CN112335486A - Method for culturing edible and medicinal fungi with high flavone content - Google Patents
Method for culturing edible and medicinal fungi with high flavone content 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
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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
- A01G18/20—Culture media, e.g. compost
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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
Abstract
The invention discloses a method for culturing edible and medicinal fungi with high flavone content, belonging to a formula of an edible and medicinal fungi culture medium and application thereof. The invention provides a method for culturing edible and medicinal fungi with high flavone content, wherein a culture medium formula comprises seabuckthorn fruit residue filtrate. The flavonoids in fructus Hippophae residue can effectively prevent hyperglycemia and hyperlipemia, and also has effects of scavenging free radicals, resisting arrhythmia, and inhibiting platelet aggregation. The content of the total flavone in the edible and medicinal fungi mycelium cultured by the method reaches 1459.40mg/100g to the maximum, and is increased by about 23.11-231.95% compared with a control group, and the biomass, the extracellular polysaccharide and the intracellular polysaccharide are also significantly increased by about 2.25-70.78%, 9.59-136.60% and 3.24-37.57% respectively compared with the control group.
Description
Technical Field
The invention relates to a formula and application of an edible and medicinal fungus liquid culture medium, and particularly provides a method for culturing edible and medicinal fungi with high flavone content.
Background
The edible and medicinal fungi refer to edible and medicinal mushroom (large-scale fungus) with large fruiting body, and is commonly called mushroom. More than 350 edible and medicinal fungi are known in China, wherein more fungi belong to the subphylum basidiomycotina, and the following fungi are common: lentinus Edodes, straw mushroom, Agaricus campestris, Auricularia, Tremella, Hericium Erinaceus, caulis Bambusae in Taeniam, Tricholoma matsutake, Russula vinifera, Ganoderma, Cordyceps, truffle, Pleurotus nebrodensis, and Boletus edulis. In the field of modern agricultural science and technology, edible and medicinal fungi are the most important components of white agriculture and are green organic food in the true sense. They are not only delicious in taste and rich in nutrition, but also rich in protein, polysaccharide, vitamin, nucleotide and unsaturated fatty acid, have very high medical care function, are deeply loved by people, are often called as health foods, and are very popular in markets at home and abroad. With the improvement of living standard of people, people have strong desire for life quality and good health and long life. Scientists predict that food focus in the 21 st century is health foods and functional foods. The medicinal fungi are outstanding as health-care food, and have wide development prospect. Therefore, the production of edible and medicinal fungi has been popularized and popularized in many places in cities and countryside of China, and is developed towards the direction of intensification and industrialization, and the yield and the output value are unprecedentedly expanded.
The edible mushrooms (Lentinus edodes) are gradually favored by residents in China due to the unique flavor and the high nutritional value thereof, become artificially cultured edible mushrooms with wide culture range and maximum yield in China in recent years, are processed into various leisure foods and seasoning products by people and are supplied to the market, and have wide consumption prospect. Medicinal fungi such as Ganoderma lucidum and Phellinus linteus have high medicinal value and culture background, and are explored and advocated from ancient times to present. The ganoderma lucidum spore powder and the extract thereof are safe and have no toxic or side effect, can be eaten for a long time, has the function of inhibiting the growth of tumors, and has pharmacological actions in various aspects such as enhancing the immunologic function, calming the nerves, reducing blood pressure and blood fat, improving the detoxification capability of a human body, clearing free radicals and resisting aging; phellinus igniarius is sweet and pungent in nature and bitter in taste, has the effects of resisting tumors, enhancing immunity and treating liver diseases, and particularly has a very obvious curative effect on treating liver cirrhosis caused by fatty liver and viral hepatitis. With the continuous and deep research on edible and medicinal fungi, more and more efficacies are gradually discovered.
