CN111996223A - Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology - Google Patents
Method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing bidirectional fermentation technology Download PDFInfo
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Abstract
The invention discloses a method for obtaining ganoderma lucidum and isatis root biological polysaccharide by utilizing a bidirectional fermentation technology, which is characterized by comprising the following steps of: the method comprises the following steps: 1) treating the medicine residue powder; 2) activating the ganoderma lucidum strain; 3) preparing a bidirectional fermentation culture medium; 4) bidirectional fermentation; 5) treating fermentation mycoplasm; 6) and (4) extracting polysaccharide. The invention has the beneficial effects that: the process is simple, the equipment requirement is not high, and the popularization is easy; residual waste produced by a Chinese medicine factory is used for fermentation, so that the recycling of residues is realized, the production cost is reduced, the economic benefit is improved, and the environmental pollution is reduced; by utilizing a bidirectional fermentation technology, obtaining biological polysaccharides of lucid ganoderma and isatis root; vacuum freeze drying is adopted, and the activity of the polysaccharide is kept to the maximum extent; the fungus residue after polysaccharide extraction is detected to contain no viable bacteria, has no toxicity to organisms, is ecological and environment-friendly, and has no residue and no pollution.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for obtaining ganoderma lucidum and isatis root biological polysaccharide by utilizing a bidirectional fermentation technology.
Background
Shandong is a big province of Chinese herbal medicine artificial planting in China, the variety of Chinese herbal medicine planting in the whole province exceeds more than 70 varieties at present, and the Shandong is a main cultivation and production area of isatis root, honeysuckle, hawthorn, salvia miltiorrhiza, scutellaria baicalensis, American ginseng and the like. Along with the large-scale planting of Chinese herbal medicines, the deep processing industry of the Chinese herbal medicines is rapidly developed, and the number of enterprises in the whole province in 2018 exceeds 250. However, at present, the method for obtaining the effective components of the Chinese herbal medicines in most enterprises mainly adopts water extraction, the obtaining efficiency is not ideal, a large amount of medicinal components still remain in the dregs of a decoction, and the dregs of a decoction are generally buried, burned or prepared into organic fertilizer to be returned to the field, so that the waste of medicinal resources is caused, and the environmental pollution is easily caused. The medical resources are recycled after the industrialized production, and the utilization efficiency of the Chinese herbal medicines is improvedThe method is particularly important, not only can reduce the production cost and improve the economic benefit, but also can reduce the environmental pollution; radix Isatidis (radix Isatidis: (radix Isatidis))Isatis TinctoriaL.) is a common medicinal plant, is used as a whole plant medicine, is a dry root of isatis indigotica fort of Brassicaceae and folium isatidis, has the effects of clearing heat, removing toxicity and cooling blood, and is used for treating diseases such as cold, microbial infection, hypertension, tumor and the like. The main components of the composition comprise biological polysaccharide, alkaloid, flavonoid compounds, organic acid and the like.
Ganoderma lucidum is a traditional rare fungus in China, and is a precious medicinal material with the effects of nourishing, strengthening body resistance and consolidating constitution. The experiment proves that the ganoderma lucidum has the effects of nourishing and strengthening, strengthening brain, diminishing inflammation, promoting urination, tonifying kidney, resisting bacteria, oxidation, cancer, tumor, immunity and the like. The chemical components of ganoderma lucidum which are researched more at present mainly comprise triterpenoids, polysaccharides, nucleosides, sterols, alkaloids, furan derivatives, amino acid polypeptides, inorganic elements, fatty acids and the like. The polysaccharide compound is one of the important chemical components contained in ganoderma lucidum, and the ganoderma lucidum polysaccharide has the functions of resisting tumor, regulating immunity, reducing blood sugar, reducing blood fat, resisting oxidation, resisting aging and the like. In recent years, the demand of ganoderma lucidum polysaccharide is increasing, but wild ganoderma lucidum is very scarce, the period of artificially cultivating solid strains of ganoderma lucidum is more than half a year, and the period of using liquid strains is more than 100 days.
The bidirectional fermentation is to inoculate a certain medicinal strain on a certain substrate, perform fermentation culture under certain conditions, and reach the fermentation end point under specific conditions, so that a large amount of hypha can be obtained in a short period (7-10 days), the radix isatidis is used as a culture medium to accelerate the growth of ganoderma lucidum hypha and shorten the fermentation time, and the water-soluble polysaccharide is extracted from the fermentation product, namely the mixed polysaccharide containing ganoderma lucidum polysaccharide and medicinal polysaccharide is obtained. Can be popularized as a combined example of a medicine and medicinal fungi, and can develop a medicine with good efficacy. At present, no literature reports the preparation method of the mixed polysaccharide.
