WO2019205662A1 - 一种蛹虫草培养基多糖及其分离纯化方法和用途 - Google Patents

一种蛹虫草培养基多糖及其分离纯化方法和用途 Download PDF

Info

Publication number
WO2019205662A1
WO2019205662A1 PCT/CN2018/120506 CN2018120506W WO2019205662A1 WO 2019205662 A1 WO2019205662 A1 WO 2019205662A1 CN 2018120506 W CN2018120506 W CN 2018120506W WO 2019205662 A1 WO2019205662 A1 WO 2019205662A1
Authority
WO
WIPO (PCT)
Prior art keywords
polysaccharide
cordyceps militaris
culture medium
militaris culture
medium
Prior art date
Application number
PCT/CN2018/120506
Other languages
English (en)
French (fr)
Inventor
黄儒强
张竞雯
王静辉
王倩
高林林
Original Assignee
华南师范大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 华南师范大学 filed Critical 华南师范大学
Priority to US16/770,983 priority Critical patent/US11111317B2/en
Publication of WO2019205662A1 publication Critical patent/WO2019205662A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Definitions

  • the invention relates to a polysaccharide of Cordyceps militaris culture medium and a separation and purification method thereof and use thereof.
  • Polysaccharide also known as polysaccharide, is a linear or branched chain polymer linked by aldose or keto sugar through a glycosidic bond. It is a polar complex macromolecule with a degree of polymerization greater than 10, and the molecular weight is generally tens of thousands. The above is one of the four basic substances that constitute life activities. In addition to being in a free state, polysaccharides in living organisms also combine with proteins or fats to form proteoglycans and lipopolysaccharides.
  • Cordyceps militaris (C. militaris) is also known as Cordyceps militaris. It is classified into Ascomycetes, nucleobacteria, and Phytophthora in the taxonomy. It is also a genus of Cordyceps militaris, which is mainly distributed in Northeast China, North China, Northwest China, etc. . Cordyceps militaris can be parasitic on larvae or carcasses of insects such as Lepidoptera, Coleoptera, and Diptera, and can be artificially cultivated by using silkworm cocoons and rice culture media.
  • Cordyceps militaris On a solid medium such as rice, under a certain temperature, humidity and light conditions, cultured 35-45 days to obtain the Cordyceps militaris fruit body; (2) inoculate the Cordyceps militaris strain in the silkworm larvae or live mites, at a certain temperature, Under the condition of humidity and light, the fruit body of Cordyceps militaris was obtained by culturing for 35-45 days; (3) Cordyceps militaris was cultured by liquid fermentation with soybean sucrose or corn sucrose as the medium.
  • Chinese Patent Application (Application No. 201110086808.2) discloses a method for extracting polysaccharide from Cordyceps militaris culture medium, using different enzyme liquids to remove protein and starch in Cordyceps militaris culture medium, and then obtaining alcoholic scutellariae medium by alcohol precipitation Polysaccharide; its operation process is complicated, the cost is high, and the polysaccharide cannot be further separated and purified, and the polysaccharide component with higher purity cannot be obtained.
  • a primary object of the present invention is to provide a polysaccharide of Cordyceps militaris.
  • Another object of the present invention is to provide a method for separating and purifying polysaccharides from the Cordyceps militaris culture medium, and extracting polysaccharides from the Cordyceps militaris waste culture medium by ultrasonic assisted water extraction and alcohol precipitation method, and separating and purifying the polysaccharide by ion exchange chromatography.
  • the polysaccharide component having higher purity and biological activity is thus isolated and isolated.
  • a further object of the present invention is to provide the use of the polysaccharide of the Cordyceps militaris culture medium described above.
  • a Cordyceps militaris medium polysaccharide comprising the following mole percent monosaccharide: 0.11% ribose, 0.11% rhamnose, 0.45% arabinose, 0.13% xylose, 14.50% mannose, 83.96% glucose, 0.73% galactose;
  • the composition of the monosaccharide is calculated by the GC-MS area normalization method. Except the solvent peak, all the peak areas are regarded as 100%, and the main peak area is calculated as a percentage of the total area.
  • the unknown sample may be measured. Containing a trace amount of a monosaccharide other than the standard, or a deviation caused by the calculation method, resulting in a molar percentage composition of the monosaccharide of less than 100%, which is normal;
  • the average molecular weight of the polysaccharide of the Cordyceps militaris medium is 2.18 k Da;
  • the polysaccharide of the Cordyceps militaris medium has a very small amount of sulfate groups, and also contains a pyranose ring and an ⁇ -type glycosidic bond.
  • the method for separating and purifying the polysaccharide of the Cordyceps militaris medium comprises the following steps:
  • Extracting polysaccharides from Cordyceps militaris culture medium The sorghum grass rice medium is dried and pulverized, and sieved to obtain dry powder of the waste material; the dry powder is added to 15-16 times of distilled water, and ultrasonic treatment is performed for 30 minutes or more at 70 ° C. The extract was refluxed for 1.5-2.0 h, and the combined extracts were extracted several times. The extract was filtered and concentrated to obtain a polysaccharide concentrate. 3-4 volumes of 95% (V/V) ethanol were added to the polysaccharide concentrate, and stirred at 4 Allow to stand overnight at °C; after centrifugation, the precipitate is dried to obtain polysaccharide extract of Cordyceps militaris culture medium;
  • step (1) The sieving described in step (1) is preferably passed through a 40 mesh sieve
  • step (1) The concentration described in step (1) is preferably concentrated at 50-55 ° C;
  • step (1) The centrifugation described in step (1) is preferably centrifuged at 5000 r/min for 15 min;
  • the concentration of the H 2 O 2 solution described in the step (2) is preferably 30% (V/V);
  • 3-1 mixing the papain solution with the polysaccharide extract of the Cordyceps militaris medium, the volume ratio of the two is 1.0: 1.5 to 1.0: 1.7, and the enzyme is 2-3 hours at 60-70 ° C;
  • the papain solution is prepared with a pH 6.0 PBS buffer, wherein the concentration of papain is preferably 250 U / ml;
  • the Sevage reagent according to the step (3) is prepared by chloroform and n-butanol in a volume ratio of 5:1;
  • the shaking culture described in the step (3) is preferably 150r/min;
  • step (3) The centrifugation described in step (3) is preferably centrifuged at 4000 r / min for 20-30 min;
  • the separation and purification of polysaccharides is not only a process of removing impurities, but also a process of separating the mixed polysaccharide into a single component.
  • Column chromatography can be divided into ion exchange chromatography and gel column chromatography. Ion exchange chromatography is fractionated according to the difference of ion charge density.
  • the charge group of anion exchanger is positively charged and the counter ion is negatively charged. Therefore, this exchanger can exchange reaction with negatively charged compounds or anions in solution.
  • the cation exchanger is the opposite.
  • the polysaccharide of the Cordyceps militaris culture medium of the present invention can be used as an antioxidant or a bacteriostatic agent, and can also be used for preparing a drug having a uric acid-lowering effect.
  • the invention obtains crude polysaccharide of Cordyceps militaris culture medium by water extraction and alcohol precipitation method, and separates and purifies crude polysaccharide by using ion exchange chromatography column, and has the following advantages and effects compared with the prior art:
  • the invention fully utilizes the culture material waste material produced by cultivating Cordyceps militaris, and conforms to the green environmental protection concept of turning waste into treasure, and establishes a complete and feasible polysaccharide extraction, separation and purification, physical and chemical properties, structure and structure of Cordyceps militaris culture medium.
  • the technical route of biological activity research has improved the residue treatment process of artificially cultivated Cordyceps sinensis industry, and provided technical guidance for the extraction, separation and purification of edible fungi polysaccharides.
  • the water extraction and alcohol precipitation method used in the invention can complete the large-flux polysaccharide extraction operation, has the advantages of low cost, good repeatability and high yield, and is suitable for industrial large-scale production.
  • the DEAE Sephrose Fast Flow used in the present invention has better physical and chemical stability and mechanical properties, large exchange capacity, can be cleaned in place, and the bed volume changes little with the ionic strength of the pH, and is suitable for carrying out due to high flow rate and load. Purification of a large number of crude products.