Seabuckthorn (Hippophae rhamnoides) is a general name of plants in the genus of seabuckthorn of the family elaeagnus, perennial deciduous shrubs or small trees, and berries as fruits mainly grow in arid and semi-arid regions, and has strong stress resistance and wide adaptability. Seabuckthorn contains more than two hundred active substances, which comprise essential amino acids and non-essential amino acids of human bodies, vitamins, trace elements and the like. The seabuckthorn fruit residues are rich in various flavonoid components, and the flavonoid compounds can effectively prevent hyperglycemia and hyperlipidemia; resisting free radical, resisting arrhythmia, and inhibiting platelet aggregation.
At present, the reduction trend of forest resources is serious, and in order to protect rare forest resources, management of forest resource cutting is strengthened or is strengthened in many inland areas. The edible and medicinal fungi are seriously dependent on forest resources, the contradiction between the edible fungi production and the forest resources is very prominent, and great pressure is brought to ecological protection and sustainable development. Therefore, in order to harmonize the relationship between forest resource management and edible and medicinal fungi industry development and ensure that forest resource cultivation and industry development are both good, the development of a novel culture medium becomes an important problem which must be solved in the development of the edible and medicinal fungi industry. The sea-buckthorn branches are the largest amount of byproducts in sea-buckthorn cultivation production, and a large amount of sea-buckthorn branches are pruned and burned every year, so that a large amount of resources are wasted, and the environment is polluted. Most of the seabuckthorn pomace after juicing and extracting the seabuckthorn oil is discarded as waste.
In order to culture edible and medicinal fungi with high flavone content and simultaneously recycle waste seabuckthorn pomace, the development of a novel culture medium is urgent.
Disclosure of Invention
Aiming at the technical problems in the background art, the invention aims to provide a method for culturing edible and medicinal fungi with high flavone content, wherein the cultured edible and medicinal fungi not only have high flavone content, but also have high biomass and polysaccharide content.
Another object of the present invention is to provide a liquid fermentation medium for culturing edible and medicinal fungi with high flavone content.
The purpose of the invention can be realized by the following technical scheme:
application of fructus Hippophae residue in culturing edible and medicinal fungi with high flavone content is provided.
As a preferred technical scheme, the sea-buckthorn pomace filtrate is adopted to prepare a liquid fermentation culture medium for culturing the edible and medicinal fungi with high flavone content; the liquid fermentation medium comprises the following components in percentage by weight: 0.5 to 1.5 percent of maltose, 1 to 3 percent of glucose, 0.1 to 0.3 percent of yeast extract, 0.1 to 0.3 percent of peptone and MgSO4·7H2O 0.02％～0.08％、KH2PO40.2-0.7 percent of the total weight of the sea-buckthorn pomace filtrate, 2-80 percent of the total weight of the sea-buckthorn pomace filtrate and the balance of water to make up 100 percent. Preferably, the amount of the sea buckthorn pomace filtrate is 2% -50%, and preferably 2% -30%; more preferably, the dosage of the sea buckthorn pomace filtrate is preferably 5% -30%; most preferably, the dosage of the sea buckthorn pomace filtrate is 10% -30% or 20% -30%.
Most preferably, the liquid fermentation medium comprises the following components: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent of seabuckthorn fruit residue filtrate, 10 to 30 percent of seabuckthorn fruit residue filtrate and the balance of water to make up to 100 percent.
As a preferred technical scheme, the sea-buckthorn pomace filtrate is prepared by drying sea-buckthorn pomace according to the mass ratio of the sea-buckthorn pomace: and (3) leaching with water at a ratio of 1:20, filtering, sucking supernatant and adjusting the pH value to 6-8.
