CN111349670A - Extraction method of plant medicinal material polysaccharide and prepared product - Google Patents
Extraction method of plant medicinal material polysaccharide and prepared product Download PDFInfo
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Abstract
The invention belongs to the technical field of traditional Chinese medicine microorganisms, and particularly relates to a method for improving the content of plant polysaccharide. The method is to improve the content of plant polysaccharide by a microbial fermentation method, wherein the microorganism is at least one of endophyte (Paraengyodontium SP) MD313901 strain and (Purpureocellurium SP) MD313902 strain, and is separated from natural plant medicinal materials. The fermentation method of the endophyte of the plant provided by the invention can obviously improve the polysaccharide content of the plant medicine, can build a product probiotic environment and improve the pharmacological activity, and can be widely used for deep fermentation production of the plant medicine.
Description
Technical Field
The patent belongs to the field of traditional Chinese medicine microbial technology application, and particularly relates to a plant endophytic strain for improving plant polysaccharide content and application thereof.
Background
Polysaccharides are widely existing substances in organisms, are natural polymer polymers formed by connecting aldose or ketose through glycosidic bonds, are important biological macromolecules in organisms, and are one of basic substances for maintaining normal operation of life activities. Plant polysaccharides, also known as plant polysaccharides, are polysaccharides with a degree of polymerization of more than 10 produced by plant cell metabolism.
Plant polysaccharide research is receiving increasing attention today and the international scientific community even proposes the 21 st century to be the century for polysaccharides. Scientific experimental research shows that many plant polysaccharides have biological activity and pharmacological activity, can be used as a broad-spectrum immunopotentiator to regulate the immune function of organisms, and can play a wide range of pharmacological actions in the aspects of tumor resistance, virus resistance, oxidation resistance, blood sugar reduction, radiation resistance and the like. Because of its wide source, strong pharmacological activity, low cytotoxicity, strong safety and small toxic and side effects, the polysaccharide has attracted extensive attention in the medical field and becomes one of the hot spots in the life science research of today.
Up to now, more than 300 kinds of polysaccharide compounds have been isolated from natural products. At present, the extraction method of plant polysaccharide mainly comprises the following steps: hot water extraction, alkaline extraction, enzymatic extraction, microwave-assisted methods, and ultrasonic methods. The enzyme method is a method combining enzyme and hot water extraction, and the enzyme mostly adopts a certain amount of pectinase, cellulase and neutral protease. The main methods include a complex enzyme method, a separate enzyme method and a single enzyme method. The enzymatic extraction of plant polysaccharide has the advantages of mild reaction conditions, high efficiency, easy removal of impurities, simple and convenient process, time saving and the like.
Endophytes (endophytes) are a large group of microorganisms that live within the tissues and organs of healthy plants at some or all stages, and they can form parasitic, symbiotic, saprophytic, etc. relationships with plants. It has been found that not only endophytes can be isolated from various plants, but also endophytes are found in the roots, stems, leaves, flowers, fruits, seeds and the like of the same plant. The endophyte is large in quantity and various in types, and comprises endophyte, endophyte actinomycetes and the like. Endophytes of plants have a great diversity of organisms, and for a plant, the number of endophytes or bacteria that can be isolated from a plant is usually several to several tens, and some may even be hundreds. The plant endophyte is also a new microbial resource with potential application value, and the research of searching new bioactive substances from the plant endophyte becomes a hotspot.
Disclosure of Invention
The plant polysaccharide has complex structure and various varieties, is often combined with lipid and protein to form polysaccharide compound, and the extraction rate of the plant polysaccharide is low and the cost is high by adopting a conventional extraction method. Aiming at the defects of the prior art, the invention provides a method for extracting plant polysaccharide, aiming at innovatively adopting the brand-new endophytic fungi strain fermentation method provided by the invention to improve the content of the plant polysaccharide in medicinal materials, remove ineffective components and toxic components and increase the medicinal activity.
The second purpose of the invention is to provide a fermentation liquor prepared by the method.
A method for extracting polysaccharide from plant medicinal material comprises subjecting plant medicinal material to microorganism fermentation treatment, wherein the microorganism comprises at least one of (Paraengyodontium SP.) MD313901 strain (also referred to as MD313901 for short), and (PurpureocelliumSP.) MD313902 strain (also referred to as MD313902 for short);
(Paraengyodontium SP.) the preservation number of the MD313901 strain is CCTCC M2018256;
(Purpureocillium SP.) the strain MD313902 has a preservation number of CCTCC M2018257.
