A method for extracting polysaccharide from medicinal plant and the product prepared thereby

Abstract

The application belongs to the technical field of traditional Chinese medicine microorganisms, and particularly relates to a method for increasing the content of plant polysaccharides. The method is used for increasing the content of plant polysaccharides through microbial fermentation, and the microorganism is at least one of a plant endophyte (Parengyodontium SP.) MD313901 strain and a (Purpureocillium SP.) MD313902 strain, which are separated from natural plant medicinal materials. The plant endophyte fermentation method can significantly increase the content of plant polysaccharides, create a product probiotic environment, improve pharmacological activity, and can be widely used in deep fermentation production of plant medicines.

Description

TECHNICAL FIELD

[0001] The patent belongs to the field of Chinese herbal medicine microbial technology application, and particularly relates to a plant endophyte strain for increasing plant polysaccharide content and application thereof. BACKGROUND

[0002] Polysaccharide is a kind of natural high-molecular polymer connected by glycosidic bond of aldehyde sugar or ketose, which is widely existed in organisms. It is an important biological macromolecule in organisms and one of the basic substances for maintaining normal operation of life activities. Plant polysaccharide, also known as plant polysaccharide, is a polysaccharide with a degree of polymerization of more than 10 produced by plant cell metabolism.

[0003] Nowadays, plant polysaccharide research is increasingly concerned, and the international scientific community even proposes that the 21st century is the century of polysaccharide. Scientific experimental research shows that many plant polysaccharides have biological activity and pharmacological activity. It can not only be used as a broad-spectrum immune promoter to regulate immune function of the body, but also can play a wide range of pharmacological effects in anti-tumor, anti-virus, anti-oxidation, blood sugar reduction, anti-radiation and other aspects. Polysaccharide has attracted widespread attention in the medical field due to its wide source, strong pharmacological activity, low cytotoxicity, strong safety and small toxic side effects, and has become one of the hotspots of life science research.

[0004] So far, more than 300 polysaccharide compounds have been isolated from natural products. At present, the extraction methods of plant polysaccharide mainly include hot water extraction, alkali extraction, enzymatic extraction, microwave-assisted method and ultrasonic method. Among them, the enzyme method is a method combining enzyme and hot water extraction, and the enzyme is mainly a certain amount of pectinase, cellulase and neutral protease. The main methods include complex enzyme method, separate enzyme method and single enzyme method. Enzymatic extraction of plant polysaccharide has many advantages such as mild reaction condition, high efficiency, easy impurity removal, simple process and time saving.

[0005] Plant endophyte (Endophyte) is a kind of microorganism living in the tissues and organs of healthy plants at certain stages or all stages, which can form parasitic, symbiotic, saprophytic and other relationships with plants. It is found that endophytes can be isolated from various plants, and endophytes are also found in roots, stems, leaves, flowers, fruits and seeds of the same plant. Plant endophytes are numerous and diverse, including endophytic fungi, endophytic bacteria and endophytic actinomycetes. For a plant, several to dozens of endophytic fungi or bacteria can be isolated from it, and even several hundred endophytic fungi or bacteria can be isolated from it. Plant endophyte is a new microbial resource with potential application value, and searching for new bioactive substances from plant endophyte has become a research hotspot. SUMMARY

[0006] Plant polysaccharide structure is complex, and there are many kinds, and often combined with lipid and protein into polysaccharide complex, the extraction rate of plant polysaccharide is low by using conventional extraction method, and the cost is high. In view of the deficiency of the prior art, the present application provides a plant medicinal material polysaccharide extraction method, which aims to innovatively use the new endophytic fungus strain fermentation method provided by the present application to improve the content of plant polysaccharide in medicinal materials, remove ineffective components and toxic components, and increase the activity of drugs.

[0007] The second object of the present application is to provide a fermentation broth prepared by the method.

[0008] A plant medicinal material polysaccharide extraction method, the plant medicinal material is subjected to microbial fermentation treatment, the microorganism comprises at least one of (Parengyodontium SP.) MD313901 strain (also referred to as MD313901 in the present application) and (Purpureocillium SP.) MD313902 strain (also referred to as MD313902 in the present application);

[0009] The preservation number of (Parengyodontium SP.) MD313901 strain is CCTCC M 2018256;

[0010] The preservation number of (Purpureocillium SP.) MD313902 strain is CCTCC M 2018257.

