CN108251490B - Fermentation medium and fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia - Google Patents
Fermentation medium and fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia Download PDFInfo
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Abstract
The invention discloses a fermentation medium for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia and a fermentation method, wherein the fermentation medium comprises, by weight, 2% -5% of corn flour, 1% -2% of soybean flour, 1% -3% of glucose, 0.1% -0.5% of potassium dihydrogen phosphate, 0.05% -0.1% of magnesium sulfate, 150-100 ppm of vitamin B, 200-300 mu mol/L of methyl jasmonate and the balance of water.
Description
Technical Field
The invention belongs to the technical field of edible and medicinal fungus production processes, and particularly relates to a fermentation medium and a fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, so that the ganoderic acid A content of the ganoderma lucidum mycelia is improved.
Background
Ganoderma lucidum is praised as 'Rucao' for strengthening body resistance and consolidating constitution by medical scientists of our country, and has been studied in large quantities for its pharmacological actions, and the studies show that Ganoderma lucidum has wide pharmacological actions, such as anti-tumor action, immunoregulation action, radioresistance and chemoresistance action, sedative and tranquilization action, cardiotonic and anti-myocardial ischemia action, blood lipid regulation action, blood glucose reduction action, asthma relief action, liver protection action, anti-anoxia and anti-aging action, etc. With the intensive research on ganoderma lucidum in modern science, more than 350 chemical components, mainly polysaccharides, terpenoids, alkaloids, peptides, amino acids, sterols, vitamins and the like, are separated so far.
Modern pharmacological research proves that the ganoderma triterpene has the effects of resisting tumor, protecting liver, resisting HIV-1 and HIV-1 protease activity, resisting histamine release, resisting aging, resisting microorganisms, reducing blood fat, reducing blood sugar and the like. The method for obtaining the ganoderma triterpene material at the present stage comprises 3 methods, namely field sporocarp or artificially cultivated sporocarp, spore, liquid fermentation mycelium and fermentation liquor. Compared with artificial cultivation of ganoderma lucidum, the ganoderma lucidum triterpene produced by the liquid submerged fermentation technology has the characteristics of no influence of seasons, short production period, relatively stable triterpene content and the like, and is the most effective method for obtaining the ganoderma lucidum triterpene.
The content of total triterpenes in ganoderma lucidum mycelia produced by a liquid submerged fermentation technology is high and can reach 2-3 times of that of cultured ganoderma lucidum fruit bodies and ganoderma lucidum spore powder, and ganoderic acid A in the total triterpenes is a natural compound with obvious pharmacological activity in ganoderma lucidum, and is a main functional component for achieving the effects of enhancing immunity, protecting liver, expelling toxin, reducing blood pressure, reducing cholesterol, resisting tumors and the like.
Disclosure of Invention
In view of the above, the invention provides a fermentation medium and a fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, so that the fermentation time is prolonged, the content of ganoderic acid A in a ganoderma lucidum mycelia fermentation product is improved, and the pharmacological activity of the ganoderma lucidum mycelia is enhanced.
The technical scheme of the invention is as follows:
a fermentation medium for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia comprises, by weight, 2% -5% of corn flour, 1% -2% of soybean flour, 1% -3% of glucose, 0.1% -0.5% of monopotassium phosphate, 0.05% -0.1% of magnesium sulfate, 150-100 ppm of vitamin B, 200-300 [ mu ] mol/L of methyl jasmonate and the balance of water.
Preferably, the pH value of the fermentation medium is 6.0-7.0.
The invention also provides a fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, which comprises the following steps: inoculating the ganoderma lucidum mycelium seed liquid into the fermentation culture medium for fermentation, wherein the fermentation temperature is 25-26 ℃, and when the pH value of the fermentation culture medium is reduced to 3.5-4.0, the fermentation is stopped.
Preferably, the fermentation time is 3-4 days.
Preferably, the inoculation amount of the ganoderma lucidum mycelium seed liquid is 10-15%.