A liquid fermentation culture medium for culturing edible and medicinal fungi with high flavone content comprises the following components in percentage by weight: 0.5 to 1.5 percent of maltose, 1 to 3 percent of glucose, 0.1 to 0.3 percent of yeast extract, 0.1 to 0.3 percent of peptone and MgSO4·7H2O 0.02％～0.08％、KH2PO40.2-0.7 percent of the total weight of the sea-buckthorn pomace filtrate, 2-80 percent of the total weight of the sea-buckthorn pomace filtrate and the balance of water to make up 100 percent. As a preferred technical scheme, the sea-buckthorn pomace filtrate is prepared by drying sea-buckthorn pomace according to the mass ratio of the sea-buckthorn pomace: and (3) leaching with water at a ratio of 1:20, filtering, sucking supernatant and adjusting the pH value to 6-8.
The liquid fermentation culture medium is applied to culturing edible and medicinal fungi with high flavone content.
A method for culturing edible and medicinal fungi with high flavone content comprises the following steps:
(1) activating strains: selecting hypha edge fungus blocks on a PDA solid slant culture medium of edible and medicinal fungi, inoculating the hypha edge fungus blocks to a CYM solid culture medium, and culturing; for example, hypha edge fungus blocks are selected from a PDA solid slant culture medium of the mushrooms and inoculated to a CYM solid culture medium for culture; or selecting hypha edge fungus blocks on a PDA solid slant culture medium of the lucid ganoderma, inoculating the hypha edge fungus blocks to a CYM solid culture medium, and culturing; or selecting hypha edge fungus blocks on a PDA solid slant culture medium of phellinus igniarius, inoculating the hypha edge fungus blocks to a CYM solid culture medium, and culturing;
(2) mother seed culture: selecting marginal mycelium blocks on a CYM solid culture medium, inoculating the marginal mycelium blocks on the CYM liquid culture medium, and performing shake culture to obtain liquid seed liquid;
(3) inoculating and culturing strains: sufficiently crushing the liquid seed liquid by using a seed crusher under the aseptic condition to obtain homogeneous seeds, inoculating the homogeneous seeds into the liquid fermentation culture medium, and culturing by shaking the seeds;
(4) and (3) hypha collection: after the culture, the liquid fermentation medium was filtered to remove the components, and the mycelia were collected.
As a preferred technical scheme, the formula of the CYM liquid culture medium comprises the following components in percentage by weight: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent, and the balance of water to make up to 100 percent;
the formula of the CYM solid culture medium comprises the following components in percentage by weight: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent, 2 percent of agar powder and the balance of water to make up to 100 percent.
As a preferred technical scheme, the formula of the PDA solid slant culture medium is as follows: the 1L culture medium contains peeled potato 200g, glucose 20g, and agar 20 g. The preparation method comprises the following steps: weighing 200g of potato, cutting into small pieces, adding 900mL of water, boiling for 20min, filtering with eight layers of gauze, adding 20g of glucose, stirring uniformly, adding 20g of agar, continuing heating, stirring uniformly, slightly cooling after the agar is dissolved, supplementing water to 1000mL, subpackaging test tubes, plugging and binding, sterilizing at 121 ℃ for about 20min, taking out the test tubes, placing the test tubes on an inclined plane, cooling and storing for later use.
As a preferred embodiment, the culture conditions of the culture in step (1) are: culturing edible and medicinal fungi hyphae in a constant temperature box at 25-28 ℃ for 5-7 days; preferably, the mushroom mycelia are cultured in a constant temperature box at 25 ℃ for 5-7 days; culturing the ganoderma lucidum mycelia in a constant temperature box at 28 ℃ for 5-7 days; culturing phellinus igniarius hyphae in a constant temperature box at 28 ℃ for 5-7 days;
as a preferable technical scheme, the conditions of the shake culture in the step (2) are as follows: carrying out shake culture on edible and medicinal fungi hyphae for 7 days at the temperature of 25-28 ℃ at the rotating speed of 150 r/min; preferably, the mushroom hyphae are cultured for 7 days in a shaking table at 25 ℃ and the rotating speed is 150 r/min; culturing Ganoderma mycelia at 28 deg.C for 7 days with shaking at 150 r/min; culturing Phellinus linteus mycelium at 28 deg.C for 7 days with shaking at 150 r/min;
as a preferred technical scheme, the inoculation amount of the liquid seed liquid in the step (3) is as follows: inoculating the liquid fermentation culture medium with an inoculation amount of 5%; the culture conditions of shake culture are as follows: carrying out shake culture on edible and medicinal fungi hyphae for 5 days at the temperature of 25-28 ℃ at the rotating speed of 150 r/min; preferably, the mushroom hyphae are cultured for 5 days in a shaking table at 25 ℃ and the rotating speed is 150 r/min; culturing Ganoderma mycelia at 28 deg.C for 5 days with shaking at 150 r/min; culturing Phellinus linteus mycelium at 28 deg.C for 5 days with shaking at 150 r/min.