[0011] The Parengyodontium SP. MD313901 strain and the Purpureocillium SP. MD313902 strain of the present application both belong to plant endophytic fungi, and are preserved in China Center for Type Culture Collection, Wuhan, on May 8, 2018, with preservation numbers of CCTCC M 2018256 and CCTCC M 2018257, respectively.

[0012] The present application innovatively finds that the use of MD313901 and MD313902 strains for fermentation of plant materials can significantly improve the extraction rate of polysaccharide components of natural medicinal materials.

[0013] As a preferred, the microorganism is a flora comprising (Purpureocillium SP.) MD313902 strain and (Parengyodontium SP.) MD313901 strain.

[0014] The application innovatively studies and finds that the (Parengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain can surprisingly improve the extraction rate of polysaccharide or glycoprotein of natural medicinal materials. In addition, the present inventors have also found that the MD313901 strain and MD313902 have good synergy, which can further improve the extraction rate of polysaccharide or glycoprotein components of natural medicinal materials. The present inventors have further found that the combined MD313901 strain and MD313902 strain has better technical effect in improving the polysaccharide extraction of two or more mixed plant medicinal materials.

[0015] In order to further improve the extraction rate of the plant medicinal materials of the application, the wall-breaking pretreatment is performed before the fermentation.

[0016] Further preferably, the wall-breaking pretreatment is biological enzyme hydrolysis.

[0017] The present inventors have found that the wall-breaking treatment of plant medicinal materials by biological enzymes and the fermentation by the microbial strains of the application can further effectively promote the release of medicinal material polysaccharide components, improve the content of medicinal material polysaccharide components, and significantly improve the extraction rate of polysaccharide.

[0018] In the application, the biological enzyme treatment and the fermentation of microbial strains are combined, the biological enzyme treatment is performed first to promote the rapid release of effective components, and then the fermentation of microbial strains is performed to improve the content of plant polysaccharide components.

[0019] In the application, the biological enzyme hydrolysis process is: the plant medicinal materials to be treated are washed, cut or homogenized, and then the wall is broken by adding microbial enzymes.

[0020] As preferred, the biological enzyme is at least one of pectinase, cellulase, hemicellulase, protease, and amylase.

[0021] As preferred, in the enzyme hydrolysis process, the amount of biological enzyme is 0.01%-5% of the weight of plant medicinal materials; preferably 3-5%.

[0022] Further preferably, the biological enzyme is pectinase and cellulase, wherein the amount of pectinase is 0.1%-5% of the weight of plant medicinal materials; and the amount of cellulase is 0.1%-5% of the weight of plant medicinal materials.

[0023] The biological enzyme treatment is performed under suitable temperature and pH conditions.

[0024] The pH of the preferred enzyme hydrolysis process is 4-8.

[0025] The temperature of the preferred enzyme hydrolysis process is 30-60℃.

[0026] The enzymatic hydrolysis time is preferably 2-8 hours.

[0027] After the enzymatic hydrolysis is completed, the enzymatic hydrolysis system does not need to be separated, and the innovative strain of the present application is directly inoculated and sterilized for fermentation.

[0028] The sterilization method can use the existing method. For example, steam sterilization is used, and the time is, for example, 30-60 minutes.

[0029] In the method of the present application, the broken wall system is fermented by the strain or microbial community described in the present application, and some components other than polysaccharides in the broken wall system can be further converted into polysaccharides, so as to further increase the content of polysaccharides in the fermentation liquor and improve the extraction rate of polysaccharides.

[0030] In addition to the innovative use of the strain, the further cooperation of the broken wall pretreatment of medicinal materials and the control of fermentation parameters can further improve the fermentation effect and further improve the extraction rate of polysaccharides.

[0031] As a preferred, the inoculation amount of microorganism in the fermentation process is 0.01-20% of the total raw material (referring to the weight of plant medicinal materials).

[0032] Further preferably, the inoculation amount of microorganism is 0.1%-10% of the total raw material (referring to the weight of plant medicinal materials). Within the preferred inoculation range, the fermentation period can be effectively shortened, and in addition, the rapid growth of the strain can be avoided, the rapid aging and death of the strain can be reduced, and the polysaccharide content of the fermentation product can be improved.

[0033] More preferably, the inoculation amount of microorganism is 0.3%-6% of the total raw material (referring to the weight of plant medicinal materials).