Preferably, the method further comprises the following steps after the fermentation is terminated: filtering the fermented culture medium, drying, and pulverizing to obtain Ganoderma mycelia.
Preferably, the ganoderma lucidum mycelium seed liquid is obtained by culturing ganoderma lucidum mycelium strains in a liquid culture medium at 25-26 ℃, and the liquid culture medium comprises, by mass, 2-4% of corn flour, 1-2% of soybean flour, 1-3% of glucose, 1% of yeast extract, 0.1-0.5% of monopotassium phosphate, 0.05-0.1% of magnesium sulfate, 150-100 ppm of vitamin B and the balance of water.
Preferably, the culturing includes activation culturing and scale-up culturing.
More preferably, the activation culture time is 3-4 days, and the period of the amplification culture is 2-3 days.
The invention also comprises the ganoderma lucidum mycelium prepared by any one of the methods, wherein the content of ganoderic acid A in the ganoderma lucidum mycelium is 60-105 mg/g.
Compared with the prior art, the invention has the following advantages:
the invention provides a fermentation medium for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, which comprises, by weight, 2% -5% of corn flour, 1% -2% of soybean flour, 1% -3% of glucose, 0.1% -0.5% of potassium dihydrogen phosphate, 0.05% -0.1% of magnesium sulfate, 150-100 ppm of vitamin B, 200-300 mu mol/L of methyl jasmonate and the balance of water.
The invention provides a fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, which is characterized in that the ganoderma lucidum mycelia are subjected to liquid submerged fermentation by using a culture medium containing methyl jasmonate, so that the fermentation culture time of the ganoderma lucidum is prolonged to 3-4 days, the mycelia are cultured after autolysis, and the ganoderic acid A is synthesized in a large amount during secondary metabolism to form components of a ganoderma lucidum mycelia structure.
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FIG. 1 shows the fermentation process of liquid fermentation of Ganoderma mycelia according to the present invention.
Detailed Description
The invention provides a fermentation medium for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, which comprises, by weight, 2% -5% of corn flour, 1% -2% of soybean meal, 1% -3% of glucose, 0.1% -0.5% of monopotassium phosphate, 0.05% -0.1% of magnesium sulfate, 150-100 ppm of vitamin B, 200-300 mu mol/L of methyl jasmonate and the balance of water.
In the invention, the fermentation medium preferably comprises 3-4% of corn flour, 1.2-1.8% of soybean flour, 1.5-2.5% of glucose, 0.2-0.4% of monopotassium phosphate, 0.06-0.08% of magnesium sulfate, 160-90 ppm of vitamin B, 220-270 mu mol/L of methyl jasmonate and the balance of water.
In the invention, the pH value of the fermentation medium is preferably 6.0-7.0, and more preferably 6.5-7.0.
According to the invention, a certain amount of methyl jasmonate is added into the fermentation medium, and the fermentation time of the ganoderma lucidum mycelia can be prolonged by optimizing the formula of the medium, so that the synthesis time of the ganoderic acid A is ensured under the condition that the yield of the mycelia is not reduced, and the content of the ganoderic acid A in the fermentation product is increased.
The components of the fermentation medium are mixed according to the proportion to prepare the fermentation medium of the liquid fermentation ganoderma lucidum mycelia.
The invention also provides a fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia, which comprises the following steps: inoculating the ganoderma lucidum mycelium seed liquid into the fermentation culture medium for fermentation, wherein the fermentation temperature is 25-26 ℃, and when the pH value of the fermentation culture medium is reduced to 3.5-4.0, the fermentation is stopped.
