CN111349568A - Endophytic fungi strain and strain as well as separation and application methods thereof - Google Patents
Endophytic fungi strain and strain as well as separation and application methods thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial preparation, and particularly relates to a plant endophytic flora and a separation and purification method thereof. The plant endophyte flora at least comprises two plant endophyte beads of (Paraengyodontium SP.) MD313901 and (Purpureocellium SP.) MD 313902. The plant endophytic flora is derived from natural plant medicinal materials, and the two strains can be derived from the same plant medicinal material or different plant medicinal materials. The preparation method of the plant endophytic flora comprises the steps of pretreatment of plant tissues, leaching of endophytic bacteria in the plant tissues, separation and purification of the endophytic bacteria in the plant tissues.
Description
Technical Field
The invention relates to the technical field of microbial preparation, in particular to a preparation method of microbial floras.
Background
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 are abundant in biodiversity, and for a single plant, the number of endophytes or bacteria that can be isolated from the endophytes or bacteria 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 first purpose of the invention is to provide a plant endophytic fungus (Paraengyodontium SP.) MD313901 strain.
It is a second object of the present invention to provide a strain of endophytic fungus (purpurococcum SP.) MD 313902.
A third object of the present invention is to provide an endophytic bacteria population of a plant comprising (paragyododotium SP.) MD313901 strain and (purpurococcum SP.) MD313902 strain.
The fourth object of the present invention is to provide a microbial inoculum comprising (paragyododotium SP.) MD313901 strain and/or (purpurococcum SP.) MD313902 strain.
A fifth object of the present invention is to provide a method for separating a plant endophytic fungus and a plant endophytic bacteria group according to the present invention.
A sixth object of the present invention is to provide a method for applying the plant endophytic fungi and the plant endophytic bacteria group of the present invention.
An endophytic fungus (Paraengyodonium SP.) MD313901 strain (also called MD313901 for short in the invention) with a preservation number of CCTCC M2018256.
The invention provides a brand-new strain, which belongs to endophytic fungi and is named as (Parangyodontium SP) MD313901, wherein the strain is preserved in China center for type culture Collection, the preservation place is Wuhan, and the preservation time is 2018, 5 and 8 days; the preservation number is CCTCC M2018256.
In addition, the invention also provides another brand new strain, which is a plant endophytic fungus (Purpureocillium SP.) MD313902 strain (also referred to as MD313902 for short in the invention) with the preservation number of CCTCC M2018257.
The brand-new endophytic strain MD313902 provided by the invention belongs to endophytic fungi and is named as (Purpureocillium SP.) MD313902, and is preserved in China center for type culture Collection, wherein the preservation place is Wuhan, and the preservation time is 2018, 5 and 8 days; the preservation number is CCTCC M2018257.
The MD313901 and MD313902 have similar functions, and can remarkably promote the extraction of polysaccharide components or glycoprotein components of natural medicinal materials through the fermentation of the strain, thereby improving the polysaccharide extraction rate.
The invention also provides a plant endophyte flora, which comprises a (Paraengyodontium SP.) MD313901 strain and a (Purpureococcum SP.) MD313902 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 invention also provides an endophyte microbial inoculum which comprises (Paraengyodontium SP.) MD313901 strain and/or (Purpureocillium SP.) MD313902 strain, and also comprises an auxiliary material which can resist survival.
The invention also provides a method for separating the (Paraengyodontium SP.) MD313901 strain and/or the (Purpureocillium SP.) MD313902 strain, which comprises the following 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 research of the invention finds that the method can effectively separate out the target strain or flora.
Preferably, the plant tissue is a natural plant medicinal material.
The plant tissue at least contains at least one strain in the flora.
Preferably, the plant tissue is at least one of polygonatum, astragalus, sea buckthorn, purslane, rhizoma polygonati, platycodon grandiflorum, subprostrate sophora, isatis root, pokeberry root, houttuynia cordata, corydalis tuber, pinellia ternate, burdock root, platycodon grandiflorum, dandelion, liquorice, white paeony root, dendrobium, angelica 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 1-2min, washed by sterile water, soaked in sodium hypochlorite with the mass concentration of 3-8% for 5-10min, washed by the sterile water and dried by the surface moisture, 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 amount 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.
The separation method of the invention effectively avoids the mixed bacteria from influencing the separation effect of the endophyte through disinfection, strictly controls the environment sterility in the whole separation process, and avoids bringing the foreign bacteria in the operation process.
The endophytic floras of the plants are uniformly distributed in the same medicinal material of different producing areas and different varieties by the same preparation method.
The invention also provides application of the (Paraengyodontium SP) MD313901 strain and/or the (Purpureocillium SP) MD313902 strain in fermentation of plant tissues, and the application is used for improving the extraction rate of polysaccharides in the plant tissues.
The research of the invention finds that the extraction rate of total polysaccharide and glycoprotein of the natural plant can be obviously improved by fermenting the natural plant with the MD313901 strain and/or the MD313902 strain.
Preferably, the application is that the plant tissue is subjected to enzymolysis treatment and then is fermented by the (paregyododotituumsp.) MD313901 strain and/or the (purpurococcum SP.) MD313902 strain to extract plant tissue polysaccharide.
The method can further convert certain components except the polysaccharide in the plant tissue into the polysaccharide, thereby further improving the content of the polysaccharide in the fermentation liquor and improving the extraction rate of the polysaccharide.