[0034] As a preferred, the fermentation temperature is 10-50℃. Controlled within the preferred range, the fermentation effect is better, and out of the preferred range, the polysaccharide extraction rate is not good.

[0035] Further preferably, the fermentation temperature is 25-30℃.

[0036] As a preferred, the fermentation time is 48h and above; preferably 5-30 days.

[0037] The fermentation process is carried out in a closed container.

[0038] The fermentation mode is preferably static fermentation.

[0039] In the present application, the plant medicinal materials to be fermented include one or more of dandelion, rhizoma polygonati, dendrobium, Chinese yam, angelica, burdock root, rape pollen, gotu kola, lophatheri, astragalus, fig, pueraria, figwort, sea buckthorn, coix seed, corn silk, phytolacca, medlar, licorice, mulberry leaf, and can be a certain part of the medicinal material or the whole plant.

[0040] A preferred method for extracting polysaccharides from plant medicinal materials in the present application is achieved by the following steps:

[0041] (1) Select high-quality medicinal materials with impurities removed, and clean them with flowing drinking water.

[0042] (2) Put the cleaned raw materials into a fermentation tank, add 0.01%-5% biological enzymes (also known as microbial enzymes), control the enzymolysis temperature to 30-60℃, add edible acid or edible alkali to adjust the pH value to 4.0-8.0, and enzymolysis for 2-8 hours. After the enzymolysis is completed, steam sterilization is performed for 30-60 minutes;

[0043] (3) After the enzymolysis solution is cooled to room temperature, add 0.01%-20% of microbial strains based on the weight of the raw materials, control the stirring speed of the fermentation tank to 50-300 rpm, and control the fermentation temperature to 15-50℃. Perform closed fermentation for 5-30 days.

[0044] (4) Collect the fermentation broth, and directly fill it or continue to process it to prepare different preparations.

[0045] Existing methods can be used to extract effective components such as polysaccharides from the fermentation broth.

[0046] Preferably, the extraction process includes water extraction and alcohol extraction.

[0047] The water extraction is performed by extracting the fermentation broth with water at a temperature of 80℃ or above.

[0048] The alcohol extraction is performed by adding alcohol to the water extract, controlling the alcohol content in the solution system to be 60-90%, and performing alcohol extraction.

[0049] After water extraction, free proteins in the water extraction product are removed, and then alcohol extraction is performed.

[0050] The present application also includes the fermentation broth obtained by the method.

[0051] Advantages

[0052] The application provides a brand new (Parengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain; and it is found innovatively that the brand new strain is helpful for improving the extraction rate of total polysaccharide and glycoprotein of natural medicinal materials. In addition, it is found by the application that the (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain have a synergistic effect when used in combination, which is helpful for further improving the extraction rate of polysaccharide of natural medicinal materials, and not only this, but also can synergistically improve the polysaccharide extraction of various mixed plant medicinal materials.

[0053] The (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain for fermentation are from natural plant medicinal materials, and they can be used alone to improve the polysaccharide content of plants, or can form a plant endophyte group to jointly improve the polysaccharide content of plants.

[0054] The (Parengyodontium SP.) MD313901 strain and the (Purpureocillium SP.) MD313902 strain can significantly improve the polysaccharide content of plant medicines, and at the same time can create a product probiotic environment and improve the pharmacological activity of plant medicines. DETAILED DESCRIPTION

[0055] The following examples are specific descriptions of the application, which are intended to further illustrate the application, and cannot be understood as limiting the protection scope of the application, and the improvements and adjustments made by the person skilled in the art based on the application also belong to the protected scope.

[0056] Separation of (Parengyodontium SP.) MD313901 strain and (Purpureocillium SP.) MD313902 strain

[0057] The separation method of the (Parengyodontium SP.) MD313901 strain and / or the (Purpureocillium SP.) MD313902 strain comprises plant tissue pretreatment, leaching, separation and purification, and the operation process is as follows:

[0058] (1) Plant tissue pretreatment

[0059] After the plant tissue is cut into segments, soaked in an ethanol solution, soaked in a sodium hypochlorite solution and then washed with sterile water, pretreated plant tissue is obtained;

[0060] (2) Plant tissue endophyte leaching

[0061] The pretreated plant tissue and the biological enzyme are ground, and the supernatant in the grinding liquid is separated;

[0062] (3) Isolation and purification of plant endophyte strains

[0063] The supernatant is coated on a culture medium, and after cultivation, the strains or colonies are selected.

