CN112626149B - Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine - Google Patents
Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine Download PDFInfo
- Publication number
- CN112626149B CN112626149B CN202110174398.0A CN202110174398A CN112626149B CN 112626149 B CN112626149 B CN 112626149B CN 202110174398 A CN202110174398 A CN 202110174398A CN 112626149 B CN112626149 B CN 112626149B
- Authority
- CN
- China
- Prior art keywords
- liquid
- polysaccharide
- phellinus igniarius
- fermentation broth
- culture
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Mycology (AREA)
- Botany (AREA)
- Pulmonology (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses phellinus igniarius with the preservation number of CGMCC NO.21068 (Sanghuangporus sanghuang) The application of the fermentation broth polysaccharide in the anti-avian influenza virus medicament comprises the steps of extracting, filtering, purifying, collecting, drying and the like of the phellinus igniarius fermentation broth polysaccharide.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of phellinus igniarius fermentation broth polysaccharide in an anti-avian influenza virus medicament.
Background
With the rapid development of the breeding industry in China, particularly the intensive breeding industry in recent years, the prevention and treatment of livestock and poultry diseases also face a plurality of problems at present. The infectious diseases of livestock and poultry can be roughly classified into viral diseases, bacterial diseases, fungal diseases, mycoplasmosis and the like according to the pathogenic characteristics of the infectious diseases. Viral diseases are difficult to treat without specific medicines and treatment methods at present. At present, the main measures taken against viral infectious diseases are therapeutic methods such as vaccine injection and antiviral drug application. Both methods have certain defects, vaccine injection is only effective for a certain virus and does not have broad spectrum, and treatment by using antiviral drugs also has certain limitations, and the vaccine injection can inhibit common viruses, but the resistance to some viruses is not ideal, and the long-term use of the vaccine injection can generate drug resistance.
Research reports that phellinus igniarius polysaccharides have been found to have various biological activities, the activities which are reported at present include that phellinus igniarius polysaccharides have activities of resisting oxidation, resisting tumors, reducing blood fat and the like, and the phellinus igniarius polysaccharides have rich raw material sources and are produced into phellinus igniarius polysaccharidesThe feed additive has the advantages of low cost, simple preparation process and the like, does not damage animals, and has the effects of improving the immunity of the animals, resisting viruses, oxidation, bacteria and the like. However, some phellinus linteus polysaccharides are extracted from the fruit body of phellinus linteus, for example, CN110642958A discloses a method for extracting phellinus linteus polysaccharides and its application, the related bacterial strain isPhellinus linteus、Phellinus baumiiAndPhellinus igniariusthe method comprises the following steps: (1) pulverizing mature Phellinus linteus fruiting body, sieving, and preparing culture medium; (2) mixing cellulose degrading bacteria and Paenibacillus polymyxaPaenibacillus polymyxaInoculating into the culture medium in the step (1), and fermenting for a certain time; (3) after fermentation is finished, solid-liquid separation is carried out on thalli and impurities which are insoluble in water, and fermentation liquor with polysaccharide dissolved is obtained; washing the solid-liquid separated precipitate, and combining the washing liquid into the fermentation liquid dissolved with the polysaccharide; (4) carrying out ultrafiltration on the mixed liquor obtained in the step (3); (5) precipitating with ethanol solution to obtain Phellinus linteus polysaccharide product. The phellinus igniarius polysaccharide product obtained by the method has high yield and purity and high oxidation resistance. CN105777924A also discloses an anticancer active polysaccharide PBPP of Phellinus linteus fruiting body and a preparation method thereof, and the related strain isPhellinus baumii(ii) a Phellinus linteus polysaccharides are extracted from Phellinus linteus mycelium, for example, CN104910289A discloses a method for continuously