The Paraengyodontium SP.MD313901 strain and Purpureocillium SP.MD313902 strain both belong to endophytic fungi, and are preserved in the China center for type culture Collection, the preservation place is Wuhan, and the preservation time is 5 months and 8 days in 2018 years; the preservation numbers are CCTCC M2018256 and CCTCC M2018257 respectively.
The invention innovatively discovers that the extraction rate of polysaccharide components of natural medicinal materials can be obviously improved by fermenting plant materials by adopting MD313901 and MD313902 strains.
Preferably, the microorganism is a group comprising (purpureococcus SP.) MD313902 strain and (paragyododotium SP.) MD313901 strain.
The innovative research of the invention finds that the (Parengyodontium SP.) MD313901 strain and the (Purpureococcum SP.) MD313902 strain can unexpectedly improve the extraction rate of polysaccharide or glycoprotein fermented by natural medicinal materials. In addition, the inventor also researches and discovers that the MD313901 strain and the MD313902 have good cooperativity, and can further improve the extraction rate of polysaccharide components or glycoprotein components of natural medicinal materials. The inventor further researches and discovers that the combined MD313901 strain and MD313902 strain has a better technical effect on the aspect of improving the extraction of the polysaccharides of two or more mixed plant medicinal materials.
In order to further improve the extraction rate of the plant medicinal materials, wall breaking pretreatment is performed before the fermentation.
Further preferably, the wall breaking pretreatment is biological enzyme enzymolysis.
The research of the invention finds that the biological enzyme is used for breaking the wall of the plant medicinal material, and then the microbial strain is used for fermentation, so that the release of polysaccharide components of the medicinal material can be further effectively promoted, the content of the polysaccharide components of the medicinal material is improved, and the extraction rate of the polysaccharide is obviously improved.
According to the invention, the biological enzyme treatment and the fermentation of the microbial strains are combined, the biological enzyme treatment is firstly carried out, the quick release of effective components is promoted, and then the fermentation of the microbial strains is carried out, so that the content of plant polysaccharide components is improved.
In the invention, the biological enzyme enzymolysis process comprises the following steps: cleaning the plant materials to be treated, cutting or homogenizing, and adding microbial enzyme to break the cell wall.
Preferably, the biological enzyme is at least one of pectinase, cellulase, hemicellulase, protease and amylase.
Preferably, in the enzymolysis process, the dosage of the biological enzyme is 0.01 to 5 percent of the weight of the plant medicinal materials; preferably 3 to 5%.
Further preferably, the biological enzyme is pectinase and cellulase, wherein the dosage of the pectinase is 0.1-5% of the weight of the plant medicinal material; the dosage of the cellulase is 0.1-5% of the weight of the plant medicinal materials.
The biological enzyme treatment is carried out by adopting conditions such as proper temperature, proper pH value and the like.
The pH value in the optimized enzymolysis process is 4-8.
The preferred temperature for the enzymatic process is 30-60 ℃.
The enzymolysis time is preferably 2-8 hours.
After the enzymolysis is finished, the enzymolysis system does not need to be separated, and the innovative strain of the invention is directly sterilized and inoculated for fermentation.
The sterilization method can adopt the existing method. For example, steam sterilization is employed for a period of, for example, 30 to 60 minutes.
The method of the invention can further convert some components except the polysaccharide in the wall-broken system into the polysaccharide by fermenting the strain or the flora of the invention on the wall-broken system, thereby further improving the content of the polysaccharide in the fermentation liquor and the extraction rate of the polysaccharide.
The invention further combines the wall breaking pretreatment of the medicinal materials and the control of fermentation parameters in addition to the use of the creative ground strains, can further improve the fermentation effect and further improve the extraction rate of the polysaccharide.
Preferably, the inoculation amount of the microorganisms is 0.01-20% of the total raw material (weight of the plant medicinal materials) in the fermentation process.
More preferably, the inoculation amount of the microorganism is 0.1-10% of the total raw material (weight of the plant medicinal materials). In the preferred inoculation range, the fermentation period can be effectively shortened, in addition, the over-fast growth of the strains can be avoided, the rapid aging and death of the strains can be reduced, and the polysaccharide content of the fermentation product can be improved.