[0064] The plant tissue is at least one of Polygonatum odoratum, Astragalus membranaceus, Hippophae rhamnoides, Portulaca oleracea, Polygonatum sibiricum, Platycodon grandiflorum, Euchresta japonica, Isatis indigotica, Phytolacca acinosa, Houttuynia cordata, Corydalis saxicola, Pinellia ternata, Sonchus arvensis, Platycodon grandiflorum, Glycyrrhiza uralensis, Radix Paeoniae Alba, Dendrobium, Angelica sinensis, Acorus gramineus.

[0065] In step (1), the plant tissue is washed with water and then washed with sterile water. After the surface moisture is dried, the plant tissue is cut into segments with a length of 0.5-2.0 cm. Then the cut material is sterilized in 70-80% ethanol for 1-2 min, washed with sterile water, soaked in 3-8% sodium hypochlorite for 5-10 min, and then washed with sterile water. After the surface moisture is dried, the pretreated plant tissue is obtained.

[0066] In step (2), the pretreated plant tissue is placed in a sterilized mortar or grinding machine, and biological enzymes are added for grinding. The grinding liquid is transferred to a conical flask, and oscillated at 100-200 rpm at 25-40°C for 1-2 hours. The supernatant and plant residues are separated.

[0067] As a preferred embodiment, the biological enzyme is one or more of pectinase, cellulase, and neutral protease.

[0068] As a preferred embodiment, the amount of biological enzyme is 0.01%-5% of the weight of the medicinal material (plant tissue).

[0069] In step (3), the supernatant obtained in the leaching process is diluted 10-20 times with sterile water under sterile conditions. 100-200 μL of the diluted solution is coated on an MS culture medium for cultivation. After a period of cultivation, colonies with different morphologies are selected. Fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium.

[0070] In step (3), the isolated fungi are purified by streaking method.

[0071] Preparation Example 1

[0072] The preparation method of the plant endophyte colony is realized through the following steps:

[0073] (1) Pretreatment of plant tissue

[0074] The plant tissues (different origin and different varieties of polygonatum multiflorum medicinal materials shown in Table 1) are washed with tap water and then washed with sterile water, and after the surface moisture is dried, the plant tissues are cut into segments with a length of 0.5-2.0 cm, and then the cut materials are placed in a mortar for sterilization for 2 min with 70% ethanol, washed with sterile water, soaked in 3% sodium hypochlorite for 10 min, and then washed with sterile water, and after the surface moisture is dried, the plant tissues are ready for use.

[0075] (2) Endophytic bacteria leaching of plant tissues

[0076] The sterilized polygonatum multiflorum tissues are placed in a sterilized mortar, and biological enzymes (cellulase, 5% by weight) are added for grinding, and the grinding liquid is transferred into a conical flask for oscillation at 25 DEG C and 200 rpm for 2 hours to obtain supernatant and plant residues.

[0077] (3) Isolation and purification of plant endophytic strains

[0078] The supernatant obtained in the leaching process is diluted 10 times with sterile water under sterile conditions, and 200 μL of the diluted solution is spread on MS medium for culture, and after a period of culture, different morphological colonies are selected, fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium. The purification is performed by streaking method.

[0079] Different origin and different varieties of polygonatum multiflorum medicinal materials are selected, and the plant endophytic bacteria are isolated as shown in Table 1.

[0080] Table 1

[0081]

[0082] The results in Table 1 show that two strains can be obtained from different origin and different varieties of polygonatum multiflorum by using the separation method of the present application.

[0083] The 16sRNA fragment of the MD313901 strain of the present application is analyzed, and has a fungal specific ITS2 fragment, and has 99% homology with Parengyodontium SP., and is named MD313901, and the MD313901 strain of (Parengyodontium SP.) has been preserved in China Center for Type Culture Collection on May 8, 2018, and the preservation number is CCTCC M2018256.

[0084] The 16sRNA fragment of the MD313902 strain of the application is analyzed, has a fungal specific ITS2 fragment, and has 99% homology with Purpureocillium SP., and is named MD313902, (Purpureocillium SP.) MD313902 strain has been preserved in China Center for Type Culture Collection on May 8, 2018, and the preservation number is CCTCC M2018257.