  • the extraction method of the invention does not affect the biological activity of the polysaccharide P1 of the Cordyceps militaris culture medium, and the obtained pure P1 polysaccharide has high purity and stable properties, and has remarkable effects in anti-oxidation, uric acid-lowering and bacteriostatic action, and is beneficial to Human metabolism; low cost, pure P1 polysaccharide can be further used in the development of health care products, pharmaceuticals, cosmetics.
  • the invention creatively combines the water extraction and alcohol extraction method of polysaccharide with the ion exchange chromatography separation and purification method for the research of the polysaccharide of the cordyceps sinensis medium, compares and obtains the optimal process parameters, and determines the DEAE.
  • Sephrose Fast Flow provides technical guidance and new ideas for the extraction, separation and purification of polysaccharides from Cordyceps militaris.
  • Figure 1 is a graph showing the elution of polysaccharides from Cordyceps militaris culture medium.
  • Figure 2 is a UV spectrum of polysaccharide P1.
  • Figure 3 is an infrared spectrum of polysaccharide P1.
  • Figure 4 is the ABTS radical scavenging ability of polysaccharide P1.
  • Figure 5 is the OH radical scavenging ability of polysaccharide P1.
  • Fig. 6 is a graph showing the results of bacteriostatic test of polysaccharide P1; wherein, the inhibition zone of Sa-to S. aureus, the inhibition zone of Pa-Pseudomonas aeruginosa, the inhibition zone of 1-polysaccharide P1, 0-control Group of inhibition zones.
  • the physical and chemical properties of the polysaccharide of Cordyceps militaris culture medium are analyzed by the Guangzhou Analytical Testing Center, and the report numbers are respectively 2018001306-1b.
  • a method for extracting and separating and purifying polysaccharides from a stalk of Cordyceps militaris comprising the steps of:
  • Phenol-sulfuric acid method for the detection of polysaccharide content precise weighing 0.1g of anhydrous glucose standard dried to constant weight at 105 ° C, placed in a 100ml volumetric flask, dissolved in distilled water and fixed to volume, shake well, formulated into 1mg / ml The standard solution is ready for use. The solution was diluted to a standard solution of different concentrations of 10, 20, 40, 60, 80, 100 ⁇ g/ml.
  • the polysaccharide component obtained by elution was concentrated, dialyzed, and freeze-dried to obtain a pure polysaccharide of Cordyceps militaris culture medium, which was named P1.
  • the polysaccharide P1 of the Cordyceps militaris culture medium obtained in Example 1 was subjected to ultraviolet spectrum analysis, and 1 mg of the polysaccharide sample was weighed to prepare a 1 mg/mL polysaccharide solution, and the ultraviolet spectrum was scanned in the range of 200-800 nm.
  • Figure 2 shows the ultraviolet spectrum of P1. The results show that P1 has a weak absorption peak at 260 nm and 280 nm, indicating that P1 contains trace amounts of proteins and nucleic acids.
  • the molecular weight analysis of the polysaccharide P1 of the Cordyceps militaris culture medium obtained in Example 1 was carried out.
  • the specific experimental methods are as follows:
  • the molecular weight was determined by gel permeation chromatography (GPC). 2 mg of the freeze-dried polysaccharide sample was weighed, dissolved in 0.02 M phosphate buffer solution, prepared into a 2.0 mg/mL solution, and filtered through a 0.22 ⁇ m sterile filter membrane, and the filtrate was taken for use.
  • GPC gel permeation chromatography
  • the monosaccharide composition analysis of the polysaccharide P1 of the Cordyceps militaris culture medium obtained in Example 1 was carried out as follows:
  • the column was analyzed by gas chromatography, and the analytical column was an HP-5MS quartz capillary column (30 m ⁇ 0.25 mm ⁇ 0.25 ⁇ m).
  • the heating procedure is as follows: the inlet temperature is 250 ° C, the initial column temperature is 100 ° C, held for 0.5 min; then it is raised to 140 ° C at 20 ° C / min for 5 min; at 3 ° C / min to 160 ° C; The °C/min speed was raised to 250 ° C for 5 min.
  • the injection volume was 1 ⁇ L; the split ratio was 10:1; the mobile phase was helium; the flow rate was 1 mL/min.
  • the monosaccharide composition results of the polysaccharides of Cordyceps militaris were measured as follows:
  • the peak at 1154 cm -1 is CO on the ring.
  • the absorption peaks, the peaks of 1081 and 1023 cm -1 are generated by the angular vibration of the alcoholic hydroxyl group. These three peaks indicate the presence of a pyranose ring in P1
  • the peak at 850 cm -1 indicates the presence of an ⁇ -type glycosidic bond in P1.
  • the absorbance at 734nm was measured and recorded as A 1 ; 2mL ABTS free radical solution was mixed with 1mL distilled water to determine 734nm The absorbance at the point is denoted as A 0 ; the absorbance at 734 nm of 2 mL of distilled water and 1 mL of the polysaccharide solution is measured and designated as A 2 .
  • Figure 4 shows the ABTS free radical scavenging ability of polysaccharide P1 of Cordyceps militaris. It can be seen from Fig. 4 that the polysaccharide component P1 of Cordyceps militaris has a significant ability to scavenge ABTS free radicals in a concentration range of 1.0 to 5.0 mg/ml, and is positively correlated with the polysaccharide concentration. When the polysaccharide concentration was 5.0 mg/ml, the ABTS radical scavenging rate of P1 was 85.4%.
  • Figure 5 shows the OH radical scavenging ability of the polysaccharide P1 of Cordyceps militaris. It can be seen from Fig. 5 that the polysaccharide component P1 of Cordyceps militaris has a significant ability to scavenge OH radicals. In the concentration range of 1.0-5.0 mg/ml, the scavenging ability of P1 with OH radicals gradually increases with the increase of solution concentration. Enhanced, at a concentration of 5.0mg / ml, the clearance rate can reach 81.03%.
  • mice Sixty male mice were randomly divided into 6 groups, 10 rats in each group, followed by blank group, model group, polysaccharide P1 high (400 mg/kg), P1 medium (200 mg/kg), and P1 low (100 mg). /kg) dose group and positive drug (50 mg/kg) control group. Every morning, the model group, the positive control group, the high, middle and low dose groups of polysaccharide P1 were intragastrically administered with jaundice 600 mg/kg (ig) + intraperitoneal injection of potassium oxonate 100 mg/kg (ip.) for one hour before the model was fasted. The blank group was given the same dose of normal saline.
  • polysaccharide P1 In the afternoon, the high, middle and low dose groups of polysaccharide P1 were intragastrically administered with polysaccharide suspension.
  • the positive control group was intragastrically administered with allopurinol suspension 50 mg/kg.
  • the model group and the blank group were given the same volume of physiology by gavage. Brine, seven days in a row
  • the low, medium and high dose groups of polysaccharide P1 could decrease the serum creatinine level of mice by 5.03%, 20.19% and 32.76%, respectively, which could decrease the serum uric acid level of mice by 39.58%, 46.46% and 53.72%, respectively.
  • the serum urea nitrogen levels in mice were decreased by 22.77%, 40.05%, and 51.59%, respectively. It is indicated that under this experimental model, Cordyceps militaris polysaccharide P1 can reduce serum creatinine, serum uric acid and serum urea nitrogen levels of hyperuricemia.
  • the common pathogenic bacteria Staphylococcus aureus (Sa) and Pseudomonas aeruginosa (Pa) were determined by perforation method for antibacterial activity.
  • the main operation process is as follows: take a petri dish with a diameter of 90mm, pour the plate and take 150 ⁇ l of the bacterial suspension with a pipette. After coating evenly, use 4mm per hole for each plate and add 40 ⁇ l of spare 1mg/ M.
  • Cordyceps sinensis medium polysaccharide P1 and the control group was added 40 ⁇ l of physiological saline per well.
  • the cells were cultured in a 37 ° C incubator for 24 hours, and the diameters of the inhibition zones on the experimental plates and the control plates were measured by a cross method, and the average value was calculated.
  • the antibacterial test results of polysaccharide P1 are shown in Fig. 6. They are the inhibition results of P1 against Staphylococcus aureus, the control group of Staphylococcus aureus, the inhibition of P1 against Pseudomonas aeruginosa, and the control group of Pseudomonas aeruginosa.
  • the diameter of the inhibition zone is shown in Table 3.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Materials Engineering (AREA)
  • Polymers & Plastics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Sustainable Development (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Toxicology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)