More preferably, the inoculation amount of the microorganism is 0.3-6% of the total raw material (weight of the plant medicinal materials).
Preferably, the fermentation temperature is 10-50 ℃. The fermentation effect is more excellent when the concentration is controlled within the preferred range, and the polysaccharide extraction rate is not favorable when the concentration is not controlled within the preferred range.
Further preferably, the fermentation temperature is 25-30 ℃.
Preferably, the fermentation time is 48h or more; preferably 5 to 30 days.
The fermentation process is carried out in a closed container.
The fermentation mode is preferably static fermentation.
In the invention, the plant medicinal materials to be fermented comprise one or more of dandelion, rhizoma polygonati, dendrobium, Chinese yam, angelica, burdock root, rape pollen, purslane, lophatherum gracile, astragalus, fig, kudzu root, figwort, sea-buckthorn, coix seed, corn stigma, pokeberry root, medlar, liquorice and mulberry leaf, and can be a certain part of the medicinal materials or the whole plant of the medicinal materials.
The invention relates to a preferable extraction method of plant medicinal material polysaccharide, which is realized by the following steps:
(1) selecting high-quality medicinal materials with impurities removed, and cleaning with flowing drinking water.
(2) Putting the cleaned raw materials into a fermentation tank, adding 0.01-5% of biological enzyme (also called microbial enzyme), controlling the enzymolysis temperature at 30-60 ℃, adding edible acid or edible alkali to adjust the pH value to 4.0-8.0, carrying out enzymolysis for 2-8 hours, and after the enzymolysis is finished, introducing steam for sterilization for 30-60 minutes;
(3) cooling the enzymolysis liquid to room temperature, adding microbial strains accounting for 0.01-20% of the weight of the raw materials, controlling the stirring speed of a fermentation tank to be 50-300rpm, controlling the fermentation temperature to be 15-50 ℃, and carrying out closed fermentation for 5-30 days.
(4) Collecting fermentation liquor, and directly filling the fermentation liquor or continuously processing the fermentation liquor to prepare different preparations.
The effective components, such as polysaccharides, in the fermentation broth can be extracted by conventional methods.
Preferably, the extraction process comprises aqueous and alcoholic extraction as follows.
The water extract is obtained by extracting the fermentation liquid with water at 80 deg.C or above.
The alcohol extraction is to add alcohol into the water extract, control the content of the alcohol in a solution system to be 60-90%, and carry out alcohol extraction.
After water extraction treatment, free protein in the water extraction product is removed, and then alcohol extraction is carried out.
The invention also comprises the fermentation liquor obtained by the method.
Advantageous effects
The invention provides a brand-new (Paraengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain; the brand new strain is innovatively found to be beneficial to improving the extraction rate of total polysaccharides and glycoproteins of natural medicinal materials. In addition, the invention also discovers that the combined use of the (paragyododium SP.) MD313901 strain and the (purpureococcus SP.) MD313902 strain has a synergistic effect, which is helpful for further improving the extraction rate of the polysaccharide of the natural medicinal materials, and can also synergistically improve the extraction of the polysaccharide of a plurality of mixed plant medicinal materials.
The plant endophytic bacteria (Paraengyodontium SP) MD313901 strain and (Purpureocillium SP) MD313902 strain for fermentation are from natural plant medicinal materials, and can be independently applied to improve the content of plant polysaccharide, can also form plant endophytic bacteria, and can jointly act to improve the content of plant polysaccharide.
The (paragyododium SP.) MD313901 strain and the (purpurococcum SP.) MD313902 strain can obviously improve the polysaccharide content of the plant medicine, and simultaneously can create a product probiotic environment and improve the pharmacological activity of the plant medicine.
Detailed Description
The following examples are intended to be illustrative of the present invention and are not to be construed as limiting the scope of the invention, which is intended to be covered by the claims.
