07g/100mL of magnesium sulfate, 0.03-0.07g/100mL of potassium chloride, 0.03-0.07g/100mL of sodium chloride, 0.001-0.003g/100mL of ferrous sulfate, 0.1-3g/100mL of cholesterol and water.
2. The culture medium for microorganisms containing cholesterol at a high concentration according to claim 1, wherein the culture medium comprises the following raw materials: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 0.1-3g/100mL of cholesterol and water;
preferably, the culture medium comprises the following raw materials: 0.2g/100mL of sodium nitrate, 0.1g/100mL of monopotassium phosphate, 0.05g/100mL of magnesium sulfate, 0.05g/100mL of potassium chloride, 0.05g/100mL of sodium chloride, 0.002g/100mL of ferrous sulfate, 1.5g/100mL of cholesterol and water.
3. The culture medium for microorganisms containing cholesterol at a high concentration according to claim 1 or 2, further comprising agar at a concentration of 1 to 2g/100 ml.
4. A method for preparing a culture medium for microorganisms containing cholesterol at a high concentration according to any one of claims 1 to 3, comprising the steps of:
s1: dissolving sodium nitrate, monopotassium phosphate, magnesium sulfate, potassium chloride, sodium chloride and ferrous sulfate in water to obtain a mixed solution; when the culture medium contains agar, heating and melting the agar, and mixing the agar with the mixed solution to obtain mixed gel;
s2: sterilizing the mixed solution or the mixed gel, and standing to obtain a sterile culture medium;
s3: adding cholesterol into the sterile culture medium, and uniformly mixing to obtain the product.
5. The method for preparing a culture medium for microorganisms having a high cholesterol concentration according to claim 4, wherein the sterilization at S2 is a high temperature and high pressure sterilization; preferably, the high-temperature and high-pressure sterilization is sterilization in an autoclave, the sterilization temperature is 115-130 ℃, and the sterilization time is 15-30 min.
6. The method for preparing a culture medium for microorganisms having a high cholesterol concentration according to claim 4, wherein the temperature of the standing at S2 is 4 to 25 ℃.
7. The method of claim 4, wherein the purity of the cholesterol at S3 is greater than 90%.
8. The method of claim 4 or 7, wherein the step of UV sterilizing the cholesterol in a hundred-class ultra-clean bench for 30-40min is further performed before the step of mixing S3.
9. The method of claim 4, wherein when the culture medium contains agar, the mixing step (S3) further comprises heating the sterilized culture medium in a microwave oven until the sterilized culture medium is melted.
10. Use of the culture medium of any one of claims 1 to 3 or the culture medium of any one of claims 4 to 9 for the microbiological degradation of cholesterol; preferably, the microorganism is aspergillus oryzae.
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