Abstract

一种蛹虫草培养基多糖及其分离纯化方法和用途，该多糖含有以下摩尔百分比的单糖：0.11％核糖、0.11％鼠李糖、0.45％阿拉伯糖、0.13％木糖、14.50％甘露糖、83.96％葡萄糖、0.73％半乳糖。该提取方法不影响蛹虫草培养基多糖P1的生物活性，所得到的多糖纯品P1纯度高、性质稳定，在抗氧化、降尿酸、抑菌方面具有显著效果，有益于人体代谢；成本较低，多糖P1纯品可进一步用于保健品、药品、化妆品的开发。

Description

一种蛹虫草培养基多糖及其分离纯化方法和用途 技术领域
本发明涉及一种蛹虫草培养基多糖及其分离纯化方法和用途。
背景技术
多糖(Polysaccharide)又称多聚糖，由醛糖或酮糖通过糖苷键相连接在一起的线性或分枝链状聚合物，是聚合度大于10的极性复杂大分子，分子量一般为数万以上，是构成生命活动的四大基本物质之一，生物体内的多糖除了以游离态存在外，也会与蛋白质或脂肪结合成蛋白多糖和脂多糖。
蛹虫草(C.militaris)又称北虫草，分类学上属于子囊菌亚门、核菌纲、球壳菌目，与北冬虫夏草同属麦角科虫草属，主要分布在我国东北、华北、西北等地区。蛹虫草可寄生于鳞翅目、鞘翅目、双翅目等昆虫的幼虫或蛹体上，能采用家蚕蛹、大米培养基等进行人工批量培育。
由于天然的蛹虫草资源有限，目前人工栽培的较多，主要有三种培养方法：(1)采集野生的蛹虫草，进行菌种的分离、纯化，然后将蛹虫草菌种接种在含蚕蛹粉的大米等固体培养基上，在一定的温度、湿度和光照条件下，培养35-45d获得蛹虫草子实体；(2)将蛹虫草菌种接种在家蚕幼虫或活蛹体内，在一定的温度、湿度和光照条件下，培养35-45d获得蛹虫草子实体；(3)以大豆粉蔗糖或玉米浆蔗糖为培养基，采用液体发酵培养蛹虫草。
我国采用人工方法培养蛹虫草子实体的生产已有相当规模，但在子实体收获的同时，也产生了大量的蛹虫草培养基下脚料，不仅造成环境的污染，也是不可忽视的资源浪费。
研究表明，蛹虫草培养基下脚料中含有虫草素、虫草多糖等生物活性物质。因此为了更好地开发利用蛹虫草资源，提高经济效益，完善蛹虫草产业链，对蛹虫草培养基下脚料的利用和研究开发已成为必要。
中国专利申请(申请号201110086808.2)公开了一种从蛹虫草培养基中提 取多糖的方法，利用不同的酶液来去除蛹虫草培养基中的蛋白、淀粉，再经醇沉得到蛹虫草培养基粗多糖；其操作过程复杂，成本较高，且不能将多糖进一步分离纯化，无法得到纯度较高的多糖组分。
发明内容
本发明的首要目的在于提供一种蛹虫草培养基多糖。
本发明的另一目的是提供上述蛹虫草培养基多糖的分离纯化方法，利用超声波辅助水提醇沉法对蛹虫草废弃培养基进行多糖提取，并通过离子交换层析法对多糖进行分离纯化，由此提取分离出纯度较高、具有生物活性的多糖组分。
本发明的再一目的在于提供上述蛹虫草培养基多糖的用途。
本发明的目的通过下述技术方案实现：
一种蛹虫草培养基多糖，包含以下摩尔百分比的单糖：0.11％核糖、0.11％鼠李糖、0.45％阿拉伯糖、0.13％木糖、14.50％甘露糖、83.96％葡萄糖、0.73％半乳糖；单糖的组成是由GC-MS面积归一化法计算出来的，是除溶剂峰外，把所有的出峰面积看作100％，计算主峰面积占总面积的百分数，可能所测的未知样品里包含了微量的标准品以外的单糖，或是由计算方法造成的偏差，导致该单糖的摩尔百分比组成不足100％，这属于正常情况；
所述蛹虫草培养基多糖的平均分子量为2.18k Da；
所述的蛹虫草培养基多糖中存在非常微量的硫酸基，还含有吡喃糖环和α-型糖苷键。
上述蛹虫草培养基多糖的分离纯化方法，包括以下步骤：
(1)提取蛹虫草培养基多糖：将蛹虫草大米培养基下脚料干燥后粉碎，过筛得下脚料干粉；称取干粉加入15-16倍质量的蒸馏水，超声波处理30min以上，在70℃下回流提取1.5-2.0h，提取若干次合并提取液，提取液过滤后浓缩，得到多糖浓缩液；在多糖浓缩液中加入3-4倍体积的95％(V/V)乙醇，搅拌后于4℃中静置过夜；离心后将沉淀烘干得到蛹虫草培养基多糖提取物；
步骤(1)所述的过筛优选过40目筛；
步骤(1)所述的浓缩优选在50-55℃下浓缩；
步骤(1)所述的离心优选5000r/min离心15min；
(2)蛹虫草培养基多糖的脱色：将蛹虫草培养基多糖提取物加蒸馏水溶解，调节pH值至8.0-8.5，滴加H 2O 2溶液至无色，50-55℃保温2h以上；
步骤(2)所述的H 2O 2溶液，浓度优选30％(V/V)；
(3)酶法结合Sevage法脱蛋白：
3-1：将木瓜蛋白酶溶液与蛹虫草培养基多糖提取液混合，两者的体积比为1.0:1.5～1.0:1.7，60-70℃酶解2-3h；
所述的木瓜蛋白酶溶液用pH值6.0的PBS缓冲液配制而成，其中木瓜蛋白酶的浓度优选250U/ml；
3-2：在酶解液中加入1/5体积的Sevage试剂，振荡培养30min以上，然后多次离心直至无蛋白沉淀析出为止，取上清液，烘干得到蛹虫草培养基粗多糖；
步骤(3)所述的Sevage试剂，是氯仿和正丁醇按体积比5:1配制而成；
步骤(3)所述的振荡培养，摇床转速优选150r/min；
步骤(3)所述的离心优选4000r/min离心20-30min；
(4)蛹虫草培养基多糖的分离纯化：将蛹虫草培养基粗多糖用蒸馏水溶解后上DEAE琼脂糖凝胶FF(DEAE Sephrose Fast Flow)离子交换层析柱，用蒸馏水进行洗脱，得到所述的蛹虫草培养基多糖P1。