The edible and medicinal fungi include, but are not limited to, mushrooms, ganoderma lucidum, phellinus igniarius, straw mushrooms, agarics, tremella, hericium erinaceus, dictyophora phalloidea, tricholoma matsutake, russula vinosa and the like. The room temperature in the invention is generally 25 +/-5 ℃.
The invention has the beneficial effects that:
the seabuckthorn fruit residues are rich in various flavonoid components, and the flavonoid compounds can effectively prevent hyperglycemia and hyperlipidemia; resisting free radical, resisting arrhythmia, and inhibiting platelet aggregation. The content of total flavonoids in the shiitake mycelia cultured by the culture medium is higher and reaches 633.78mg/100g, compared with a control group, the content is improved by about 115.96%, and the content of biomass, extracellular polysaccharide and intracellular polysaccharide is also obviously improved by 70.78%, 96.50% and 37.57% respectively. The content of total flavone in medicinal fungus-ganoderma lucidum mycelium is up to 1640mg/100g, which is increased by about 231.95% compared with the control group, and extracellular polysaccharide and intracellular polysaccharide are also significantly increased by 113.86% and 29.16% respectively compared with the control group. The content of total flavone in medicinal fungus-Phellinus igniarius mycelium is up to 1199.04mg/100g, which is increased by about 114.11% compared with the control group, and extracellular polysaccharide and intracellular polysaccharide are also significantly increased by 136.59% and 16.12% respectively compared with the control group.
According to the method for culturing the edible and medicinal fungi with high flavone content, maltose, glucose, yeast extract, peptone and the like are added to provide nutrition for the edible and medicinal fungi hyphae, and the seabuckthorn fruit residues are rich in various active substances and can effectively enhance the growth and metabolism of the edible and medicinal fungi hyphae. And KH2PO4As a pH buffer toAnd acidic substances generated in the growth process of edible and medicinal fungi hyphae, so that the hyphae are prevented from being inhibited by too low pH in the later period.
Drawings
FIG. 1 is a glucose standard curve
FIG. 2 shows a rutin standard curve
Detailed description of the invention
The technical solution of the present invention will be described in detail below with reference to specific examples.
Example 1 preparation of liquid fermentation Medium and other Medium for culturing high-flavone-content edible-medicinal fungus mycelia
The composition comprises the following components in percentage by weight: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent of the total weight of the extract, 2 to 80 percent of the sea-buckthorn pomace filtrate and the balance of water to make up to 100 percent. The specific amounts of the sea buckthorn pomace filtrate are exemplified by 2%, 5%, 10%, 20% and 30%.
The preparation method of the sea-buckthorn pomace filtrate comprises the following steps: the dried seabuckthorn fruit residues are mixed according to the mass ratio: leaching with water at room temperature for 30min at a ratio of 1:20, filtering, collecting supernatant, and adjusting pH to about 7.
Mixing the components, stirring uniformly, subpackaging 100mL of liquid culture medium into 250mL of conical flask, covering with sealing membrane, sterilizing at 115 deg.C for 30min, taking out, cooling and storing.