Disclosure of Invention
The invention aims to provide a method for obtaining ganoderma lucidum and isatis root biological polysaccharide by utilizing a bidirectional fermentation technology aiming at the defects in the prior art.
The technical scheme is as follows: a method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing a bidirectional fermentation technology comprises the following steps:
1) treating the medicine residue powder: drying radix Isatidis and/or folium Isatidis residue, cutting into segments, and grinding into powder;
2) activating the ganoderma lucidum strain: inoculating Ganoderma strain to PDA culture medium, activating, culturing at constant temperature of 25-30 deg.C for 5-10 days, selecting good-growing mycelium, inoculating to PDA solid plate containing 20% medicinal residue powder extractive solution, and performing amplification culture for 5-7 days until the mycelium grows over the plate;
3) preparing a bidirectional fermentation culture medium: mixing 50-100% of the residue powder, 0-48% of the corncob powder, and 0-0.5% of MgSO40-0.5% (NH)4)2SO4And 0-1% KH2PO4After mixing, adjusting the pH value to about 6.5, adding a proper amount of water to make the final concentration 65%, uniformly stirring and compacting, and then sterilizing by high-pressure steam at 121 ℃ for later use, wherein the liquid culture medium is a PDA liquid culture medium prepared by 5-20% of decoction dreg powder water extract;
4) bidirectional fermentation: inoculating the Ganoderma of the expanded culture into the culture medium of step 3, and fermenting in solid or liquid state with solid fermentation inoculum size of 10% and liquid fermentation inoculum size of 2-10%, and culturing at 28 deg.C in dark place for 7-14 days;
5) and (3) fermentation mycoplasm treatment: when the hyphae grow over the whole culture medium and the appearance is thick and white, the fermentation is finished, when the liquid fermentation is carried out, the hypha balls grow over the whole fermentation liquid, the hypha balls are spherical, and when the appearance is thick and white, the fermentation is finished; drying and grinding the solid fermentation product in an oven at 65 ℃; separating mycelium pellets from a liquid culture medium, carrying out rotary evaporation on a clear liquid rotary evaporator, collecting a product, mixing the product with the mycelium pellets, drying in an oven at 65 ℃, and grinding into powder; or discarding the clear liquid, drying the mycelium pellets in a 65 ℃ oven and grinding the mycelium pellets into powder;
6) polysaccharide extraction: adding distilled water into the fermentation product dry powder according to the mass ratio of 1: 30, heating and extracting, separating supernatant, and concentrating to obtain supernatant with the volume of 1/2-1/6. Adding 95% ethanol into the concentrated solution until the final concentration of ethanol is 80%, stirring, standing to precipitate polysaccharide, centrifuging to collect precipitate, and vacuum drying to obtain biological polysaccharide.
In the step of processing the dregs, the dregs of the radix isatidis and/or the folium isatidis are dried, cut into segments and ground into powder of 10-80 meshes.
Preparing the decoction dreg powder extracting solution: taking a certain weight of the decoction dreg powder, adding distilled water with the same weight, putting the mixture into a sealed container, boiling the mixture for 20 minutes, and filtering the mixture by 16 layers of gauze to obtain the traditional Chinese medicine decoction.
The invention has the beneficial effects that:
1. the process is simple, the equipment requirement is not high, the growth speed of ganoderma lucidum mycelia on the isatis root culture medium is increased by 20 percent compared with that of the common culture medium, the fermentation time is shortened, and the method is easy to popularize.
2. The residual waste produced by a Chinese medicine factory is used for fermentation, so that the extraction residue of the isatis root is recycled, the production cost is reduced, the economic benefit is improved, and the environmental pollution is reduced.
3. By utilizing the bidirectional fermentation technology and simultaneously utilizing the medicinal properties and functions of the isatis root and the lucid ganoderma, the biological polysaccharide of the lucid ganoderma and the isatis root is obtained through the deep interaction of the isatis root and the lucid ganoderma.
4. Vacuum freeze drying is adopted, and the activity of the polysaccharide is kept to the maximum extent.
5. The bacterial residues after polysaccharide extraction are detected to contain no viable bacteria and no toxicity to organisms, the organic matter content is more than 80%, the bacterial residues can be directly used as organic fertilizers and returned to the field, plants can easily absorb the organic fertilizers, and the whole production process is ecological and environment-friendly, and has zero residue and zero pollution.