多糖的分离纯化不仅是一个除杂的过程，同时也是将混合多糖分离为单一组分的过程。柱层析法可分为离子交换层析和凝胶柱层析。离子交换层析是依据离子电荷密度的不同而进行分级分离，阴离子交换剂的电荷基团带正电，反离子带负电，因此这种交换剂可以与溶液中的负电荷化合物或阴离子进行交换反应，而阳离子交换剂则反之。
本发明的蛹虫草培养基多糖可以作为抗氧化剂或抑菌剂应用，也可以用于制备具有降尿酸功效的药物。
本发明通过水提醇沉法得到蛹虫草培养基粗多糖，利用离子交换层析柱进行粗多糖的分离纯化，相对于现有技术具有如下的优点及效果：
(1)本发明充分利用了培养蛹虫草而产生的培养基下脚料，符合变废为宝的绿色环保理念，建立了一条完整可行的蛹虫草培养基多糖提取、分离纯化、理化性质、结构和生物活性研究的技术路线，完善了人工培养蛹虫草产业的残渣处理工艺，为食用菌多糖的提取分离纯化提供了技术指导。
(2)本发明所用的水提醇沉法可以完成大通量的多糖提取操作，成本较低，重复性好，得率高，适用于工业化大规模生产。
(3)本发明所用的DEAE Sephrose Fast Flow理化稳定性和机械性能更好，交换容量大，可以在位清洗，床体积随pH的离子强度变化很小，由于流速和载量高，适合于进行大量粗产品的纯化工作。
(4)采用本发明的提取方法不影响蛹虫草培养基多糖P1的生物活性，所得到的多糖纯品P1纯度高、性质稳定，在抗氧化、降尿酸、抑菌方面具有显著效果，有益于人体代谢；成本较低，多糖P1纯品可进一步用于保健品、药品、化妆品的开发。
(5)本发明创造性地将多糖的水提醇沉提取方法与离子交换层析分离纯化方法结合起来用于蛹虫草培养基下脚料多糖的研究，比较并得到较优的工艺参数，并确定DEAE Sephrose Fast Flow作为层析柱填料，为蛹虫草培养基下脚料多糖的提取分离纯化提供技术指导和新思路。
附图说明
图1是蛹虫草培养基多糖的洗脱曲线图。
图2是多糖P1的紫外光谱图。
图3是多糖P1的红外光谱图。
图4是多糖P1的ABTS自由基清除能力。
图5是多糖P1的OH自由基清除能力。
图6是多糖P1的抑菌实验结果图；其中，Sa-对金黄色葡萄球菌的抑菌圈，Pa-对绿脓杆菌的抑菌圈，1-多糖P1产生的抑菌圈，0-对照组的抑菌圈。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
本发明中，蛹虫草培养基多糖的理化性质分析由中国广州分析测试中心进行，报告编号分别为2018001306-1b。
实施例1
一种从蛹虫草培养基下脚料中提取并分离纯化多糖的方法，包括以下步骤：
(1)提取蛹虫草培养基多糖：称取55g蛹虫草大米培养基下脚料充分干燥后粉碎，过40目筛，称取50g干粉加入800ml的蒸馏水，超声30min，在70℃下回流提取1.5h，提取3次合并滤液，真空抽滤后于55℃下浓缩至100ml，得到多糖浓缩液；在多糖浓缩液中加入400ml的95％乙醇，不断搅拌使多糖均匀沉淀，于4℃中静置过夜；5000r/min离心15min后将沉淀烘干得到蛹虫草培养基多糖提取物；
(2)蛹虫草培养基多糖的脱色：将蛹虫草培养基多糖提取物加蒸馏水溶解配成浓度为0.05g/ml的多糖提取液，加入NaOH调节pH值至8.0，滴加30％的H 2O 2至无色，50℃下保温2h；
(3)酶法结合Sevage法脱蛋白：
精确称取木瓜蛋白酶0.1g，用pH值6.0的PBS缓冲液溶解成终浓度为250U/ml的溶液，与蛹虫草培养基多糖提取液混合，酶液与蛹虫草培养基多糖提取液的体积比为1.0:1.5，64℃下酶解3h；
在酶解液中加入1/5体积的Sevage试剂(氯仿:正丁醇＝5:1)，置于摇床150r/min振荡30min后4000r/min离心20min，重复离心多次直到无蛋白沉淀析出，合并上清液，50℃下烘干得到蛹虫草培养基粗多糖；
(4)蛹虫草培养基多糖的分离纯化：
称取0.1g蛹虫草培养基粗多糖溶于10ml蒸馏水中，上DEAE Sephrose Fast Flow离子交换层析柱，用蒸馏水进行洗脱，流速为0.5ml/min，每10min收集1管，以苯酚-硫酸法逐管检测多糖含量，依据洗脱曲线(图1)合并，得到蛹虫草培养基多糖P1。
苯酚-硫酸法检测多糖含量具体操作：精密称取105℃干燥至恒重的无水葡萄糖标准品0.1g，置于100ml容量瓶中加蒸馏水溶解并定容，摇匀，配成1mg/ml的标准品溶液备用。取该溶液分别稀释成10、20、40、60、80、100μg/ml不同浓度的标准溶液。分别吸取上述溶液各1ml置于试管中，加入6％苯酚溶液0.5ml并混匀，再加入2.5ml浓硫酸混匀，室温静置20min，以蒸馏水为空白对照，于490nm处测定吸光度，以葡萄糖浓度为横坐标，OD值为纵坐标，绘制标准曲线。未知样品用标准曲线法测定多糖含量。
将洗脱得到的多糖组分浓缩、透析后冷冻干燥，得到蛹虫草培养基多糖纯品，命名为P1。
实施例2
对实施例1得到的蛹虫草培养基多糖P1进行紫外光谱分析，各称取1mg多糖样品，配制1mg/mL多糖溶液，扫描在200-800nm范围内紫外图谱。
图2为P1的紫外光谱图，结果显示，P1在260nm、280nm处有微弱的吸收峰，表明P1含有微量的蛋白质和核酸。
实施例3
对实施例1得到的蛹虫草培养基多糖P1进行多糖分子量分析，具体实验方法如下：
采用凝胶渗透色谱法(GPC)测定分子量。称取冷冻干燥的多糖样品2mg，加0.02M磷酸缓冲液溶解，配制成2.0mg/mL溶液，用0.22μm无菌滤膜过滤，取过滤清液待用。