[0085] Preparation Example 2

[0086] The preparation method of the plant endophyte population is realized through the following steps:

[0087] (1) Plant tissue pretreatment

[0088] The plant tissue (different producing areas and different varieties of Platycodon grandiflorum plant medicinal materials shown in Table 2) is washed with tap water and then washed with sterile water. After the surface moisture is dried, it is cut into segments with a length of 0.5-2.0 cm. Then the cut material is placed in a mortar for 1 min of disinfection with 80% ethanol by volume. It is washed with sterile water, then soaked with 8% sodium hypochlorite by mass for 5 min, and then washed with sterile water. After the surface moisture is dried, it is ready for use.

[0089] (2) Plant tissue endophyte leaching

[0090] The disinfected plant tissue is placed in a disinfected mortar and ground with biological enzymes (a mixture of pectinase and cellulase at a ratio of 1:1, with a dosage of 2%). The grinding liquid is transferred to a conical flask and shaken at 40°C and 100 rpm for 1 hour to obtain supernatant and plant residue.

[0091] (3) Isolation and purification of plant endophyte strains

[0092] The supernatant obtained in the leaching process is diluted 20 times with sterile water under sterile conditions. 100 μL of each is spread on MS medium for culture. After a period of culture, different morphological colonies are selected, fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium. Purification is performed by streaking method.

[0093] Different producing areas and different varieties of Platycodon grandiflorum plant medicinal materials are separated by the method of the present case, purified by streak culture method, and their plant endophytes are separated. The results are shown in Table 2.

[0094] Table 2

[0095] Strain Platycodon grandiflorum Ait. from Anhui Platycodon grandiflorum Ait. from Yunnan Platycodon grandiflorum Ait. from Sichuan Platycodon grandiflorum Ait. from Heilongjiang MD313901 √ √ √ √

[0096] The results in Table 2 show that different producing areas and different varieties of Platycodon grandiflorum can also obtain a strain by the separation method of the present patent.

[0097] Preparation Example 3

[0098] The preparation method of the plant endophyte population is realized through the following steps:

[0099] (1) Pretreatment of plant tissue

[0100] The plant tissue (different varieties of plant medicinal materials of Phellodendron amurense Rupr. shown in Table 2) is washed with tap water and then washed with sterile water. After the surface moisture is dried, the plant tissue is cut into segments with a length of 0.5-2.0 cm. Then the cut plant tissue is sterilized with 75% ethanol for 1.5 min, washed with sterile water, soaked with 5% sodium hypochlorite for 8 min, and then washed with sterile water. After the surface moisture is dried, the plant tissue is ready for use.

[0101] (2) Extraction of plant endophyte

[0102] The sterilized plant tissue is placed in a sterilized mortar, and biological enzymes (a mixture of pectinase, cellulase and neutral protease in a ratio of 1:1:1, with a dosage of 0.1%) are added for grinding. The grinding liquid is transferred into a conical flask and shaken at 35°C and 200 rpm for 1.5 hours to obtain supernatant and plant residue.

[0103] (3) Isolation and purification of plant endophyte strains

[0104] The supernatant obtained in the extraction process is diluted 20 times with sterile water, and 200 μL of the diluted solution is spread on MS medium for culture under sterile conditions. After a period of culture, different morphological colonies are selected, and fungi are cultured on PDA (potato dextrose agar) medium, and bacteria are cultured on beef extract peptone medium. The colonies are purified by streaking method.

[0105] Different varieties of plant medicinal materials of Phellodendron amurense Rupr. are taken, and the plant endophytes are separated by streak culture method. The results are shown in Table 2.

[0106] Table 3

[0107]

[0108] The results in Table 3 show that two strains can be obtained from different varieties of Phellodendron amurense Rupr. by the separation method of the present application.

[0109] In the following examples, the total raw material mass refers to the weight of the medicinal material, unless otherwise specified.

[0110] I. Example of improving the polysaccharide content of single medicinal material by microbial strain fermentation:

[0111] Example 1:

[0112] 1. Enzymatic disruption of dandelion medicinal material

[0113] Take 10 kg of dandelion medicinal materials, remove impurities and rinse with tap water, pour into a fermentation tank, add purified water to cover the medicinal materials, add 0.1% pectinase (total raw material mass fraction) and 0.1% cellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.8, control the temperature at 48°C, and enzymolysis for 2 hours, then sterilize by steam for 40 min.

[0114] 2. Fermentation

[0115] After cooling to room temperature, add 0.3% (total raw material mass fraction) Parengyodontium SP. MD313901 strain, and stand for fermentation at 30°C for 6 days, filter, and collect the medicinal liquid for treatment.