Preparation of CYM liquid medium: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent, and the balance of water to make up to 100 percent; sterilizing by conventional method.
Preparation of CYM solid medium: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent of agar powder, 2 percent of agar powder and the balance of water to make up to 100 percent; sterilizing by conventional method.
Preparing a PDA solid slant culture medium: the 1L culture medium contains peeled potato 200g, glucose 20g, and agar 20 g. The preparation method comprises the following steps: weighing 200g of potato, cutting into small pieces, adding 900mL of water, boiling for 20min, filtering with eight layers of gauze, adding 20g of glucose, stirring uniformly, adding 20g of agar, continuing heating, stirring uniformly, slightly cooling after the agar is dissolved, supplementing water to 1000mL, subpackaging test tubes, plugging and binding, sterilizing at 121 ℃ for about 20min, taking out the test tubes, placing the test tubes on an inclined plane, cooling and storing for later use.
Example 2 cultivation of high flavone content Lentinus edodes with the liquid fermentation Medium prepared in example 1
The method for culturing the shiitake mushrooms with high flavone content by adopting the liquid fermentation culture medium prepared in the example 1 comprises the following steps:
(1) activating strains: selecting hypha marginal fungus blocks on a PDA solid slant culture medium of a mushroom strain F2, inoculating the hypha marginal fungus blocks to a CYM solid culture medium, and culturing in a constant temperature incubator at 25 ℃ for about 6 days.
(2) Mother seed culture: selecting 8 marginal mycelium blocks with the diameter of 6mm on a CYM solid culture medium, inoculating the marginal mycelium blocks into a 250mL triangular flask, filling 100mL CYM liquid culture medium into the flask, and culturing for 7 days in a shaker at the temperature of 25 ℃ at the rotating speed of 150r/min to obtain liquid seed liquid.
(3) Inoculating strains: and (3) fully smashing the liquid seed liquid by using a seed beater under the aseptic condition of a superclean workbench to obtain homogeneous seeds. The medium was inoculated at 5% (v/v) into a 250mL Erlenmeyer flask containing 100mL of liquid medium. Culturing by shaking bacteria: culturing in a shaker at 25 deg.C for 5 days at 150 r/min.
(4) And (3) hypha collection: after completion of the culture, the liquid medium was filtered to remove the components, and the mycelia were collected.
Example 3 cultivation of Ganoderma lucidum with high flavone content Using the liquid fermentation Medium prepared in example 1
The method for culturing the ganoderma lucidum with high flavone content by adopting the liquid fermentation culture medium prepared in the embodiment 1 comprises the following steps:
(1) activating strains: selecting hypha marginal fungus blocks on a PDA solid slant culture medium of the ganoderma lucidum strain G20, inoculating the hypha marginal fungus blocks to a CYM solid culture medium, and culturing in a constant temperature incubator at 28 ℃ for about 6 days.
(2) Mother seed culture: selecting 8 marginal mycelium blocks with the diameter of 6mm on a CYM solid culture medium, inoculating the marginal mycelium blocks into a 250mL triangular flask, filling 100mL CYM liquid culture medium into the flask, and culturing for 7 days in a shaking table at the temperature of 28 ℃ at the rotating speed of 150r/min to obtain liquid seed liquid.
(3) Inoculating strains: and (3) fully smashing the liquid seed liquid by using a seed beater under the aseptic condition of a superclean workbench to obtain homogeneous seeds. The medium was inoculated at 5% (v/v) into a 250mL Erlenmeyer flask containing 100mL of liquid medium. Culturing by shaking bacteria: culturing in a shaking table at 28 deg.C for 5 days at 150 r/min.
(4) And (3) hypha collection: after completion of the culture, the liquid medium was filtered to remove the components, and the mycelia were collected.