Detailed Description
A method for obtaining biological polysaccharide of lucid ganoderma and isatis root by utilizing a bidirectional fermentation technology comprises the following steps:
1) treating the medicine residue powder: drying radix Isatidis and/or folium Isatidis residue, cutting into segments, and grinding into powder;
2) activating the ganoderma lucidum strain: inoculating Ganoderma strain to PDA culture medium, activating, culturing at constant temperature of 25-30 deg.C for 5-10 days, selecting good-growing mycelium, inoculating to PDA solid plate containing 20% medicinal residue powder extractive solution, and performing amplification culture for 5-7 days until the mycelium grows over the plate;
3) preparing a bidirectional fermentation culture medium: 50 to 100 percent of the medicine residue powder,0-48% of corncob powder, 0-0.5% of MgSO40-0.5% (NH)4)2SO4And 0-1% KH2PO4After mixing, adjusting the pH value to about 6.5, adding a proper amount of water to make the final concentration 65%, uniformly stirring and compacting, and then sterilizing by high-pressure steam at 121 ℃ for later use, wherein the liquid culture medium is a PDA liquid culture medium prepared by 5-20% of decoction dreg powder water extract;
4) bidirectional fermentation: inoculating the Ganoderma of the expanded culture into the culture medium of step 3, and fermenting in solid or liquid state with solid fermentation inoculum size of 10% and liquid fermentation inoculum size of 2-10%, and culturing at 28 deg.C in dark place for 7-14 days;
5) and (3) fermentation mycoplasm treatment: when the hyphae grow over the whole culture medium and the appearance is thick and white, the fermentation is finished, when the liquid fermentation is carried out, the hypha balls grow over the whole fermentation liquid, the hypha balls are spherical, and when the appearance is thick and white, the fermentation is finished; drying and grinding the solid fermentation product in an oven at 65 ℃; separating mycelium pellets from a liquid culture medium, carrying out rotary evaporation on a clear liquid rotary evaporator, collecting a product, mixing the product with the mycelium pellets, drying in an oven at 65 ℃, and grinding into powder; or discarding the clear liquid, drying the mycelium pellets in a 65 ℃ oven and grinding the mycelium pellets into powder;
6) polysaccharide extraction: adding distilled water into the fermentation product dry powder according to the mass ratio of 1: 30, heating and extracting, separating supernatant, and concentrating to obtain supernatant with the volume of 1/2-1/6. Adding 95% ethanol into the concentrated solution until the final concentration of ethanol is 80%, stirring, standing to precipitate polysaccharide, centrifuging to collect precipitate, and vacuum drying to obtain biological polysaccharide.
In the step of processing the dregs, the dregs of the radix isatidis and/or the folium isatidis are dried, cut into segments and ground into powder of 10-80 meshes.
Preparing the decoction dreg powder extracting solution: taking a certain weight of the decoction dreg powder, adding distilled water with the same weight, putting the mixture into a sealed container, boiling the mixture for 20 minutes, and filtering the mixture by 16 layers of gauze to obtain the traditional Chinese medicine decoction.
The first embodiment is as follows: solid state two-way fermentation
Weighing 230g of radix isatidis residue, crushing the radix isatidis residue into 40-mesh residue by a grinder, adding 200ml of distilled water into 30g of powder, covering the powder, boiling the powder for 20 minutes, filtering the powder by 16 layers of gauze, and preparing the filtrate into a PDA solid culture medium plate. Adding 200g of powder into a beaker, adding 372ml of water, uniformly stirring, standing at normal temperature for 1 hour, adjusting pH =6.5, subpackaging into a culture bottle, compacting, scraping off redundant residues on the inner wall of the bottle, and sterilizing with high-pressure steam for later use. Inoculating Ganoderma strain to common PDA plate, activating, culturing at 28 deg.C for 7 d, inoculating to PDA plate containing radix Isatidis water extractive solution, and performing amplification culture for 6 days until mycelium grows over the whole plate. Inoculating the plate for enlarged culture into a culture bottle according to the mass ratio of 10 g/100 g, and culturing at 28 ℃ in a dark place for 13 days to ensure that hyphae overgrow the whole matrix, the hyphae are dense and white, and a small amount of tawny fungus skin is present, so that the plate is pollution-free. Taking out the fermentation product, drying and grinding. Weighing 100 g of dry powder, adding 3L of distilled water, boiling and extracting for 1 hour at 90 ℃, filtering and filtering by using filter paper to retain clear liquid, continuously adding 30 times of distilled water into residues, boiling and extracting for 1 hour at 90 ℃, and filtering by using filter paper to retain clear liquid. Mixing the two extractive solutions, concentrating with a multistage concentrator at 95 deg.C and inlet flow rate of 80 mL/min to 500 mL, centrifuging at 4000 rpm for 15 min, and discarding the precipitate. Adding 2.7L 95% ethanol into the concentrated solution until the final concentration of ethanol is 80%, centrifuging at 4000 rpm for 15 min, collecting precipitate, washing the precipitate with 95% ethanol twice, transferring the obtained precipitate to an evaporating dish, and vacuum freeze drying to obtain light-colored powder, i.e. radix Isatidis and Ganoderma mixed polysaccharide.