色谱条件：柱温35℃；0.02mol/L磷酸盐缓冲液(pH值7.0)为流动相，流速0.6ml/min，进样量20μL；TSK凝胶保护柱(40mm×6.0mm)，TSKG-4000K凝胶柱(300mm×7.8mm)和TSKG-2500K凝胶柱(300mm×7.8mm)；Waters 2414示差折光检测器检测。配制一系列不同分子量的葡聚糖溶液(700,400,200,100,50,30,10,5kD)作为标样，做标准曲线。样品的分子量根据其相应的洗脱体积对照标准曲线进行计算。
结果表明，蛹虫草培养基多糖P1的平均分子量为2.18k Da。
实施例4
对实施例1得到的蛹虫草培养基多糖P1进行单糖组成分析，具体方法如下：
称取多糖样品10mg，加入5mL三氟乙酸(4M)，110℃水解2h。水解液于50℃真空旋转蒸发至干，用色谱纯甲醇清洗3次(加入色谱纯甲醇，再进行旋干，反复3次，直至旋干物质再无三氟乙酸味道)，得多糖水解物。
在多糖水解物中依次加入10mg盐酸羟胺、1mg内标肌醇和2mL吡啶，密 封，90℃水浴30min后加入2m L醋酸酐90℃水浴30min，加入2m L水终止反应。加入2m L二氯甲烷萃取，重复2次，合并二氯甲烷相，加入无水硫酸钠干燥，过0.22μm有机微孔滤膜，备用。
采用气相色谱仪分析，分析柱为HP-5MS石英毛细管柱(30m×0.25mm×0.25μm)。升温程序如下：进样口温度为250℃，初始柱温100℃，保持0.5min；然后以20℃/min升至140℃，保持5min；以3℃/min速度升至160℃；再以10℃/min速度升到250℃，保持5min。进样体积为1μL；分流比为10:1；流动相为氦气；流速为1mL/min。
各种单糖标准品(鼠李糖、阿拉伯糖、核糖、木糖、甘露糖、葡萄糖和半乳糖)按照相同步骤进行实验，按照相同的检测程度，将处理后的标准品单糖进行气相色谱分析。
测得蛹虫草培养基多糖的单糖组成结果如下表：
表1蛹虫草培养基多糖P1的单糖组成
Figure PCTCN2018120506-appb-000001
实施例5
对实施例1得到的蛹虫草培养基多糖P1进行傅里叶红外光谱分析：
称取2mg多糖样品，将其与干燥后的KBr(溴化钾)于研钵中混匀研磨，经压片机压成片，采用傅里叶变换红外光谱仪，在400-4000cm -1的波数范围内扫描。
图3为P1的红外光谱图，在3402、3401cm -1处的峰分别是P1的O-H伸缩振动产生的，而2929cm -1处的峰是由C-H振动产生的，1622、1642cm -1处的峰分别是P1的C＝O伸缩振动产生的，这些都是多糖的特征峰，说明P1属于多糖类物质。
此外，在P1的红外光谱中，1240cm -1处的弱峰是由S＝O的伸缩振动所产生的，这说明P1中存在非常微量的硫酸基，1154cm -1处的峰是环上C-O的吸收峰，1081、1023cm -1的峰是由醇羟基的变角振动产生的，这三个峰说明P1中存在吡喃糖环，850cm -1处的峰表示P1中存在α-型糖苷键。
实施例6
对实施例1得到的蛹虫草培养基多糖P1进行抗氧化能力测定：
(1)ABTS自由基清除能力测定：
取7mmol/L ABTS水溶液和2.45mmol/L过硫酸钾水溶液各5mL混合，置于暗处反应12h，产生ABTS自由基，将ABTS自由基溶液稀释并使其在734nm波长条件下的吸光值为0.70±0.02。将1mL不同质量浓度的蛹虫草培养基多糖P1溶液与2mL ABTS自由基溶液混合均匀，10min后测其在734nm处的吸光值，记作A 1；2mL ABTS自由基溶液与1mL蒸馏水混合后测定734nm处的吸光值，记作A 0；测定2mL蒸馏水与1mL多糖溶液在734nm处的吸光值，记作A 2。
清除率(％)：Y＝[1-(A 1-A 2)/A 0]×100％
图4为蛹虫草培养基多糖P1的ABTS自由基清除能力。由图4可知，在浓度1.0～5.0mg/ml范围内，蛹虫草培养基多糖组分P1具有显著的清除ABTS自由基能力，且与多糖浓度呈正相关。当多糖浓度为5.0mg/ml时，P1的ABTS自由基清除率为85.4％。
(2)OH自由基清除能力测定：
分别在试管中加入不同浓度的蛹虫草培养基多糖P1溶液1mL，再加入1mL 6mmol/L FeSO 4溶液，1mL 6mmol/L H 2O 2溶液和1mL 6mmol/L水杨酸-乙醇溶液，37℃水浴1h，510nm处测定其吸光度A i。用蒸馏水代替样品溶液测得A 0，用蒸馏水代替H 2O 2溶液测得A j。
清除率(％)＝[1-(A i-A j)/A 0]×100％
图5为蛹虫草培养基多糖P1的OH自由基清除能力。由图5可知，蛹虫草培养基多糖组分P1具有显著的清除OH自由基能力，在质量浓度1.0～5.0mg/ml范围内，P1随着溶液浓度的增加，对OH自由基的清除能力逐渐增强，在浓度为5.0mg/ml时，清除率可达81.03％。
实施例7
对实施例1得到的蛹虫草培养基多糖P1进行降尿酸研究：
(1)高尿酸血症模型的建立
雄性小鼠60只，饲养一周后，随机分为6组，每组10只，依次为空白组、模型组、多糖P1高(400mg/kg)、P1中(200mg/kg)、P1低(100mg/kg)剂量组以及阳性药物(50mg/kg)对照组。每天上午模型组、阳性对照组、多糖P1高中低剂量组灌胃给予黄嘌呤600mg/kg(ig)+腹腔注射氧嗪酸钾100mg/kg(ip.)造模前一个小时禁食不禁水，空白组给予同等剂量生理盐水。下午多糖P1高、中、低剂量组采用多糖混悬液进行灌胃给药，阳性对照组灌胃给予别嘌醇混悬液50mg/kg，模型组和空白组则灌胃给予同等体积的生理盐水，连续七天
(2)体内生化指标的测定