The ganoderma lucidum mycelium seed solution is obtained by culturing ganoderma lucidum mycelium strains in a liquid culture medium at 25-26 ℃, and the liquid culture medium is obtained by diluting a plate culture medium for preserving the ganoderma lucidum mycelium strains. The liquid culture medium comprises, by mass, 2-4% of corn flour, 1-2% of soybean flour, 1-3% of glucose, 1% of yeast extract, 0.1-0.5% of monopotassium phosphate, 0.05-0.1% of magnesium sulfate, 150-100 ppm of vitamin B and the balance of water; preferably 2.5 to 3.5 percent of corn flour, 1.5 percent of soybean meal, 1.5 percent of glucose, 1 percent of yeast extract, 0.2 to 0.4 percent of monopotassium phosphate, 0.06 to 0.08 percent of magnesium sulfate, 160 to 90ppm of vitamin B and the balance of water. The plate culture medium is a culture medium for conventional ganoderma lucidum mycelium preservation in the field, such as PDA culture medium.
The source of the ganoderma lucidum mycelium strain is not specially limited, and the ganoderma lucidum mycelium strain is preserved in the conventional field or sold on the market.
In the present invention, the culture preferably includes activation culture and amplification culture which are performed sequentially. The culture medium used for the activation culture and the amplification culture is preferably a liquid culture medium as described in the above technical means.
The activation culture is to dilute a plate culture medium with ganoderma lucidum mycelia with water and inoculate the plate culture medium in a liquid culture medium, wherein the dilution multiple is preferably about 1 square centimeter, the plate culture medium with the culture medium is diluted and inoculated in 100-300 ml of the liquid culture medium, more preferably about 1 square centimeter, the plate culture medium with the culture medium is diluted and inoculated in 150ml of the liquid culture medium, the diluted liquid culture medium is subjected to shake culture, the shaking speed is 100-120 r/min, more preferably 110 r/min, the activation culture time is preferably 3-4 days, the activation culture is preferably performed in a stirring tank, the stirring tank is subjected to shake culture in a shaker, and particularly, the stirring tank is preferably 30L stirring tank, and activated ganoderma lucidum mycelia are obtained after the shake culture.
The method comprises the steps of carrying out amplification culture on activated ganoderma lucidum mycelia, preferably carrying out first-stage amplification culture and second-stage amplification culture, preferably carrying out amplification in a fermentation tank step by step according to the volume of the fermentation tank from small to large, preferably adopting a 300-500L seed tank for the first-stage amplification culture, and adopting a 1-2 ton seed tank for the second-stage amplification culture, preferably carrying out 2-3 days for each stage of amplification culture, wherein after 2-3 days of fermentation, the quantity of the mycelia in a liquid culture medium is too large, the nutrition provided by the liquid culture medium cannot support the growth of the mycelia, so that aged mycelia are autolyzed in the culture solution to reduce the extraction rate of the mycelia.
Inoculating the seed liquid of the ganoderma lucidum mycelia into the fermentation culture medium for fermentation. The inoculation amount is preferably 10-15%, and more preferably 12-13%. The fermentation is aerobic fermentation. The aeration ratio in the fermentation process is preferably 0.5-1.0, and more preferably 0.6-0.8; the stirring speed in the fermentation process is 100-120 r/min, and more preferably 110 r/min.
The pH value of the fermentation medium is gradually reduced along with the fermentation, and the fermentation is preferably stopped when the pH value of the fermentation medium is reduced to 3.8-3.9. In this case, the fermentation time of the ganoderma lucidum mycelia is preferably 3 to 4 days. However, in the conventional liquid culture medium without methyl jasmonate, the autolysis phenomenon occurs when the fermentation time of the ganoderma lucidum mycelia is less than 3 days, so that insufficient time is available for synthesizing the ganoderic acid A in the ganoderma lucidum mycelia, and the content of the ganoderic acid A in the fermentation product is very low.
After the fermentation is terminated, the fermentation medium with the ganoderma lucidum mycelia is taken out, filtered, dried and crushed to obtain the ganoderma lucidum mycelia product. The filtration is preferably plate-and-frame filter pressing, and the pressure is preferably 50-60 kg, and more preferably 55 kg. The drying is preferably vacuum drying, and the temperature of the vacuum drying is preferably 60-65 ℃, and more preferably 62-64 ℃. The particle size of the crushed powder is preferably 60-80 meshes, and more preferably 65-70 meshes.