preparing Phellinus linteus mycelium polysaccharides of which the related species arePhellinus linteusBelonging to the technical field of biological engineering. The method comprises the following steps: extracting defatted Phellinus linteus mycelium with 95 deg.C hot water under reflux for 8 hr, centrifuging, concentrating, precipitating with ethanol, removing protein, dialyzing, and freeze drying to obtain water-extracted Phellinus linteus mycelium polysaccharide; dissolving the water extraction residue in 1% ammonium oxalate solution, reflux extracting at 95 deg.C for 8 hr, centrifuging, concentrating, precipitating with ethanol, removing protein, dialyzing, and freeze drying to obtain acid-extracted Phellinus Linteus mycelium polysaccharide; dissolving the acid extraction residue in 1.25mol/L sodium hydroxide and 0.05% sodium borohydride solution, extracting at room temperature for 3h, centrifuging, concentrating, adding acetic acid, precipitating with ethanol, removing protein, dialyzing, and freeze drying to obtain alkali-extracted Phellinus linteus mycelium polysaccharide. The phellinus igniarius mycelium polysaccharide continuously prepared by the method has obvious free radical scavenging capacity and antioxidant activity, and the extraction preparation of the inventionThe method is simple and easy to implement, has low cost, can be used for large-scale production, and can be applied to other fungus mycelia such as ganoderma lucidum, cordyceps sinensis, grifola frondosa and the like. CN110627917A also discloses that the phellinus igniarius mycelium is used as an extraction raw material, so that the cost is greatly saved, the method is simple in step and convenient to operate, the polysaccharide in the phellinus igniarius mycelium can be effectively extracted, and the polysaccharide yield is high. However, the extraction of phellinus linteus polysaccharide from phellinus linteus fermentation liquor is not reported, and the application of phellinus linteus polysaccharide as an immunopotentiator, especially in the aspect of preventing and treating avian influenza virus is not reported.
Disclosure of Invention
In order to solve the problems, the invention discloses application of phellinus igniarius fermentation broth polysaccharide in an anti-avian influenza virus medicament.
The invention is realized by the following technical scheme:
phellinus igniarius with preservation number of CGMCC NO.21068Sanghuangporus sanghuang) Application of fermentation liquor polysaccharide in anti-avian influenza virus medicine
Preferably, the phellinus igniarius fermentation broth polysaccharide is extracted by the following method:
(1) extraction: concentrating the Phellinus linteus fermentation broth obtained after shaking culture for 8-10d to 1/20-1/10 of the original volume, adding 2-5 times of 75-95% ethanol, standing for precipitating with ethanol for 12-48h, centrifuging at 5000 rpm for 10-30 min to obtain an ethanol precipitate, freeze-drying, pulverizing, and sieving with 40-80 mesh sieve;
(2) and (3) filtering: dissolving appropriate amount of above dry powder of alcohol precipitate with 50-100 times volume of distilled water, dissolving with ultrasonic oscillation at 30-60 deg.C for 1-3 hr, dissolving for 15-30 min under magnetic stirring, and centrifuging at 5000 rpm for 10-30 min; dissolving the filter residue with 10-50 times of distilled water for two times, centrifuging, mixing the supernatant for 3 times, concentrating, and freeze drying to obtain polysaccharide crude extract of Phellinus linteus fermentation liquid, and labeling as EPS;
(3) and (3) purification: dissolving EPS sample with 100 times of distilled water, centrifuging at 8000 rpm for 10-30 min to remove insoluble substances, and purifying supernatant by Sephadex G-100;
(4) collecting: collecting the purified sample, and drying to obtain phellinus igniarius polysaccharides;
wherein the Phellinus linteus strain (Sanghuangporus sanghuang) The preservation number is CGMCC NO. 21068.
Preferably, the phellinus linteus fermentation broth is obtained by the following method:
(1) the plate strain culture method comprises the following steps: sterile operation, taking out the area of 0.25cm from the slant mother seeds2Transferring the strain block to the center of a plate culture medium cooled to below 28 ℃, putting the plate culture medium in a constant-temperature incubator at 25-28 ℃, keeping out of the sun, inverting, and standing for 6-10 days to obtain the plate strain.