Example 4 cultivation of Phellinus linteus with high flavone content using the liquid fermentation Medium prepared in example 1
The liquid fermentation culture medium prepared in example 1 is used for culturing phellinus linteus with high flavone content, and comprises the following steps:
(1) activating strains: selecting hypha edge fungus blocks on a PDA solid slant culture medium of phellinus igniarius WT, inoculating the hypha edge fungus blocks to a CYM solid culture medium, and culturing the hypha edge fungus blocks in a constant temperature incubator at the temperature of 28 ℃ for about 6 days.
(2) Mother seed culture: selecting 8 marginal mycelium blocks with the diameter of 6mm on a CYM solid culture medium, inoculating the marginal mycelium blocks into a 250mL triangular flask, filling 100mL CYM liquid culture medium into the flask, and culturing for 7 days in a shaking table at the temperature of 28 ℃ at the rotating speed of 150r/min to obtain liquid seed liquid.
(3) Inoculating strains: and (3) fully smashing the liquid seed liquid by using a seed beater under the aseptic condition of a superclean workbench to obtain homogeneous seeds. The medium was inoculated at 5% (v/v) into a 250mL Erlenmeyer flask containing 100mL of liquid medium. Culturing by shaking bacteria: culturing in a shaking table at 28 deg.C for 5 days at 150 r/min.
(4) And (3) hypha collection: after completion of the culture, the liquid medium was filtered to remove the components, and the mycelia were collected.
Comparative example 1
A liquid fermentation culture medium for culturing edible and medicinal fungus mycelium comprises (by weight) maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, and MgSO4·7H2O 0.05％、KH2PO40.46 percent, and the balance of water to make up 100 percent.
Mixing the components, stirring uniformly, subpackaging 100mL of liquid culture medium into 250mL of conical flask, covering with sealing membrane, sterilizing at 115 deg.C for 30min, taking out, cooling and storing.
The liquid fermentation medium was used to culture Lentinus edodes, Ganoderma lucidum and Phellinus linteus, respectively, in the same manner as in examples 2, 3 and 4, respectively.
And (4) analyzing results:
taking the hyphae obtained in the experimental examples 2-4 and the comparative example 1, measuring the total flavone content, wherein the method for measuring the flavone is as follows:
(1) preparation of a standard curve: accurately weighing 23.2mg of rutin standard, dissolving with 70% ethanol, and dissolving in 50mL volumetric flask to obtain standard solution. Accurately sucking standard rutin solution 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0mL, placing in 25mL volumetric flask, adding 0.72mol/L NaNO2Shaking the solution 1mL, standing for 6min, adding 0.47mol/L Al (NO)3)3Shaking the solution 1mL, standing for 6min, adding 1.00mol/L NaOH solution 10mL, diluting with 70% ethanol to desired volume, and standing for 10 min. The absorbance was measured at 510nm and the absorbance (y) was linearly regressed with the actual concentration (x) of rutin (see figure 2 for the standard rutin curve).
(2) The extraction method of the total flavonoids in the sample comprises the following steps: spreading the mycelium on non-woven fabric, and oven drying at 60 deg.C in oven. Accurately weighing 0.05g of mycelium powder, adding 5mL of 70% ethanol, and placing the mixture into an ultrasonic cleaning machine for ultrasonic treatment for 1h to obtain an extract. Respectively taking 0, 0.1, 0.2, 0.4, 0.6 and 0.8mL of rutin standard solution in a 5mL centrifuge tube, and adding 0.72mol/L of NaNO20.2mL of the solution was shaken up and allowed to stand for 6 min. 0.47mol/L Al (NO) was added3)30.2mL of the solution was shaken up and allowed to stand for 6 min. 2mL of 1.00mol/L NaOH solution is added, and 70% ethanol is added to the solution to reach 5 mL.
(3) And (3) total flavone content determination: sucking 1mL of extract liquid, placing the extract liquid in a 5mL centrifuge tube, preparing a solution according to a method for drawing a standard curve, adjusting zero by taking a blank reagent as a reference solution, measuring absorbance at 510nm, measuring for 3 times, taking an average value, and calculating the content of the total flavonoids in each tube through a linear regression equation.