Example two: liquid two-way fermentation
Weighing 230g of radix isatidis residue, crushing the radix isatidis residue into 30-mesh residue by a grinder, adding 200ml of distilled water into 30g of powder, covering the powder, boiling the powder for 20 minutes, filtering the powder by 16 layers of gauze, and preparing the filtrate into a PDA solid culture medium plate. Adding 200g of isatis root powder into 2000 mL of PDA culture medium, uniformly stirring, standing at normal temperature for 1 hour, adjusting pH =6.5, subpackaging into culture bottles, and sterilizing with high-pressure steam for later use. Inoculating Ganoderma strain to common PDA plate, activating, culturing at 28 deg.C for 7 d, inoculating to PDA plate containing radix Isatidis water extractive solution, and performing amplification culture for 6 days until mycelium grows over the whole plate. Inoculating the plate for enlarged culture into a culture bottle according to the mass ratio of 2 g/100 g, standing and culturing for 12 hours at 28 ℃ in a dark place, carrying out shaking culture at 150rpm, and growing mycelium pellets over the whole culture medium after 8 days, wherein the mycelium pellets are thick, white and pollution-free. Sieving to separate mycelium pellet, oven drying mycelium pellet, and grinding. Weighing 50 g of dry powder, adding 1500 mL of distilled water, boiling and extracting for 1 hour at 90 ℃, filtering and filtering by using filter paper to retain clear liquid, continuously adding 30 times of distilled water into residues, boiling and extracting for 1 hour at 90 ℃, and filtering by using filter paper to retain clear liquid. Mixing the two extractive solutions, concentrating with multi-stage concentrator at 95 deg.C and inlet flow rate of 80 mL/min to volume of 300 mL, centrifuging at 4000 rpm for 15 min, and discarding the precipitate. Adding 1600 mL of 95% ethanol into the concentrated solution until the final concentration of ethanol is 80%, centrifuging at 4000 rpm for 15 min, collecting precipitate, washing the precipitate with 95% ethanol twice, transferring the obtained precipitate to an evaporation dish, and freeze-drying in vacuum to obtain light-colored powder, i.e. ganoderan. The method can obtain relatively pure intracellular polysaccharide of Ganoderma, but the yield is low.
Example three: liquid two-way fermentation
Weighing 330 g of radix isatidis dregs, crushing the dregs into 30-mesh residues through a grinder, adding 3300 ml of distilled water into 30g of powder, covering the residues, boiling the residues for 20 minutes, performing suction filtration, and using filtrate for preparing a PDA liquid culture medium. Activating Ganoderma strain on common PDA plate, inoculating into PDA culture solution containing radix Isatidis extract, and fermenting at 28 deg.C and 150rpm for 4 days to obtain secondary strain. Adding 3L of PDA culture solution containing radix Isatidis extract into a small fermentation tank, covering, sterilizing in a high pressure steam sterilization pot, cooling to about 30 deg.C, inoculating secondary bacteria solution at 8%, performing liquid submerged fermentation with pH controlled at about 6.5, rotating at 250rpm within 24 hr, rotating at 160 rpm after 24 hr, fermenting at constant temperature of 0.05 MPa and 28 deg.C for 7 days, wherein the mycelium pellet density reaches above 80% of that of the lens, and ending fermentation to obtain fermentation liquid. Filtering the fermentation liquid with a 250-mesh screen, evaporating the solvent of the clear liquid by using a rotary evaporator, and drying and grinding the residual substances and mycelium pellets in a mixing oven. Taking 50 g of fermentation product dry powder, adding 1500 mL of distilled water, boiling and extracting for 1 hour at 90 ℃, filtering and filtering by using filter paper to retain clear liquid, continuously adding 30 times of distilled water into residues, boiling and extracting for 1 hour at 90 ℃, and filtering by using filter paper to retain clear liquid. Mixing the two extractive solutions, concentrating with multi-stage concentrator at 95 deg.C and inlet flow rate of 80 mL/min to 200mL, centrifuging at 4000 rpm for 15 min, and discarding the precipitate. Adding 1600 mL of 95% ethanol into the concentrated solution until the final concentration of the ethanol is 80%, centrifuging at 4000 rpm for 15 min, collecting the precipitate, washing the precipitate with 95% ethanol twice, transferring the obtained precipitate to an evaporation dish, and freeze-drying in vacuum to obtain light-colored powder, namely the mixed polysaccharide of radix Isatidis and Ganoderma.