(Paraengyodontium SP.) MD313901 strain, (Purpureocillium SP.) MD313902 strain isolation
The method for separating the (paragyododium SP.) MD313901 strain and/or the (purpurococcum SP.) MD313902 strain comprises the steps of plant tissue pretreatment, leaching, separation and purification: the operation process is as follows:
(1) plant tissue pretreatment
Cutting plant tissues into sections, soaking the plant tissues in an ethanol solution, soaking the plant tissues in a sodium hypochlorite solution, and then washing the plant tissues with sterile water to obtain pretreated plant tissues;
(2) plant tissue endophyte leaching
Grinding the pretreated plant tissue and the biological enzyme, and separating supernatant in grinding liquid;
(3) separation and purification of endophytic bacterial strain of plant
And (3) coating the supernatant on a culture medium, and selecting the strain or flora after culture.
The plant tissue is at least one of rhizoma Polygonati Odorati, radix astragali, fructus Hippophae, herba Portulacae, rhizoma Polygonati, radix Platycodi, radix Sophorae Tonkinensis, radix Isatidis, radix Phytolaccae, herba Houttuyniae, rhizoma corydalis, rhizoma Pinelliae, radix Arctii, radix Platycodi, herba Taraxaci, Glycyrrhrizae radix, radix Paeoniae alba, herba Dendrobii, radix Angelicae sinensis, and rhizoma Acori Graminei.
In the step (1), the plant tissue is washed clean by water and then washed by sterile water, the plant tissue is cut into sections with the length of 0.5-2.0cm after the surface moisture is dried, then the cut material is placed in ethanol with the volume concentration of 70-80% for disinfection for 1-2 min, washed by sterile water, soaked by sodium hypochlorite with the mass concentration of 3-8% for 5-10min, washed by sterile water and dried, and the pretreated plant tissue is obtained.
In the step (2), the pretreated plant tissue is placed in a sterilized mortar or a grinder, biological enzyme is added for grinding, grinding liquid is transferred into a conical flask for shaking for 1-2 hours at the temperature of 25-40 ℃ and at the speed of 200rpm, and supernatant and plant residues are obtained by separation.
Preferably, the biological enzyme is one or more of pectinase, cellulase and neutral protease.
Preferably, the dosage of the biological enzyme is 0.01-5% of the weight of the medicinal materials (plant tissues).
In the step (3), diluting the supernatant obtained in the leaching process by 10-20 times with sterile water under the aseptic condition, respectively taking 100-.
In the step (3), the fungus obtained by separation is purified by a scribing method.
Preparation example 1
The preparation method of the plant endophytic flora is realized by the following steps:
(1) plant tissue pretreatment
Cleaning plant tissues (adopting polygonatum odoratum plant medicinal materials of different producing areas and different varieties shown in table 1) with tap water, washing with sterile water, air-drying surface water, cutting into sections with the length of 0.5-2.0cm, sterilizing the cut materials with ethanol with the volume concentration of 70% for 2min, washing with sterile water, soaking with sodium hypochlorite with the mass concentration of 3% for 10min, washing with sterile water, and air-drying surface water for later use;
(2) plant tissue endophyte leaching
Placing the sterilized rhizoma Polygonati Odorati tissue in a sterilized mortar, adding biological enzyme (cellulase, 5%), grinding, and shaking the grinding solution in a conical flask at 25 deg.C and 200rpm for 2 hr to obtain supernatant and plant residue.
(3) Separation and purification of endophytic bacterial strain of plant
Diluting the supernatant obtained in the leaching process with sterile water by 10 times under aseptic condition, respectively coating 200 μ L of the supernatant on MS culture medium for culturing, selecting colonies with different forms after culturing for a period of time to form colonies, culturing the fungi in PDA (potato dextrose agar) culture medium, and culturing the bacteria in beef extract peptone culture medium. Purifying by scribing.
Selecting different producing areas and different varieties of polygonatum odoratum plant medicinal materials, and separating the endophytic bacteria of the polygonatum odoratum plant medicinal materials as shown in table 1.
TABLE 1
The results in table 1 show that different species of Yuzhu in different producing areas can obtain two kinds of strains by the separation method.
The 16sRNA fragment of the MD313901 strain of the invention is analyzed, has a fungus-specific ITS2 fragment, has 99 percent of homology with Parangyodontium SP., is named as MD313901, (Parangyodontium SP.) MD313901 strain which is preserved in China Center for Type Culture Collection (CCTCC) M2018256 in 2018, 5 and 8 days.