第七天下午给药1h后称体重，断头取血，室温放置40min后，以3000r/min离心10min，吸取上层血清，采用试剂盒检测血清中尿酸(UA)、血清肌酐(CREA)和血清尿素氮(BUN)值，考察多糖P1对高尿酸血症模型小鼠UA、CREA和BUN及肾功能的影响。
测得蛹虫草培养基多糖P1的降尿酸作用结果如下表：
表2蛹虫草培养基多糖P1的降尿酸作用
Figure PCTCN2018120506-appb-000002
由表2可以看出，与正常组比，模型组小鼠的血清肌酐、血清尿酸和血清尿素氮水平均显著升高了，表明造模成功。
与模型组相比，多糖P1的低中高剂量组可使小鼠血清肌酐水平分别下降5.03％、20.19％、32.76％，可使小鼠血清尿酸水平分别下降39.58％、46.46％、53.72％，可使小鼠血清尿素氮水平分别下降22.77％、40.05％、51.59％。说明在该实验模型下，蛹虫草培养基多糖P1能够降低高尿酸血症的血清肌酐、血清尿酸和血清尿素氮水平。
实施例8
对实施例1得到的蛹虫草培养基多糖P1进行抑菌研究：
对常见的致病菌金黄色葡萄球菌(Sa)、绿脓杆菌(Pa)采用打孔法测定抑菌活性。主要操作过程如下：取直径为90mm的培养皿，倒平板后用移液枪取150μl的菌悬液，涂布均匀后每一平板用8mm打孔器打4孔，分别加入40μl备用的1mg/ml蛹虫草培养基多糖P1，对照组则每孔加入40μl生理盐水。置于37℃恒温培养箱内培养24h，用十字交叉法分别测量实验平板及对照平板上各抑菌圈的直径，并计算其平均值。
多糖P1的抑菌实验结果如图6所示，分别是P1对金黄色葡萄球菌的抑菌结果、金黄色葡萄球菌对照组、P1对绿脓杆菌的抑菌结果、绿脓杆菌对照组，测得抑菌圈的直径如表3。
表3抑菌圈直径(cm)
Figure PCTCN2018120506-appb-000003
由表3可知，蛹虫草培养基多糖P1对金黄色葡萄球菌、绿脓杆菌均有不同程度的抑制作用，其中对金黄色葡萄球菌的抑制作用比绿脓杆菌的更强。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种蛹虫草培养基多糖，其特征在于含有以下摩尔百分比的单糖：0.11％核糖、0.11％鼠李糖、0.45％阿拉伯糖、0.13％木糖、14.50％甘露糖、83.96％葡萄糖、0.73％半乳糖。
  2. 根据权利要求1所述的蛹虫草培养基多糖，其特征在于：平均分子量为2.18k Da。
  3. 根据权利要求1所述的蛹虫草培养基多糖，其特征在于：含有硫酸基、吡喃糖环和α-型糖苷键。
  4. 权利要求1-3任一项所述的蛹虫草培养基多糖的分离纯化方法，其特征在于包括以下步骤：
    (1)提取蛹虫草培养基多糖：将蛹虫草大米培养基下脚料干燥后粉碎，过筛得下脚料干粉；称取干粉加入15-16倍质量的蒸馏水，超声波处理30min以上，在70℃下回流提取1.5-2.0h，提取若干次合并提取液，提取液过滤后浓缩，得到多糖浓缩液；在多糖浓缩液中加入3-4倍体积的95％(V/V)乙醇，搅拌后于4℃中静置过夜；离心后将沉淀烘干得到蛹虫草培养基多糖提取物；
    (2)蛹虫草培养基多糖的脱色：将蛹虫草培养基多糖提取物加蒸馏水溶解，调节pH值至8.0-8.5，滴加H 2O 2溶液至无色，50-55℃保温2h以上；
    (3)酶法结合Sevage法脱蛋白：
    3-1：将木瓜蛋白酶溶液与蛹虫草培养基多糖提取液混合，两者的体积比为1.0:1.5～1.0:1.7，60-70℃酶解2-3h；
    3-2：在酶解液中加入1/5体积的Sevage试剂，振荡培养30min以上，然后多次离心直至无蛋白沉淀析出为止，取上清液，烘干得到蛹虫草培养基粗多糖；
    (4)蛹虫草培养基多糖的分离纯化：将蛹虫草培养基粗多糖用蒸馏水溶解后上DEAE琼脂糖凝胶FF离子交换层析柱，用蒸馏水进行洗脱，得到多糖。
  5. 根据权利要求4所述的分离纯化方法，其特征在于：步骤(1)所述的浓缩是在50-55℃下浓缩。
  6. 根据权利要求4所述的分离纯化方法，其特征在于：步骤(2)所述的H 2O 2溶液，浓度为30％(V/V)。
  7. 根据权利要求4所述的分离纯化方法，其特征在于：步骤(3)所述的木瓜蛋白酶溶液用pH值6.0的PBS缓冲液配制而成，其中木瓜蛋白酶的浓度为250U/ml。
  8. 根据权利要求4所述的分离纯化方法，其特征在于：步骤(3)所述的Sevage试剂，是氯仿和正丁醇按体积比5:1配制而成。
  9. 权利要求1-3任一项所述的蛹虫草培养基多糖作为抗氧化剂或抑菌剂的应用。
  10. 权利要求1-3任一项所述的蛹虫草培养基多糖在制备具有降尿酸功效的药物中的应用。
PCT/CN2018/120506 2018-04-27 2018-12-12 一种蛹虫草培养基多糖及其分离纯化方法和用途 WO2019205662A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/770,983 US11111317B2 (en) 2018-04-27 2018-12-12 Cordyceps militaris medium polysaccharide, method for separating and purifying same, and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810389081.7A CN108546306B (zh) 2018-04-27 2018-04-27 一种蛹虫草培养基多糖及其分离纯化方法和用途
CN201810389081.7 2018-04-27