The polysaccharide content of the mycelia obtained in the experimental examples 2-4 and the comparative example 1 was measured, and the polysaccharide measurement method was as follows:
(1) and (3) standard curve preparation: accurately weighing 100mg of pre-dried analytically pure glucose, quantifying in a volumetric flask, and preparing into 1mg/mL glucose standard solution; adding 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of glucose standard solution into the reaction system respectively; a standard curve is drawn with the glucose content as abscissa and 490nm as ordinate. (glucose standard curve is shown in FIG. 1)
(2) The extraction method and the determination of the extracellular polysaccharide of the sample are that 0.5mL of culture solution which is cultured for 7 days and filtered to remove hyphae is evenly mixed with 95 percent ethanol with 4 times of volume; precipitating with ethanol at-20 deg.C overnight; centrifuging at 3000g for 10min, discarding supernatant, and oven drying precipitate; dissolving the precipitate with 0.5mL of 1.00mol/L NaOH, and carrying out water bath at 60 ℃ for 1 h; sampling and detecting the polysaccharide content by a phenol-sulfuric acid method.
(3) The extraction method and the determination of the intracellular polysaccharide of the sample comprise the following steps: repeatedly washing the mycelium cultured for 7 days with water, and oven drying at 60 deg.C; grinding the dried mycelium, and dissolving 0.1g of the mycelium with 15mL of 1.00mol/L NaOH; mixing, and water-bathing at 65 deg.C for 3 hr, or shaking; centrifuging at 10000g for 10min, collecting supernatant, and detecting polysaccharide content by phenol-sulfuric acid method.
(4) And (3) determining the polysaccharide content by a phenol-sulfuric acid method: 0.5mL sample and 0.5mL 0.53mol/L phenol are mixed evenly, then 3mL concentrated sulfuric acid is added, reaction is carried out for 30min at room temperature, the light absorption value at 490nm is measured, and a glucose standard curve is substituted to calculate the polysaccharide content.
The measurement results of total flavonoids of shiitake mushrooms according to example 2 and comparative example 1 show that: the addition of the sea buckthorn pomace filtrate with different contents in the basic culture medium can obviously increase the flavone content in the mushroom hyphae, and meanwhile, the contents of biomass, extracellular polysaccharide and intracellular polysaccharide are also obviously improved, and specific results are shown in tables 1 and 2.
TABLE 1 Total flavone content of Lentinus edodes mycelium
TABLE 2 Mushroom hypha biomass and polysaccharide content
The results of the measurement of total flavonoids in ganoderma lucidum according to example 3 and comparative example 1 show that: the addition of the sea buckthorn pomace filtrate with different contents into the basic culture medium can obviously increase the flavone content in the ganoderma lucidum mycelium, and simultaneously, the contents of biomass, extracellular polysaccharide and intracellular polysaccharide are also obviously improved, and the specific results are shown in tables 3 and 4.
TABLE 3 Total flavone content of Ganoderma lucidum mycelia
TABLE 4 Ganoderma lucidum hypha biomass and polysaccharide content
The results of the Phellinus linteus total flavone assay according to example 4 and comparative example 1 show that: the addition of the sea buckthorn pomace filtrate with different contents into the basic culture medium can obviously increase the flavone content in phellinus igniarius hyphae, and obviously improve the contents of biomass, extracellular polysaccharide and intracellular polysaccharide, and the specific results are shown in tables 5 and 6.
TABLE 5 Phellinus Linteus hypha Total flavone content
TABLE 6 Phellinus Linteus mycelium Biomass and polysaccharide content
Claims (10)
1. Application of fructus Hippophae residue in culturing edible and medicinal fungi with high flavone content is provided.