The 16sRNA fragment of the MD313902 strain of the present invention was analyzed to have a fungal-specific ITS2 fragment, which has 99% homology with purporococcus SP. and was designated as MD313902, (purporococcus SP.) the MD313902 strain was deposited at 2018, 8 days via the chinese collection center for type cultures, with a deposit number of CCTCC M2018257.
Preparation example 2
The preparation method of the plant endophytic flora is realized by the following steps:
(1) plant tissue pretreatment
Washing plant tissues (different producing areas and different varieties of platycodon grandiflorum plant medicinal materials shown in table 2) with tap water, washing with sterile water, air-drying surface water, cutting into sections with the length of 0.5-2.0cm, sterilizing the cut materials with 80 vol% ethanol for 1min, washing with sterile water, soaking with 8 wt% sodium hypochlorite for 5min, washing with sterile water, and air-drying surface water for later use;
(2) plant tissue endophyte leaching
Placing the sterilized plant tissue in a sterilized mortar, adding biological enzyme (mixed enzyme composed of pectase and cellulase 1:1, dosage is 2%) for grinding, transferring the grinding solution into a conical flask, and shaking at 40 deg.C and 100rpm for 1 hr to obtain supernatant and plant residue.
(3) Separation and purification of endophytic bacterial strain of plant
Diluting the supernatant obtained in the leaching process with sterile water by 20 times under aseptic condition, respectively coating 100 μ L of the supernatant on MS culture medium for culturing, selecting colonies of different forms after culturing for a period of time to form colonies, culturing the fungi in PDA (potato dextrose agar) culture medium, and culturing the fungi in beef extract peptone culture medium. Purifying by scribing.
According to the method, platycodon grandiflorum plant medicinal materials in different producing areas and different varieties are separated by the method, purified by a streak culture method, and separated from endophytes of the platycodon grandiflorum plants, and the results are shown in table 2.
TABLE 2
| Bacterial strain | Radix Platycodi produced by Anhui province | Radix Platycodi produced from Yunnan province | Radix Platycodi produced in Sichuan province | Radix Platycodi produced by Heilongjiang |
| MD313901 | √ | √ | √ | √ |
The results in table 2 show that different species of platycodon grandiflorum in different production areas can obtain a strain by the separation method.
Preparation example 3
The preparation method of the plant endophytic flora is realized by the following steps:
(1) plant tissue pretreatment
Washing plant tissues (different species of pokeberry root plant medicinal materials shown in Table 2) with tap water, then washing with sterile water, air-drying surface water, cutting into sections with the length of 0.5-2.0cm, then placing the cut materials in 75% ethanol with volume concentration for disinfection for 1.5min, washing with sterile water, soaking with 5% sodium hypochlorite for 8min, then washing with sterile water, and air-drying surface water for later use;
(2) plant tissue endophyte leaching
Placing the sterilized plant tissue in a sterilized mortar, adding biological enzyme (mixture of pectinase, cellulase and neutral protease at a ratio of 1:1:1, and the dosage is 0.1%) for grinding, transferring the grinding solution into a conical flask, and shaking at 35 deg.C and 200rpm for 1.5 hr to obtain supernatant and plant residue.
(3) Separation and purification of endophytic bacterial strain of plant
Diluting the supernatant obtained in the leaching process with sterile water by 20 times under aseptic condition, respectively coating 200 μ L of the supernatant on MS culture medium for culturing, selecting colonies of different forms after culturing for a period of time to form colonies, culturing the fungi in PDA (potato dextrose agar) culture medium, and culturing the fungi in beef extract peptone culture medium. Purifying by scribing.
Different species of phytolacca acinosa medicinal materials are taken, purified by a streak culture method, and endophytes of the phytolacca acinosa medicinal materials are separated, and the results are shown in table 2.
TABLE 3
The results in Table 3 show that two species of Phytolacca acinosa can be obtained by the separation method of the present invention.
In the following cases, the total raw material mass refers to the weight of the herbs except for special statements.
An embodiment for improving the polysaccharide content of a single medicinal material by fermenting microbial strains comprises the following steps:
example 1:
1. and (3) carrying out enzymolysis wall breaking on the dandelion medicinal material:
taking 10kg of dandelion, removing impurities, washing with tap water, pouring into a fermentation tank, adding purified water to submerge the dandelion, adding 0.1% of pectinase (mass fraction of the total raw materials) and 0.1% of cellulase (mass fraction of the total raw materials), fully stirring and mixing, adjusting the pH value to 6.8, controlling the temperature to be 48 ℃, performing enzymolysis for 2 hours, and then introducing steam for sterilization for 40 minutes.