(2) The liquid strain culture method comprises the following steps: sterile operation, 500 mL triangular flask, liquid loading amount of 200mL, inoculating 6-8 blocks of uniform age, and area of 0.25cm2Performing shake culture on activated plate strain at 28 deg.C and 150 rpm in dark for 6 d to obtain liquid strain;
(3) the liquid fermentation culture method comprises the following steps: and (3) performing aseptic operation, namely inoculating the liquid strain into a liquid fermentation tank, wherein the inoculation amount is 5-15%, the temperature is 25-30 ℃, the rotation speed is 150 rpm, performing vibration culture in a dark place for 6-10 days, stopping fermentation, and filtering by using a gauze with 60-200 meshes to obtain the phellinus igniarius fermentation broth.
Preferably, the liquid seed culture medium comprises, in percent: 1-2% of glucose, 0.2-0.5% of yeast extract and KH2PO4 0.05-0.1%，MgSO4·7H2O0.02-0.05%, pH is natural;
the liquid fermentation medium comprises the following components in percentage: corn flour 0.3-0.4%, glucose 1-2%, peptone 0.1-0.3%, yeast extract 0.2-0.5%, KH2PO4 0.05-0.1%，MgSO4·7H20.02-0.08% of O, 1-3% of bran and the pH value of 5-7.
Advantageous effects
The invention provides the application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus drugs, which widens the way for preventing and treating avian influenza virus and has very important significance.
The method adopts a liquid fermentation form to obtain phellinus igniarius fermentation liquor, and extracts phellinus igniarius fermentation liquor polysaccharide in the phellinus igniarius fermentation liquor through alcohol extraction and gel column filtration and purification, wherein polysaccharide components EPS1 and EPS2 obtained after the polysaccharide is purified have good effect on resisting avian influenza H5N1 strains.
Half poisoning concentration CC of phellinus igniarius fermentation broth polysaccharide EPS15017.324ug/mL, maximum safe concentration CmaxIt was 7.6 ug/mL. Half poisoning concentration CC of phellinus igniarius fermentation broth polysaccharide EPS2502.511ug/mL, maximum safe concentration CmaxIt was 2.0 ug/mL. The challenge experiment on the (AIV) H5N1 strain was performed on the MDCK cell level using the sample EPS1 within the obtained maximum safe concentration range, and the inhibition ratio of the virus was 30.4%, when the SI was 1.64. When the challenge experiment of the (AIV) H5N1 strain is carried out on the sample EPS2 at the MDCK cell level within the obtained maximum safe concentration range, the inhibition rate of the virus reaches 100 percent, and the SI is 2.686.
Information on strain preservation
Preservation time: 1 month and 4 days 2021;
the preservation unit: china general microbiological culture Collection center;
the preservation number is: CGMCC No. 21068;
the address of the depository: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing;
and (3) classification and naming:Sanghuangporus sanghuang。
drawings
FIG. 1 shows the elution diagram of Phellinus linteus fermentation broth polysaccharide (EPS) Sephadex G-100 by column chromatography.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The strain adopted by the application is obtained by collecting fresh sporocarp from live standing tree of mulberry in mountain area of Anshun city, Guizhou province, and then carrying out tissue separation and strain purification, and the strain is classified and named as phellinus igniarius (A)Sanghuangporus sanghuang) Has been preserved in China general microbiological culture Collection management Committee (CGMCC) at 1 month and 4 days 2021The preservation number of the center is CGMCC NO. 21068.