[0116] 3. Extraction and content determination of dandelion polysaccharide

[0117] The collected medicinal liquid after fermentation is added with 8 times the amount of purified water of the medicinal materials, heated at 100°C for reflux for 2 times, each for 2 hours, filtered, concentrated to an appropriate concentration, free protein is removed by Sevag method, then anhydrous ethanol is added to make the solution concentration reach 80%, and the precipitate is dried to obtain dandelion total polysaccharide.

[0118] The content of dandelion polysaccharide is determined by phenol-sulfuric acid method, and the determination result shows that the content of dandelion polysaccharide after fermentation reaches 60.4% of the dry weight of dandelion.

[0119] Comparative Example 1

[0120] Compared with Example 1, the only difference is that the strain fermentation described in step 2 is not performed, and the specific operation is as follows:

[0121] Take 10 kg of dandelion medicinal materials, remove impurities and rinse with tap water, pour into a fermentation tank, add purified water to cover the medicinal materials, add 0.1% pectinase (total raw material mass fraction) and 0.1% cellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 6.8, control the temperature at 48°C, and enzymolysis for 2 hours, then sterilize by steam for 40 min. Collect the medicinal liquid for treatment.

[0122] The collected medicinal liquid after enzymolysis is added with 8 times the amount of purified water of the medicinal materials, heated at 100°C for reflux for 2 times, each for 2 hours, filtered, concentrated to an appropriate concentration, free protein is removed by Sevag method, then anhydrous ethanol is added to make the solution concentration reach 80%, and the precipitate is dried to obtain dandelion total polysaccharide.

[0123] The content of dandelion polysaccharide is determined by phenol-sulfuric acid method, and the determination result shows that the content of dandelion polysaccharide after fermentation reaches 60.4% of the dry weight of dandelion.

[0124] Compared with Example 1, the medicinal material is only subjected to wall breaking treatment and is not subjected to microbial fermentation. It is found through comparison between Example 1 and Comparative Example 1 that the dandelion polysaccharide of Example 1 is 2.7 times higher.

[0125] Example 2:

[0126] 1. Wall breaking of the medicinal material of burdock root:

[0127] 10 kg of burdock root medicinal material is taken, impurities are removed, and the burdock root medicinal material is washed clean with tap water and poured into a fermentation tank. Purified water is added to cover the medicinal material, 1% pectinase (total raw material mass fraction) and 2% cellulase (total raw material mass fraction) are added, and the mixture is fully stirred and mixed. The pH value is adjusted to 6.8, the temperature is controlled at 50°C, and enzymolysis is performed for 1.5 hours. Then, steam sterilization is performed for 30 minutes.

[0128] 2. Fermentation:

[0129] After cooling to room temperature, 0.3% (total raw material mass fraction) of Purpureocillium SP. MD002 strain is added, and the mixture is left to stand for fermentation at 30°C for 5 days. Filtration is performed, and the medicinal liquid is collected for treatment.

[0130] 3. Extraction and content determination of burdock root polysaccharide

[0131] The medicinal liquid collected after fermentation is added with 10 times the amount of purified water of the medicinal material, heated at 90°C for reflux for 2 times, each for 2 hours, filtered, and concentrated to an appropriate concentration. Free proteins are removed by the Sevag method, and then anhydrous ethanol is added to make the concentration of the solution reach 80%. Centrifugal precipitation is performed twice, and the precipitate is dried to obtain burdock root polysaccharide.

[0132] The content of burdock root polysaccharide is determined by the phenol-sulfuric acid method. The determination result shows that the content of burdock root polysaccharide after fermentation reaches 28.3% of the dry weight of burdock root. Compared with the burdock root polysaccharide of the case where step 2 fermentation (direct extraction after wall breaking treatment) is not performed, the content is 2.1 times higher.

[0133] II. Example of microbial strain fermentation for improving the content of polysaccharide in the medicinal material of the composition:

[0134] Example 3:

[0135] 1. Wall breaking of the composition;

[0136] 5 kg of dendrobium and 5 kg of spadeleaf are taken as the composition, impurities are removed, and the composition is washed clean with tap water and poured into a fermentation tank. Purified water is added to cover the medicinal material, 3% pectinase (total raw material mass fraction) and 2% cellulase (total raw material mass fraction) are added, and the mixture is fully stirred and mixed. The pH value is adjusted to 7.0, the temperature is controlled at 45°C, and enzymolysis is performed for 3 hours. Then, steam sterilization is performed for 30 minutes.