2. The use of claim 1, wherein the sea buckthorn pomace filtrate is used to prepare a liquid fermentation medium for culturing high flavone content edible medicinal fungi; the liquid fermentation medium comprises the following components in percentage by weight: 0.5 to 1.5 percent of maltose, 1 to 3 percent of glucose, 0.1 to 0.3 percent of yeast extract, 0.1 to 0.3 percent of peptone and MgSO4·7H2O 0.02％～0.08％、KH2PO40.2-0.7 percent of the total weight of the sea-buckthorn pomace filtrate, 2-80 percent of the total weight of the sea-buckthorn pomace filtrate and the balance of water to make up 100 percent.
3. The use according to claim 2, wherein the liquid fermentation medium comprises the following components: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent of seabuckthorn fruit residue filtrate, 10 to 30 percent of seabuckthorn fruit residue filtrate and the balance of water to make up to 100 percent.
4. The use of claim 2 or 3, wherein the fructus Hippophae residue filtrate is prepared by drying fructus Hippophae residue according to the mass ratio of fructus Hippophae residue: and (3) leaching with water at a ratio of 1:20, filtering, sucking supernatant and adjusting the pH value to 6-8.
5. A liquid fermentation culture medium for culturing edible and medicinal fungi with high flavone content is characterized by comprising the following components in percentage by weight: 0.5 to 1.5 percent of maltose, 1 to 3 percent of glucose, 0.1 to 0.3 percent of yeast extract, 0.1 to 0.3 percent of peptone and MgSO4·7H2O 0.02％～0.08％、KH2PO40.2-0.7 percent of the total weight of the sea-buckthorn pomace filtrate, 2-80 percent of the total weight of the sea-buckthorn pomace filtrate and the balance of water to make up 100 percent.
6. The liquid fermentation medium of claim 5, wherein the sea buckthorn pomace filtrate is prepared by drying sea buckthorn pomace according to the mass ratio of the sea buckthorn pomace: and (3) leaching with water at a ratio of 1:20, filtering, sucking supernatant and adjusting the pH value to 6-8.
7. Use of the liquid fermentation medium of claim 5 or 6 for culturing high-flavone-content edible and medicinal fungi.
8. A method for culturing edible and medicinal fungi with high flavone content is characterized by comprising the following steps:
(1) activating strains: selecting hypha edge fungus blocks on a PDA solid slant culture medium of edible and medicinal fungi, inoculating the hypha edge fungus blocks to a CYM solid culture medium, and culturing.
(2) Mother seed culture: selecting marginal mycelium blocks on a CYM solid culture medium, inoculating the marginal mycelium blocks on the CYM liquid culture medium, and performing shake culture to obtain liquid seed liquid;
(3) inoculating and culturing strains: sufficiently crushing the liquid seed solution by using a seed crusher under the aseptic condition to obtain a homogeneous seed, inoculating the homogeneous seed into the liquid fermentation culture medium of claim 5 or 6, and culturing by shaking;
(4) and (3) hypha collection: after the culture, the liquid fermentation medium was filtered to remove the components, and the mycelia were collected.
9. The method according to claim 8, wherein the formula of the CYM liquid culture medium comprises, in weight percent: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O0.05％、KH2PO40.46 percent, and the balance of water to make up to 100 percent;
the formula of the CYM solid culture medium comprises the following components in percentage by weight: maltose 1%, glucose 2%, yeast extract 0.2%, peptone 0.2%, MgSO4·7H2O 0.05％、KH2PO40.46 percent of agar powder, 2 percent of agar powder and the balance of water to make up to 100 percent％。
10. The edible and medicinal fungus of claim 1, 2, 5, 7 or 8 is Lentinus edodes, Ganoderma, Phellinus linteus, Volvariella volvacea, Agaricus campestris, Auricularia, Tremella, Hericium erinaceus, Dictyophora Indusiata, Tricholoma matsutake, Tricholoma giganteum or Pleurotus rubrum.
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