Morphological characteristics of fruiting bodies: the basil fruits are perennial, have no handle cover shape, are usually single-grown, have sour taste when fresh, are woody and are woody after being dried; the pileus is shaped like a horseshoe, extends outwards to 5cm, has a width of 11 cm and a base thickness of 6 cm; the surface of the pileus is yellow brown to grey brown, is smooth by fine villi, and has obvious ring areas and ring ditches; dull edge, bright yellow; the surface of the opening is yellow when fresh, brown after dried and has a slight refraction reaction; the sterile edge is obvious, and the width reaches 3 mm; the orifices are polygonal to circular, and 6-7 orifices per millimeter; the edge of the orifice is thin and the whole edge is thin; the mushroom flesh is yellow, has lighter color than the mushroom tube, is made of hardwood with suppository texture, has obvious ring area, has black lines and has the thickness of 5 cm; the mushroom tube is brown, and the length of the mushroom tube is 4 mm.
Microstructural features: a hypha system II; all diaphragms are united without locks; hyphal tissue darkened in KOH. The germ hyphae in the mushroom meat are colorless to light yellow, thin-walled to slightly thick-walled, multi-branched and frequently separated, and the diameter is 2-3 mu m; the skeleton hyphae in the mushroom meat are golden yellow, thick, have wide or narrow inner cavities, are not branched, are more separated, are nearly regularly arranged and have the diameter of 3-4 mu m. The germ hyphae in the fungus tube are colorless to light yellow, thin wall to thick wall, few branches, more partitions and 2-2.5 μm in diameter; the skeleton hyphae in the fungus tube are golden yellow, thick, have wide or narrow inner cavities, are not branched or separated, are arranged nearly regularly, and have the diameter of 2.5-3.7 mu m; the seta of the fructification layer is common and mostly in the shape of a drum, dark brown, thick wall, sharp tail end and 17-30 multiplied by 7-11 mu m in size; the sub-parenchyma layer is free of cysts and pseudo-cysts; the basilar barrel has 4 small stems, and the base has a simple partition with a size of 6-9X 4-5 μm. Basidiospores are wide oval, yellow, thick-walled, smooth, IKI-, CB-, with a size of (3.4-)3.6-4.6(-4.8) × (2.8-)3-3.5(-3.7) μm, an average length L = 4.03 μm, an average width W = 3.18 μm, and an aspect ratio Q = 1.27 (n = 30/1).
And (3) identifying the strain molecules: extracting DNA from the sporocarp and the mycelium by using a kit, amplifying and sequencing by using ITS5/ITS4 primers, wherein the common rDNA-ITS sequence of the sporocarp and the mycelium is as follows:
CGATGGGTACTGCGGAGGTCATTATCGAGTTTTGAAAGCGAGACCTGCTGCTGGTGCGAAATCGCGCATGTGCACGGTCTTCGCGCTCAAATCCAACTCAAACCCCTGTGCACCTTATATATCGCGAGTCGAAGTTAGTAGCCTGAGGTCTTGTAAGTAATTAGTAGAAGGGCGAAAGCGAGTCTTGCTCGTTAGGTAGCCTTTCGAAAATGAAAGCGAGTGCGTCGGGTGAAGACTTCGGCTTGTCGTTACAAAACACCTTATATTGTCTTTGTGAATGTAATGCTCCTTGTGGGCGAAAATAAATACAACTTTCAACAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCGCCCCTTGGTATTCCGAGGGGCATGCCTGTTTGAGTGTCATGTTTATCTCAAACCGCTCGTCTTTCTTAATTGAAGGGCTTGAGGTTTGGACTTGGAGGTTTACTGCTGGCGCCTTTCGAGGGGTCGGCTCCTCTTAAATACATTAGCTGGGCTTTGGCTCGCGTTTACGGTGTAATAGTTGATTCCATTCACCAACGAGCGCTTGCCTGACGAGCTTGCTTCTAGCCGTCCGCGTCGTCGGACAAGGAGTCACCTCCTTCTTGACACCTTTGACCTCAAATCAGGTAGGATTACCCGCCGACTTACTAATTAAAGGGGGGGGGGGGGGGAAGAAATTGAA
1. culture medium formula
Slant stock culture (PDA): potato (peeled) 20%, glucose 2%, agar 2%, pH natural.