2. Fermentation of
Cooling to room temperature, adding 0.3% (total raw material mass fraction) Parangyodontium SP.MD313901 strain, standing and fermenting at 30 deg.C for 6 days, filtering, and collecting medicinal liquid for treatment.
3. Extraction and content determination of dandelion polysaccharide
Adding 8 times of purified water into the liquid medicine collected after fermentation, heating and refluxing for 2 times at 100 ℃, each time for 2 hours, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to make the concentration of the solution reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the dandelion total polysaccharide.
The measurement of the polysaccharide content of the dandelion adopts a phenol-sulfuric acid method, and the measurement result shows that the polysaccharide content of the dandelion after fermentation reaches 60.4 percent of the dry weight of the dandelion.
Comparative example 1
Compared with the example 1, the difference is that the strain fermentation in the step 2 is not carried out, and the specific operation is as follows:
taking 10kg of dandelion, removing impurities, washing with tap water, pouring into a fermentation tank, adding purified water to submerge the dandelion, adding 0.1% of pectinase (mass fraction of the total raw materials) and 0.1% of cellulase (mass fraction of the total raw materials), fully stirring and mixing, adjusting the pH value to 6.8, controlling the temperature to be 48 ℃, performing enzymolysis for 2 hours, and then introducing steam for sterilization for 40 minutes. Collecting the liquid medicine for treatment.
Adding 8 times of purified water into the liquid medicine collected after enzymolysis, heating and refluxing for 2 times at 100 ℃, each time for 2 hours, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to make the concentration of the solution reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the dandelion total polysaccharide.
The measurement of the polysaccharide content of the dandelion adopts a phenol-sulfuric acid method, and the measurement result shows that the polysaccharide content of the dandelion after fermentation reaches 22.4 percent of the dry weight of the dandelion.
Compared with the example 1, the comparative example 1 has the advantages that the medicinal materials are only subjected to wall breaking treatment, and microbial fermentation is not carried out. The comparison between example 1 and comparative example 1 shows that the polysaccharide of dandelion in example 1 is 2.7 times higher.
Example 2:
1. wall breaking of the burdock root medicinal material:
taking 10kg of burdock root medicinal material, removing impurities, washing with tap water, pouring into a fermentation tank, adding purified water to submerge the medicinal material, adding 1% of pectinase (total raw material mass fraction) and 2% of cellulase (total raw material mass fraction), fully stirring and mixing, adjusting the pH value to 6.8, controlling the temperature to 50 ℃, performing enzymolysis for 1.5 hours, and then introducing steam for sterilization for 30 min.
2. Fermentation:
cooling to room temperature, adding 0.3% (total raw material mass fraction) of Purpureocillium SP.MD002 strain, standing at 30 deg.C for fermenting for 5 days, filtering, and collecting medicinal liquid for treatment.
3. Extraction and content determination of burdock root polysaccharide
Adding 10 times of purified water into the liquid medicine collected after fermentation, heating and refluxing for 2 times at 90 ℃, each time for 2 hours, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to enable the concentration of the solution to reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the burdock root polysaccharide.
The measurement of the content of the burdock root polysaccharide adopts a phenol-sulfuric acid method, and the measurement result shows that the content of the burdock root polysaccharide after fermentation reaches 28.3 percent of the dry weight of the burdock root. Compared with the burdock root polysaccharide in the case without fermentation (directly extracted after wall breaking treatment) in the step 2 of the embodiment, the burdock root polysaccharide is 2.1 times higher.
Secondly, the embodiment of improving the polysaccharide content in the medicinal materials of the composition by fermenting the microbial strains comprises the following steps:
example 3:
1. breaking the wall of the composition;
weighing 5kg of dendrobe and 5kg of purslane as a composition, removing impurities, washing with tap water, pouring into a fermentation tank, adding purified water to submerge medicinal materials, adding 3% of pectinase (total raw material mass fraction) and 2% of cellulase (total raw material mass fraction), fully stirring and mixing, adjusting the pH value to 7.0, controlling the temperature to be 45 ℃, performing enzymolysis for 3 hours, and then introducing steam for sterilization for 30 minutes.