In the fermented ganoderma lucidum mycelia obtained by the fermentation method, the content of ganoderic acid A can reach 60-105 mg/g, so that the pharmaceutical activity of the ganoderma lucidum mycelia is greatly improved, and the effects of enhancing immunity, protecting liver, expelling toxin, reducing blood pressure, reducing cholesterol, resisting tumors and the like are improved.
The present invention will be described in detail with reference to examples for better understanding the objects, technical solutions and advantages of the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
1) Selecting ganoderma lucidum mycelium strains:
2) and shaking table planting: keeping the temperature at 25 ℃, diluting the strain into 300ml of liquid culture medium by using a flat plate strain with the culture medium of about 1 square centimeter, placing the strain on an oscillator, and stirring for 3 days, wherein the stirring speed is controlled to be 100 revolutions per minute; the liquid culture medium comprises 2% of corn flour, 1% of soybean flour, 1% of glucose, 1% of yeast extract, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 150 ppm of vitamin B and the balance of water.
3) Maintaining the temperature at 25 ℃, sucking the culture medium stirred in the step 2) into a 300L seed tank with liquid culture medium under negative pressure, and culturing for 2 days;
4) and second-stage amplification: keeping the temperature at 25 ℃, sucking the liquid culture medium stirred in the step 3) into a 1.5-ton seeding tank with the liquid culture medium under negative pressure, and culturing for 2 days to obtain ganoderma lucidum mycelium seed liquid;
5) fermenting with methyl jasmonate culture medium by sucking Ganoderma mycelia seed solution 1000L obtained in step 4) into a 7-ton fermentation tank under negative pressure at 25 deg.C, adding into 5000L fermentation culture medium containing methyl jasmonate (254 μmol/L), and fermenting for 3 days;
the methyl jasmonate culture medium comprises, by weight, 2% of corn flour, 1% of soybean meal, 1% of glucose, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 150 ppm of vitamin B, 254 mu mol/L% of methyl jasmonate and the balance of deionized water.
The pH of the liquid medium after fermentation was 3.8.
6) And plate and frame filter pressing: taking out the liquid culture medium fermented in the step 5), and performing pressure filtration and drying under the pressure of 50kg to obtain a solid primary product;
7) drying and crushing: drying the solid primary product obtained in the step 6) in vacuum at the drying temperature of 60 ℃, and crushing after drying to obtain the high-content ganoderic acid A ganoderma lucidum mycelium product with the particle size of 60 meshes, wherein the yield is 2%.
Example 2
1) Selecting ganoderma lucidum mycelium strains:
2) and shaking table planting: keeping the temperature at 26 ℃, diluting the strain into 150ml of liquid culture medium by using a flat plate strain with the culture medium about 1 square centimeter, placing the strain on an oscillator, and stirring for 4 days, wherein the stirring speed is controlled to be 120 revolutions per minute; the liquid culture medium comprises, by mass, 4% of corn flour, 2% of soybean flour, 1% -3% of glucose, 1% of yeast extract, 0.5% of monopotassium phosphate, 0.1% of magnesium sulfate, 1100 ppm of vitamin B and the balance of water.
3) Maintaining the temperature at 26 ℃, sucking the culture medium stirred in the step 2) into a 300L seed tank with liquid culture medium under negative pressure, and culturing for 3 days;
4) and second-stage amplification: keeping the temperature at 26 ℃, sucking the liquid culture medium stirred in the step 3) into a 1.5-ton seeding tank with the liquid culture medium under negative pressure, and culturing for 3 days to obtain ganoderma lucidum mycelium seed liquid;
5) fermenting with methyl jasmonate culture medium by maintaining the temperature at 26 deg.C, sucking Ganoderma mycelia seed solution 1000L obtained in step 4) into a 7 ton fermentation tank under negative pressure, adding into 5000L fermentation culture medium containing methyl jasmonate (300 μmol/L), and fermenting for 3 days;
the methyl jasmonate culture medium comprises, by weight, 5% of corn flour, 2% of soybean meal, 3% of glucose, 0.5% of monopotassium phosphate, 0.1% of magnesium sulfate, 1100 ppm of vitamin B, 300 mu mol/L% of methyl jasmonate and the balance of deionized water.