The liquid strain culture medium comprises the following components in percentage: 2% of glucose, 0.5% of yeast extract and KH2PO4 0.1%，MgSO4·7H2O0.05% and natural pH.
The liquid fermentation medium comprises the following components in percentage: corn flour 0.3%, glucose 2%, peptone 0.1%, yeast extract 0.5%, KH2PO4 0.1%，MgSO4·7H2O0.05%, bran 1%, pH 6.
2. Culture method
The plate strain culture method comprises the following steps: sterile operation, taking out the area of 0.25cm from the slant mother seeds2Transferring the strain block to the center of a plate culture medium cooled to below 28 ℃, putting the plate culture medium in a constant-temperature incubator at 25-28 ℃, keeping out of the sun, inverting, and standing for 6-10 days to obtain the plate strain.
The liquid strain culture method comprises the following steps: sterile operation, 500 mL triangular flask, liquid loading amount of 200mL, inoculating 6-8 blocks with consistent age and area of about 0.25cm2Performing shake culture on the activated plate strain at 28 deg.C and 150 rpm in dark for 6 d to obtain liquid strain.
The liquid fermentation culture method comprises the following steps: performing aseptic operation, inoculating 10%, performing shaking culture at 28 deg.C and 150 rpm in dark for 10d, terminating fermentation, and filtering with 200 mesh gauze to obtain fermentation broth.
3. Preparation of Phellinus linteus extracellular crude polysaccharide sample
Collecting Phellinus linteus fermentation broth obtained after shaking culture for 10d, concentrating to 1/10 of original volume, adding 5 times of 95% ethanol, standing, precipitating with ethanol for 24h, centrifuging at 5000 rpm for 5min to obtain ethanol precipitate, freeze drying, pulverizing, and sieving with 80 mesh sieve.
Dissolving appropriate amount of above dry powder of alcohol precipitate with 100 times volume of distilled water, dissolving with ultrasonic oscillation at 50 deg.C for 3 hr, dissolving with magnetic stirring for 30 min, and centrifuging at 5000 rpm for 10 min. And (3) continuously dissolving the filter residue twice by using distilled water with the volume of 40 times, centrifuging, combining 3 times of centrifuged supernatant, concentrating, and freeze-drying to obtain a polysaccharide crude extract of the phellinus igniarius fermentation broth, wherein the polysaccharide crude extract is marked as EPS.
4. Separation and purification of phellinus igniarius fermentation broth polysaccharide sample
Taking a proper amount of EPS sample, dissolving the EPS sample by using 100 times of distilled water, centrifuging the EPS sample for 10 min at 8000 rpm to remove insoluble substances, and performing fractional purification on supernatant through Sephadex G-100.
4.1Sephadex pretreatment and column packing
Weighing about 18.0G of Sephadex G-100 Sephadex dry powder in a big beaker, adding 600 mL of distilled water, uniformly mixing, heating in a water bath at 100 ℃ for swelling for 1-3h, fully swelling the gel, killing microorganisms in the gel, and removing bubbles in the gel. The upper layer of water and floating gel particles are poured off to make the lower layer of resin uniform in consistency. A gel column with the size of 26 mm multiplied by 46 cm is selected, and distilled water is adopted for washing and balancing. The chromatographic column is vertically fixed on an iron support, firstly distilled water with about 1/3 column volumes is added to remove dead zones and bubbles in the plastic tube, and then the swelled gels are continuously filled while being stirred, so that the swelled gels flow into the column along the tube wall to naturally settle, bubbles are avoided, and the bubbles are removed or reloaded if the bubbles exist. And opening the lower opening to flow out the aqueous solution slowly, continuously pouring the gel when the liquid level is close to the gel surface, and slightly stirring the surface layer by using a glass rod to avoid forming a boundary layer between the two added gels. After loading, balancing with distilled water for 2-3 h until the volume of the column bed is unchanged, and then loading and separating can be carried out.