Publications (1)

Publication Number Publication Date
WO2019205662A1 true WO2019205662A1 (zh) 2019-10-31

Family

ID=63512687

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/120506 WO2019205662A1 (zh) 2018-04-27 2018-12-12 一种蛹虫草培养基多糖及其分离纯化方法和用途

Country Status (3)

Country Link
US (1) US11111317B2 (zh)
CN (1) CN108546306B (zh)
WO (1) WO2019205662A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265012A (zh) * 2021-05-31 2021-08-17 天津天丰泽田生物科技有限公司 一种蚯蚓多糖的提取方法及其在农业抗菌中的应用

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109651524A (zh) * 2018-12-06 2019-04-19 桐乡市博奥生物科技有限公司 一种基于固体发酵的北冬虫夏草多糖提取纯化工艺
CN112522114B (zh) * 2020-12-10 2024-01-26 杭州慧农生物科技有限公司 蛹虫草菌糠提取液和灵芝发酵产物及其制备方法和应用
CN112890194A (zh) * 2021-02-05 2021-06-04 山西省农业科学院食用菌研究所 一种大同黄花菜营养元浆及其制备工艺
CN114099521A (zh) * 2021-11-12 2022-03-01 吉林农业大学 一种调节肺部淋巴细胞分化的蛹虫草中性寡糖及其制备方法和应用
CN114190542A (zh) * 2021-12-06 2022-03-18 广东省农业科学院蚕业与农产品加工研究所 一种降尿酸养生汤包料及其制备方法
CN115716883B (zh) * 2022-11-18 2023-09-26 新乡医学院 一种蛹虫草中性多糖及提取方法及其应用
CN116425901B (zh) * 2023-06-13 2023-08-18 西南民族大学 苦笋多糖及其制备方法和用途