[0137] 2. Fermentation

[0138] After cooling to room temperature, 1% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain and 1% (total raw material mass fraction) of Purpureocillium SP. MD313902 strain are added, and fermentation is carried out at 30°C for 10 days, filtration is carried out, and the filtrate is collected for treatment.

[0139] 3. Extraction and content determination of polysaccharides in the composition

[0140] The filtrate collected after fermentation is added with 10 times the amount of purified water of the medicinal material, and low-temperature vacuum heating reflux is carried out at 65°C for 2 hours each time, filtration is carried out, the extract is concentrated to an appropriate concentration, free proteins are removed by the Sevag method, anhydrous ethanol is then added to make the concentration of the solution reach 80%, and the precipitate is obtained by centrifugal precipitation twice, and the precipitate is dried to obtain polysaccharides in the composition.

[0141] The content determination of polysaccharides in the composition is carried out by the phenol-sulfuric acid method, and the determination result shows that the content of polysaccharides in the composition after fermentation reaches 30.2% of the dry weight of the composition, and compared with the case without the fermentation step 2 (after the cell wall breaking treatment in step 1, the extraction in step 3 is directly carried out), it is found that the polysaccharide content is 2 times higher.

[0142] Example 4:

[0143] 1. Cell wall breaking of the composition:

[0144] 5 kg of Huangqi and 5 kg of Dangshen medicinal materials are taken, impurities are removed, and the medicinal materials are washed clean with tap water, and then poured into a fermentation tank, purified water is added to cover the medicinal materials, 4% of pectinase (total raw material mass fraction) and 1% of cellulase (total raw material mass fraction) are added, and the mixture is fully stirred and mixed, the pH value is adjusted to 7.0, the temperature is controlled at 45°C, and enzymatic hydrolysis is carried out for 3 hours, and then steam sterilization is carried out for 30 min.

[0145] 2. Fermentation

[0146] After cooling to room temperature, 2% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain and 2% (total raw material mass fraction) of Purpureocillium SP. MD313902 strain are added, and fermentation is carried out at 30°C for 10 days, filtration is carried out, and the filtrate is collected for treatment.

[0147] 3. Extraction and content determination of polysaccharides in the composition

[0148] The collected composition liquid after fermentation is added with 10 times the amount of purified water of the medicinal material, vacuum heating reflux at 70°C for 2 times, 2 hours each time, filtered, obtained the extract, concentrated to the appropriate concentration, removed free protein by Sevag method, then added anhydrous ethanol to make the solution concentration reach 80%, centrifugal precipitation twice, dried the precipitate to obtain the composition polysaccharide.

[0149] The composition polysaccharide content determination adopts the phenol-sulfuric acid method, and the determination result shows that the composition polysaccharide content after fermentation reaches 51.4% of the dry weight of the composition medicinal material, and compared with the case without the fermentation step (2) of the present case (after the cell wall breaking treatment of step (1), directly extract in step (3)), it is found that the polysaccharide content is 1.4 times higher.

[0150] Example 5

[0151] 1. Breaking the cell wall of the composition:

[0152] Take 5 kg of Rhizoma Polygonati, 5 kg of Dioscorea, 5 kg of Phytolacca acinosa and 5 kg of Pueraria as the composition, remove impurities and wash clean with tap water, pour into the fermentation tank, add purified water to cover the medicinal material, add 1% pectinase (total raw material mass fraction) and 4% cellulase (total raw material mass fraction), mix thoroughly, adjust the pH value to 7.0, control the temperature at 50°C, and enzymolysis for 4 hours, then sterilize by steam for 60 min.

[0153] 2. Fermentation

[0154] After cooling to room temperature, add 3% (total raw material mass fraction) of Parengyodontium SP. MD313901 strain and 3% (total raw material mass fraction) of Purpureocillium SP. MD313902 strain, and place at 25°C for 15 days, filter, and collect the liquid for processing.

[0155] 3. Extraction and content determination of composition polysaccharide

[0156] The collected composition liquid after fermentation is added with 10 times the amount of purified water of the medicinal material, vacuum heating reflux at 70°C for 2 times, 2 hours each time, filtered, obtained the extract, concentrated to the appropriate concentration, removed free protein by Sevag method, then added anhydrous ethanol to make the solution concentration reach 80%, centrifugal precipitation twice, dried the precipitate to obtain the composition polysaccharide.