2 fermentation
Cooling to room temperature, adding 1% (total raw material mass fraction) of Paraengyodontium SP.MD313901 strain and 1% (total raw material mass fraction) of Purpureocillium SP.MD313902 strain, standing and fermenting at 30 deg.C for 10 days, filtering, and collecting medicinal liquid for treatment.
3. Extraction and content determination of composition polysaccharide
Adding 10 times of purified water into the liquid medicine collected after fermentation, heating and refluxing for 2 times at low temperature of 65 ℃ in vacuum for 2 hours each time, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to enable the concentration of the solution to reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the composition polysaccharide.
The measurement of the polysaccharide content of the composition adopts a phenol-sulfuric acid method, and the measurement result shows that the polysaccharide content of the composition after fermentation reaches 30.2% of the dry weight of the composition, and the polysaccharide content is 2 times higher than that of the case without the fermentation step of the step 2 (the step 3 is directly extracted after the wall breaking treatment of the step 1).
Example 4:
1. wall breaking of the composition:
taking 5kg of astragalus and 5kg of codonopsis pilosula, removing impurities, washing with tap water, pouring into a fermentation tank, adding purified water to submerge the medicinal materials, adding 4% of pectinase (total raw material mass fraction) and 1% of cellulase (total raw material mass fraction), fully stirring and mixing, adjusting the pH value to 7.0, controlling the temperature to be 45 ℃, performing enzymolysis for 3 hours, and then introducing steam for sterilization for 30 minutes.
2. Fermentation of
Cooling to room temperature, adding 2% (total raw material mass fraction) Paraengyodontium SP.MD313901 strain and 2% (total raw material mass fraction) Purpureocillium SP.MD313902 strain, standing and fermenting at 30 deg.C for 10 days, filtering, and collecting medicinal liquid for treatment.
Extracting and content measuring of composition polysaccharide
Adding 10 times of purified water into the fermented composition liquid medicine, heating and refluxing for 2 times at low temperature of 70 ℃ in vacuum for 2 hours each time, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to make the concentration of the solution reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the composition polysaccharide.
The content of the polysaccharide in the composition is measured by a phenol-sulfuric acid method, and the measurement result shows that the content of the polysaccharide in the composition after fermentation reaches 51.4% of the dry weight of medicinal materials of the composition, and the content of the polysaccharide is 1.4 times higher than that in the case that the fermentation step in the step 2 (the step 3 is directly extracted after the wall breaking treatment in the step 1) is not carried out in the embodiment.
Example 5:
1. wall breaking of the composition:
weighing 5kg of rhizoma polygonati, 5kg of Chinese yam, 5kg of pokeberry root and 5kg of kudzu root as a composition, removing impurities, washing the composition with tap water, pouring the composition into a fermentation tank, adding purified water to submerge the medicinal materials, adding 1% of pectinase (total raw material mass fraction) and 4% of cellulase (total raw material mass fraction), fully stirring and mixing, adjusting the pH value to 7.0, controlling the temperature to be 50 ℃, performing enzymolysis for 4 hours, and introducing steam for sterilization for 60 minutes.
2. Fermentation of
Cooling to room temperature, adding 3% (total raw material mass fraction) Paraengyodontium SP.MD313901 strain and 3% (total raw material mass fraction) Purpureocillium SP.MD313902 strain, standing and fermenting at 25 deg.C for 15 days, filtering, and collecting medicinal liquid for treatment.
3. Extraction and content determination of composition polysaccharide
Adding 10 times of purified water into the fermented composition liquid medicine, heating and refluxing for 2 times at low temperature of 70 ℃ in vacuum for 2 hours each time, filtering to obtain an extracting solution, concentrating to a proper concentration, removing free protein by a Sevag method, adding absolute ethyl alcohol to make the concentration of the solution reach 80%, performing centrifugal precipitation twice, and drying the precipitate to obtain the composition polysaccharide.
The measurement of the polysaccharide content of the composition adopts a phenol-sulfuric acid method, and the measurement result shows that the polysaccharide content of the composition after fermentation reaches 42.0% of the dry weight of the composition, and the polysaccharide content is 2.1 times higher than that of the case without the fermentation step of the step 2 (the step 3 is directly extracted after the wall breaking treatment of the step 1).