Claims (3)
1. The method for obtaining the biological polysaccharide of the radix isatidis by utilizing the bidirectional fermentation technology is characterized by comprising the following steps of: the method comprises the following steps:
1) treating the medicine residue powder: drying radix Isatidis and/or folium Isatidis residue, cutting into segments, and grinding into powder;
2) activating the ganoderma lucidum strain: inoculating Ganoderma strain to PDA culture medium, activating, culturing at constant temperature of 25-30 deg.C for 5-10 days, selecting good-growing mycelium, inoculating to PDA solid plate containing 20% medicinal residue powder extractive solution, and performing amplification culture for 5-7 days until the mycelium grows over the plate;
3) preparing a bidirectional fermentation culture medium: mixing 50-100% of the residue powder, 0-48% of the corncob powder, and 0-0.5% of MgSO40-0.5% (NH)4)2SO4And 0-1% KH2PO4After mixing, adjusting the pH value to about 6.5, adding a proper amount of water to make the final concentration 65%, uniformly stirring and compacting, and then sterilizing by high-pressure steam at 121 ℃ for later use, wherein the liquid culture medium is a PDA liquid culture medium prepared by 5-20% of decoction dreg powder water extract;
4) bidirectional fermentation: inoculating the Ganoderma of the expanded culture into the culture medium of step 3, and fermenting in solid or liquid state with solid fermentation inoculum size of 10% and liquid fermentation inoculum size of 2-10%, and culturing at 28 deg.C in dark place for 7-14 days;
5) and (3) fermentation mycoplasm treatment: when the hyphae grow over the whole culture medium and the appearance is thick and white, the fermentation is finished, when the liquid fermentation is carried out, the hypha balls grow over the whole fermentation liquid, the hypha balls are spherical, and when the appearance is thick and white, the fermentation is finished; drying and grinding the solid fermentation product in an oven at 65 ℃; separating mycelium pellets from a liquid culture medium, carrying out rotary evaporation on a clear liquid rotary evaporator, collecting a product, mixing the product with the mycelium pellets, drying in an oven at 65 ℃, and grinding into powder; or discarding the clear liquid, drying the mycelium pellets in a 65 ℃ oven and grinding the mycelium pellets into powder;
6) polysaccharide extraction: adding distilled water into the fermentation product dry powder according to the mass ratio of 1: 30, heating and extracting, separating supernatant, and concentrating to obtain supernatant with the volume of 1/2-1/6; adding 95% ethanol into the concentrated solution until the final concentration of ethanol is 80%, stirring, standing to precipitate polysaccharide, centrifuging to collect precipitate, and vacuum drying to obtain biological polysaccharide.
2. The method for obtaining the biological polysaccharide of the radix isatidis by utilizing the bidirectional fermentation technology as claimed in claim 1, wherein the method comprises the following steps: in the step of processing the dregs, the dregs of the radix isatidis and/or the folium isatidis are dried, cut into segments and ground into powder of 10-80 meshes.
3. The method for obtaining the biological polysaccharide of the radix isatidis by utilizing the bidirectional fermentation technology as claimed in claim 1, wherein the method comprises the following steps: preparing the decoction dreg powder extracting solution: taking a certain weight of the decoction dreg powder, adding distilled water with the same weight, putting the mixture into a sealed container, boiling the mixture for 20 minutes, and filtering the mixture by 16 layers of gauze to obtain the traditional Chinese medicine decoction.
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| CN113249227A (en) * | 2021-05-11 | 2021-08-13 | 聊城大学 | Novel lucid ganoderma strain and cultivation application thereof based on medicinal residue fungus bag |
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