[0157] The composition polysaccharide content determination adopts the phenol-sulfuric acid method, and the determination result shows that the composition polysaccharide content after fermentation reaches 51.4% of the dry weight of the composition medicinal material, and compared with the case without the fermentation step (2) of the present case (after the cell wall breaking treatment of step (1), directly extract in step (3)), it is found that the polysaccharide content is 1.4 times higher.

[0158] Example 6

[0159] Compared with Example 1, the only difference is that the strains used are MD313901 strain of (Parengyodontium SP.) and MD313902 strain of (Purpureocillium SP.) (the strain ratio of the two is 1:1; the total inoculation amount of the strains is 0.3% of the weight of the dandelion medicinal material), and other parameters are the same as those in Example 1.

[0160] The dandelion polysaccharide content is determined by the phenol-sulfuric acid method, and the determination result shows that the polysaccharide content of the fermented dandelion reaches 66.3% of the dry weight of the dandelion.

[0161] It is found by comparing Example 1 and Example 6 that the combination of the two strains can achieve a synergistic effect.

[0162] Example 7

[0163] Compared with Example 1, the only difference is that the step 1 of the wall breaking treatment is not performed, the dandelion medicinal material is directly cleaned by removing impurities and washing with tap water, and then poured into a fermentation tank, inoculated with the strains after sterilization, and then subjected to the step 2 of fermentation, and then subjected to the step 3 of polysaccharide extraction. The polysaccharide content in the fermentation liquid is tested by the method of Example 1 to be 40.6%.

[0164] Example 8

[0165] Compared with Example 1, the only difference is that the step 1 of the wall breaking treatment is not performed, the dandelion medicinal material is directly cleaned by removing impurities and washing with tap water, and then poured into a fermentation tank, inoculated with the strains after sterilization, and then subjected to the step 2 of fermentation, and then subjected to the step 3 of polysaccharide extraction. The polysaccharide content in the fermentation liquid is tested by the method of Example 1 to be 40.6%.

[0166] It is found by comparing Example 1, Example 7 and Example 8 that the polysaccharide content of the fermented material after the wall breaking treatment is obviously improved. In addition, it is found by the present application that the wall breaking technology used is biological enzyme wall breaking, and the wall breaking method of ultrafine grinding is selected for comparison in the experimental implementation process. Compared with the wall breaking technology of ultrafine grinding, the biological enzyme wall breaking is more gentle and the wall breaking effect is more sufficient, and has no effect on the active ingredients.

Claims

1. A method for extracting polysaccharides from plant medicinal materials, characterized in that, Microbial fermentation treatment of medicinal plants, wherein the microorganisms include Parengyodontium SP . MD313901 strain, Purpureocillium SP. At least one of the MD313902 strains; Parengyodontium SP The accession number for strain MD313901 is CCTCC M 2018256; Purpureocillium SP. The accession number for strain MD313902 is CCTCC M 2018257; The plant materials are pre-treated by cell wall breaking before being subjected to microbial fermentation; The cell wall disruption pretreatment is enzymatic hydrolysis. The bioenzyme is one or more of pectinase, cellulase, hemicellulase, neutral protease, and amylase. The medicinal plant mentioned is a medicinal part or the whole plant of one of the following: pokeweed, burdock root, dendrobium, purslane, astragalus, codonopsis, polygonatum, yam, and kudzu root.

2. The method for extracting polysaccharides from medicinal plants as described in claim 1, characterized in that, The microorganisms mentioned are those containing Purpureocillium SP. MD313902 strain and Parengyodontium SP The microbial community of strain MD313901.

3. The method for extracting polysaccharides from medicinal plants as described in claim 1, characterized in that, During enzymatic hydrolysis, the amount of biological enzyme used is 0.01%-5% of the weight of the medicinal plant material; The pH range for the enzymatic hydrolysis process is 4-8.

4. The method for extracting polysaccharides from medicinal plants according to any one of claims 1 to 3, characterized in that, During fermentation, the inoculation amount of microorganisms is 0.01 to 20% of the total raw material mass.

5. The method for extracting polysaccharides from medicinal plants as described in claim 4, characterized in that, The fermentation temperature is 10–50℃, and the fermentation time is 48 hours or more.

6. A fermentation product obtained by the method for extracting polysaccharides from medicinal plants as described in any one of claims 1 to 5.

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