Example 6
Compared with the example 1, the difference is only that the adopted strains are (Paraengyodontium SP.) MD313901 strain and (Purpureococcum SP.) MD313902 strain (the ratio of the two strains is 1:1, the total inoculation amount of the strains is 0.3 percent of the weight of dandelion herb), and other parameters are the same as the example 1.
The measurement of the polysaccharide content of the dandelion adopts a phenol-sulfuric acid method, and the measurement result shows that the polysaccharide content of the dandelion after fermentation reaches 66.3 percent of the dry weight of the dandelion.
By comparing examples 1 and 6, it was found that a synergistic effect can be achieved by the combination of the two species.
Example 7
Compared with the example 1, the difference is that the wall breaking treatment of the step 1 is not carried out, impurities of dandelion medicinal materials are directly removed, the dandelion medicinal materials are washed clean by tap water, poured into a fermentation tank, inoculated with the strain after sterilization, fermented in the step 2, and then extracted by the step 3. The content of polysaccharide in the fermentation broth was tested to be 40.6% using the method of example 1.
Example 8
Compared with example 1, the difference is only that the superfine grinding wall breaking method in step 1 replaces the enzymatic wall breaking method in example 1. Other operations and parameters were the same as those in example 1. Using the method of example 1, the polysaccharide content of the fermentation broth was tested to be 56.0%.
Through comparison of example 1, example 7 and example 8, it is found that the polysaccharide content of the fermentation product after the wall-breaking treatment is obviously improved by comparing the fermentation experiment of the medicinal material which is subjected to the wall-breaking treatment in advance with the fermentation experiment of the non-wall-breaking treatment. In addition, the invention also researches and discovers that the adopted wall breaking technology is biological enzyme wall breaking, and an ultramicro crushing wall breaking method is selected for comparison in the experiment implementation process, so that compared with the ultramicro crushing wall breaking technology, the biological enzyme wall breaking is milder, the wall breaking effect is more sufficient, and no influence is caused on active ingredients.
Claims (10)
1. A method for extracting polysaccharide from plant medicinal materials is characterized in that the plant medicinal materials are subjected to microbial fermentation treatment, and the microorganisms comprise at least one of (Paraengyodontium SP.) MD313901 strain and (Purpureocellidium SP.) MD313902 strain;
(Paraengyodontium SP.) the preservation number of the MD313901 strain is CCTCC M2018256;
(Purpureocillium SP.) the strain MD313902 has a preservation number of CCTCC M2018257.
2. The method for extracting polysaccharides from plant materials according to claim 1, wherein the microorganism is a flora comprising (Purpureocillium SP.) MD313902 strain and (Paraengyodontium SP.) MD313901 strain.
3. The method for extracting polysaccharide from plant material as claimed in claim 1, wherein the plant material is subjected to microbial fermentation and pre-treatment of wall breaking.
4. The method for extracting polysaccharide from plant materials as claimed in claim 3, wherein the wall-breaking pretreatment is bio-enzyme enzymolysis.
5. The method for extracting plant polysaccharide as claimed in claim 4, wherein the biological enzyme is one or more of pectinase, cellulase, hemicellulase, neutral protease and amylase.
6. The method for extracting plant medicinal material polysaccharide as claimed in claim 5, wherein in the enzymolysis process, the dosage of the biological enzyme is 0.01% -5% of the weight of the plant medicinal material;
the pH value in the enzymolysis process is 4-8.
7. The method for extracting polysaccharides from plant materials according to any one of claims 1 to 6, wherein the amount of the microorganism inoculated during the fermentation is 0.01 to 20% of the total raw material mass.
8. The method for extracting polysaccharides from plant materials according to claim 7, wherein the fermentation temperature is 10-50 ℃ and the fermentation time is 48 hours or more.
9. The method for extracting polysaccharide from plant material according to any one of claims 1 to 8, wherein the plant material is a medicinal part or a whole plant of one of dandelion, rhizoma polygonati, dendrobium, yam, angelica, burdock root, rape pollen, purslane, lophatherum gracile, astragalus, fig, kudzu root, figwort root, sea buckthorn, coix seed, corn stigma, pokeberry root, wolfberry fruit, licorice and mulberry leaf.
10. A fermentation product obtained by the method of any one of claims 1 to 9.
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