The pH of the liquid medium after fermentation was 4.0.
6) And plate and frame filter pressing: taking out the liquid culture medium fermented in the step 5), and carrying out filter pressing and drying under the pressure of 60kg to obtain a solid primary product;
7) drying and crushing: drying the solid primary product obtained in the step 6) in vacuum at 65 ℃, and crushing after drying to obtain a high-content ganoderic acid A ganoderma lucidum mycelium product with the particle size of 80 meshes, wherein the yield is 2.3%.
Example 3
1) Selecting ganoderma lucidum mycelium strains:
2) and shaking table planting: keeping the temperature at 26 ℃, diluting the strain into 200ml of liquid culture medium by using a flat plate strain with the culture medium of about 1 square centimeter, placing the strain on an oscillator, and stirring for 3 days, wherein the stirring speed is controlled to be 110 revolutions per minute; the liquid culture medium comprises, by mass, 3% of corn flour, 2% of soybean flour, 2% of glucose, 1% of yeast extract, 0.3% of monopotassium phosphate, 0.08% of magnesium sulfate, 175 ppm of vitamin B and the balance of water.
3) Maintaining the temperature at 25 ℃, sucking the culture medium stirred in the step 2) into a 300L seed tank with liquid culture medium under negative pressure, and culturing for 2 days;
4) and second-stage amplification: keeping the temperature at 25 ℃, sucking the liquid culture medium stirred in the step 3) into a 1.5-ton seeding tank with the liquid culture medium under negative pressure, and culturing for 2 days to obtain ganoderma lucidum mycelium seed liquid;
5) fermenting with methyl jasmonate culture medium by maintaining the temperature at 26 deg.C, sucking Ganoderma mycelia seed solution 1000L obtained in step 4) into a 7 ton fermentation tank under negative pressure, adding into 5000L fermentation culture medium containing methyl jasmonate (200 μmol/L), and fermenting for 3 days;
the methyl jasmonate culture medium comprises, by weight, 4% of corn flour, 2% of soybean meal, 2% of glucose, 0.3% of monopotassium phosphate, 0.07% of magnesium sulfate, 180 ppm of vitamin B, 300 mu mol/L% of methyl jasmonate and the balance of deionized water.
The pH of the liquid medium after fermentation was 3.9.
6) And plate and frame filter pressing: taking out the liquid culture medium fermented in the step 5), and carrying out filter pressing and drying under the pressure of 60kg to obtain a solid primary product;
7) drying and crushing: drying the solid primary product obtained in the step 6) in vacuum at 63 ℃, and crushing after drying to obtain a high-content ganoderic acid A ganoderma lucidum mycelium product with the particle size of 70 meshes, wherein the yield is 2.1%.
Comparative example
The other steps were the same as example 1 except that the fermentation medium in the above step 5) did not contain methyl jasmonate. And 5) performing autolysis of the ganoderma lucidum mycelia when the ganoderma lucidum mycelia are fermented to the 3 rd day. Before the ganoderma lucidum mycelium autolysis, the maximum yield of the obtained ganoderma lucidum mycelium product is 1.3%.
Example 4
The content of ganoderic acid A in the ganoderma lucidum mycelia of examples 1 to 3 was measured
The method for detecting the ganoderic acid comprises the following steps:
1. ganoderic acid a sample:
a ganoderic acid A (ganoderic acid A) control product is commercially available, and the mass fraction is 95.0%.