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 (Paraengyodontium SP) MD313901 strain and the (Purpureocellidium SP) MD313902 strain has a synergistic effect, and is helpful for further improving the extraction rate of the polysaccharide of the natural medicinal materials.
The separation method has good screening temperature, reproducibility and technical stability.
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 in any way, and those skilled in the art who have the benefit of this disclosure will appreciate numerous modifications and variations there from.
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 the table 1) with tap water, washing with sterile water, airing the surface water, cutting into sections with the length of 0.5-2.0cm, placing the cut materials in ethanol with the volume concentration of 70% for disinfection for 2min, washing with sterile water, soaking with sodium hypochlorite with the mass concentration of 3% for 10min, washing with sterile water, airing the 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 disclosed by the invention is analyzed, has a fungus-specific ITS2 fragment, has 99% homology with Parangyodontium SP., is named as MD313901, (Parangyodontium SP.) the MD313901 strain is preserved in the China center for type culture Collection (CCTCCM 2018256) in 2018, 5 and 8 days.
The 16sRNA fragment of the MD313902 strain disclosed by the invention is analyzed, has a fungus-specific ITS2 fragment, has 99% homology with Purpureocillium SP., is named as MD313902, (Purpureocillium SP.) the MD313902 strain is preserved in China center for type culture Collection (CCTCCM 2018257) in 5/8 days in 2018.
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 diluted supernatant on MS culture medium for culturing, selecting colonies with different forms after culturing for a period of time, culturing the colonies with different forms in PDA (potato dextrose agar) culture medium, and culturing the bacteria 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.
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 with mass concentration 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, 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 diluted supernatant on MS culture medium for culturing, selecting colonies with different forms after culturing for a period of time, culturing the colonies with different forms in PDA (potato dextrose agar) culture medium, and culturing the bacteria 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.
Application 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 (total raw material mass fraction) and 0.1% of cellulase (total raw material mass fraction), 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 min.
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% of the dry weight of the dandelion, and the polysaccharide content is 2.7 times higher than that of the case without the fermentation step (step 2, the wall breaking treatment in step 1 and the extraction in step 3).
Application example 2:
1. enzymolysis 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, and the content of the polysaccharide is higher than 2.1 times compared with the case that the fermentation step of the step 2 (the step 3 is directly carried out after the wall breaking treatment of the step 1) in the case is not carried out.
Application example 3:
1. enzymatic wall breaking of compositions
Weighing 5kg of dendrobe and 5kg of purslane 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 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).
Application example 4:
1. enzymatic 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:
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.
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 enable the concentration of the solution to reach 80%, performing centrifugal precipitation twice, and drying precipitates 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.
Application example 5:
1. enzymatic 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:
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 enable the concentration of the solution to reach 80%, performing centrifugal precipitation twice, and drying precipitates 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).
Claims (10)
1. An endophytic fungus (Paraengyodonium SP) MD313901 strain, which is characterized in that the preservation number is CCTCC M2018256.
2. An endophytic fungus (Purpureocillium SP.) MD313902 strain with a preservation number of CCTCC M2018257.
3. An endophyte flora comprising a (Parengyodontium SP.) MD313901 strain and a (Purpureococcum SP.) MD313902 strain.
4. An endophyte inoculant, which is characterized by comprising (Paraengyodontium SP.) MD313901 strain and/or (Purpureococcum SP.) MD313902 strain, and also comprising an auxiliary material which is resistant to survival.
5. A method for separating (Paraengyodontium SP.) MD313901 strain and/or (Purpureocillium SP.) MD313902 strain is characterized by comprising the following steps of pretreatment of plant tissues, extraction of endophytes of the plant tissues, separation and purification of the endophytes: 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 microbial enzyme, and separating supernatant in the grinding liquid;
(3) separation and purification of endophytic bacterial strain of plant
And coating the supernatant on a culture medium, and selecting and purifying the strain or flora after culture.
6. The separation method according to claim 5, wherein in the step (1), the plant tissue is washed clean with water, then washed with sterile water, air-dried, cut into segments with a length of 0.5-2.0cm, then the cut material is sterilized with ethanol with a volume concentration of 70-80% for 1-2min, washed with sterile water, soaked with sodium hypochlorite with a mass concentration of 3-8% for 5-10min, then washed with sterile water, and air-dried to obtain the pretreated plant tissue;
in the step (2), the pretreated plant tissues are placed in a sterilized mortar or a grinder, biological enzyme is added for grinding, grinding liquid is transferred into a conical flask for shaking at the temperature of 25-40 ℃ and 200rpm for 1-2 hours, and supernatant and plant residues are obtained by separation;
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-;
and purifying the separated fungi by adopting a marking method.
7. The isolation method according to claim 5 or 6, wherein the plant tissue comprises at least one species of said bacterial population; preferably 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.
8. The separation method of claim 7, wherein the biological enzyme is one or more of pectinase, cellulase and neutral protease, and the amount is 0.01-5% by weight of the plant tissue.
9. Use of a (paragyododotium SP.) MD313901 strain and/or a (purpureococcus SP.) MD313902 strain for the fermentation of plant tissue for increasing the polysaccharide extraction rate of the plant tissue.
10. Use according to claim 9, wherein the plant tissue is subjected to an enzymatic treatment and then to fermentation.
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