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102417544A (zh) * 2011-09-28 2012-04-18 陕西师范大学 一种紫阳富硒绿茶含硒多糖及其制备方法和用途
CN102731666A (zh) * 2011-04-07 2012-10-17 大连工业大学 一种从蛹虫草培养基中提取多糖的方法
CN104497160A (zh) * 2014-12-31 2015-04-08 华南师范大学 一种蛹虫草多糖提取物及其制备方法与应用
CN104725521A (zh) * 2015-03-31 2015-06-24 广东省微生物研究所 一种皱盖假芝单峰多糖f212及其制备方法和应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5582828A (en) * 1995-03-15 1996-12-10 Ching-Yuang Lin Active fractions of Cordyceps sinensis and method of isolation thereof
TWI257928B (en) * 2002-12-27 2006-07-11 Ind Tech Res Inst Method for the separation of polysaccharides
CN101906127B (zh) * 2010-07-23 2016-03-16 上海国宝企业发展中心 一种从北冬虫夏草培养基残基中提取虫草素和虫草多糖的方法
CN103864945B (zh) * 2014-02-21 2016-01-20 湖北省农业科学院农产品加工与核农技术研究所 一种强化提取北虫草大米培养基中多糖的方法
CN105732832A (zh) * 2014-12-10 2016-07-06 黑龙江众生生物工程有限公司 一种蛹虫草多糖的制备方法
CN105085704A (zh) * 2015-09-15 2015-11-25 上海瑞丰农业科技有限公司 一种蛹虫草活性多糖的制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102731666A (zh) * 2011-04-07 2012-10-17 大连工业大学 一种从蛹虫草培养基中提取多糖的方法
CN102417544A (zh) * 2011-09-28 2012-04-18 陕西师范大学 一种紫阳富硒绿茶含硒多糖及其制备方法和用途
CN104497160A (zh) * 2014-12-31 2015-04-08 华南师范大学 一种蛹虫草多糖提取物及其制备方法与应用
CN104725521A (zh) * 2015-03-31 2015-06-24 广东省微生物研究所 一种皱盖假芝单峰多糖f212及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHEN XIAOLI, ET AL.: "Structural Characterization of a Polysaccharide from Cultured Cordyceps Militaris with Antioxidant Activity", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, vol. 37, no. 6, 31 December 2016 (2016-12-31), pages 155 - 159 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113265012A (zh) * 2021-05-31 2021-08-17 天津天丰泽田生物科技有限公司 一种蚯蚓多糖的提取方法及其在农业抗菌中的应用
CN113265012B (zh) * 2021-05-31 2022-07-12 西藏田硕农业科技有限公司 一种蚯蚓多糖的提取方法及其在农业抗菌中的应用

Also Published As

Publication number Publication date
CN108546306B (zh) 2021-08-03
CN108546306A (zh) 2018-09-18
US20200369789A1 (en) 2020-11-26
US11111317B2 (en) 2021-09-07

Similar Documents

Publication Publication Date Title
WO2019205662A1 (zh) 一种蛹虫草培养基多糖及其分离纯化方法和用途
US9284585B2 (en) Method for increasing yield of total flavonoids in Ganoderma lucidum mycelium
US20220042058A1 (en) Exopolysaccharide from Rhodopseudomonas palustris and Method for Preparing and Use Thereof
CN102370671A (zh) 灵芝子实体中的有效部位、提取方法及其用途和制剂
CN108503724B (zh) 蛹虫草培养基多糖及其分离纯化方法和应用
CN109970538A (zh) 一类海洋真菌来源的二倍半萜类化合物及其制备方法和在制备抗炎药物中的应用
CN111019008A (zh) 一种抗炎活性桑黄多糖shp及其制备方法
CN110742900B (zh) 一种具有免疫调节活性的小球藻胞外多糖复合物及其制备方法和应用
CN108641006B (zh) 一种从蛹虫草培养基下脚料中提取并分离纯化多糖的方法
CN115634241A (zh) 一种山东灵芝提取物的制备方法及其在制备降血糖药物中的应用
JP2004091780A (ja) Ａｎｔｒｏｄｉａｃａｍｐｈｏｒａｔａ菌体の多糖類
CN115490780A (zh) 马尾藻岩藻多糖粗提物的提取方法及应用
CN105274152B (zh) 姜黄素的生物转化方法、产物及应用
CN113861303A (zh) 一种从德氏乳杆菌和嗜热链球菌发酵酸奶中分离出的胞外多糖及其应用
CN115947876B (zh) β-D-半乳葡聚糖及其制备和用途
CN109536432A (zh) 一种稻瘟菌分泌蛋白的提取方法及应用shotgun技术研究其分泌蛋白质组的方法
AU2021104232A4 (en) Application of sanghuangporus sanghuang fermentation broth polysaccharides in medicaments of anti-avian influenza virus
CN108531462A (zh) 一种禽腺病毒精纯化的方法
CN112759661B (zh) 金樱子多糖制备方法、鉴定方法和应用
CN111704679B (zh) 一种猴头菌发酵菌丝体β-葡聚糖及其制备方法和应用
EP4206232A1 (en) Heteropoly oligosaccharide and application thereof in improving plant disease resistance
TWI666314B (zh) 利用半連續式培養增加矽藻產量的方法
CN117323352A (zh) 一种具有足细胞保护活性的冬虫夏草提取物及其制备方法和应用
CN115043719A (zh) 一种真菌来源的聚酮类化合物及其制备方法与应用
CN114853918A (zh) 一种具有抗氧化降脂活性的紫花苜蓿根部多糖制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18916827

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18916827

Country of ref document: EP

Kind code of ref document: A1