2. Preparation of control solutions
Precisely weighing ganoderic acid A11.16mg dried under reduced pressure to constant mass, placing in a 25ml volumetric flask, adding methanol to dissolve and dilute to scale, and shaking uniformly to obtain a reference solution with mass concentration of 446.4 μ g/m L.
3. Preparation of test sample solution
Pulverizing Ganoderma mycelium powder, sieving with No. 1 sieve, weighing 1g, placing in conical flask with plug, adding methanol solution 25ml, ultrasonic treating for 2 times, 40min each time, filtering, mixing filtrates, placing on water bath kettle, evaporating to dryness, dissolving residue with methanol, transferring to 5m L volumetric flask, fixing volume to scale, shaking, collecting filtrate, and filtering with 0.45 μm microporous membrane.
4. Chromatographic conditions
The chromatographic column adopts WelchXitimateTMAn 18C chromatographic column (4.6mm × 250mm, 5 μm) with acetonitrile as a mobile phase, 0.01 vol% acetic acid (volume ratio 37: 63), a flow rate of 1m L/min, a column temperature of 30 ℃, a sample introduction amount of 10 μ L, an evaporation diffuser drift tube temperature of 105 ℃, an air flow rate of 2.8L/min, a blank solvent of methanol, a control, and a test solution of 10 μ L were precisely aspirated and injected into a high performance liquid chromatograph, and the resulting HP L C chromatogram showed the content of ganoderic acid A. the results are shown in Table 1:
TABLE 1 content of ganoderic acid A in Ganoderma lucidum mycelia of examples 1 to 3
| Content (mg/g) | Example 1 | Example 2 | Example 3 | Comparative example |
| Ganoderic acid A | 98.2 | 101.5 | 72.6 | 31.3 |
As can be seen from Table 1, the fermentation medium of the invention can prolong the fermentation time of the ganoderma lucidum mycelia, ensure the synthesis time of the ganoderic acid A under the condition of not reducing the yield of the mycelia, and improve the content of the ganoderic acid A in the fermentation product.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (5)
1. A fermentation method for improving ganoderic acid A in liquid fermentation ganoderma lucidum mycelia is characterized in that a fermentation medium comprises, by weight, 2% -5% of corn flour, 1% -2% of soybean meal, 1% -3% of glucose, 0.1% -0.5% of monopotassium phosphate, 0.05% -0.1% of magnesium sulfate, 150-100 ppm of vitamin B, 200-300 mu mol/L of methyl jasmonate and the balance of water, wherein the pH value of the fermentation medium is 6.0-7.0;
the fermentation steps are as follows: inoculating the ganoderma lucidum mycelium seed liquid into the fermentation culture medium for fermentation, wherein the fermentation temperature is 25-26 ℃, and when the pH value of the fermentation culture medium is reduced to 3.5-4.0, the fermentation is stopped;
the fermentation time is 3-4 days;
the inoculation amount of the ganoderma lucidum mycelium seed liquid is 10-15%;
the method also comprises the following steps after the fermentation is stopped: filtering the fermented culture medium, drying, and pulverizing to obtain Ganoderma mycelia.
2. The method according to claim 1, wherein the ganoderma lucidum mycelium seed liquid is obtained by culturing ganoderma lucidum mycelium strains in a liquid culture medium at 25-26 ℃, and the liquid culture medium comprises, by mass, 2-4% of corn flour, 1-2% of soybean flour, 1-3% of glucose, 1% of yeast extract, 0.1-0.5% of monopotassium phosphate, 0.05-0.1% of magnesium sulfate, 150-100 ppm of vitamin B, and the balance of water.
3. The method of claim 2, wherein the culturing comprises activation culturing and scale-up culturing.
4. The method according to claim 3, wherein the activation culture time is 3-4 days, and the period of the amplification culture is 2-3 days.
5. The ganoderma lucidum mycelium prepared by the method according to any one of claims 1 to 4, wherein the content of ganoderic acid A in the ganoderma lucidum mycelium is 60 to 105 mg/g.
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