4.2. Eluting and collecting
The sample loading amount is 5 mL, distilled water is used as eluent, and the flow rate is controlled to be 0.5-1 mL/min. Collecting eluate for 10 min, collecting 40 tubes, measuring polysaccharide and protein content in each odd-numbered collection tube, measuring light absorption value, drawing peak diagram, combining polysaccharide peak parts according to separation result, freeze drying, and storing.
4.3. Regeneration and preservation of Sephadex column
Soaking the used sephadex in a mixed solution of 0.1 mol/L NaOH and 0.5 mol/L NaCl for 0.5-1 h, and repeatedly washing the sephadex with distilled water to be neutral for later use. And if the test is finished, continuously soaking the mixture in 50% ethanol, 75% ethanol, 95% ethanol, absolute ethanol and diethyl ether in sequence, then draining the mixture by using a Buchner funnel, repeating the steps twice, drying the mixture at 37 ℃, and storing the dried mixture for a long time.
5 results and analysis
5.1. Separating and purifying dextran gel column of phellinus igniarius fermentation broth polysaccharide
Passing through Sephadex G-100 column, separating and purifying to obtain EPS sample with two absorption peaks, collecting 7-13 tubes and combining to obtain EPS1, and collecting 30-32 tubes and combining to obtain EPS 2. According to the results of polysaccharide content measurement and soluble protein content measurement, a small amount of protein is present in EPS1, which may belong to the polysaccharide peptide component, and is the main component in the EPS sample, and the polysaccharide content is 59.53%. Almost no protein was detected in EPS2 where the sugar peak appeared, with a polysaccharide content of 7.07%.
EXAMPLE 2 test content against avian influenza Virus
1 materials and methods
1.1 test samples
Phellinus igniarius fermentation broth polysaccharides EPS1 and EPS2 are prepared and provided by applied fungi key laboratories in Shandong province.
1.2 test strains and cells
Avian Influenza Virus (AIV) H5N1 strain. MDCK cells.
1.3 Experimental conditions
And a biosafety tertiary laboratory.
1.4 measurement of cell proliferation
The cell proliferation was determined using the classical MTT method.
The MTT method comprises the following operation steps: 1 hour before the measurement, the supernatant in the wells of the 96-well plate was discarded, and 100. mu.L of MTT solution containing 5 mg/mL was added to each well, followed by incubation at 37 ℃. After the incubation time is over, quickly turning over the 96-well plate, discarding MTT solution, washing the 96-well plate with PBS (PBS), discarding PBS liquid, adding cell lysate (DMSO: ethanol =1:1, V/V), shaking the plate 100 μ L per well for 3-5min at room temperature, and placing the plate in an enzyme linked immunosorbent assay detector to measure OD value at 492nm after the purple crystal is completely dissolved. And calculating the cell inhibition rate and the virus inhibition rate by formulas.
A: OD value of test group, B: OD value of cell control, C: OD values of virus control group.
1.5 maximum safe concentration determination of different extracts of Phellinus linteus strains at MDCK cell level
96-well cell culture plates were prepared that grew good monolayers of MDCK cells. The extract stock solution was filtered through a 0.22 μm filter and diluted to 5 concentration gradients by 2-fold dilution using a maintenance solution as a diluent. Adding 100 μ L/well of different concentrations of the maintenance solution containing sample, repeating for 3 wells at each concentration, setting normal cell control (adding maintenance solution only), standing at 37 deg.C and 5% CO2And (5) continuously culturing for 72 hours in the incubator. The test was terminated by MTT method and the OD value was measured. And when the OD value of the sample adding group is not significantly lower than the OD value of the cell control group, the maximum safe concentration of the extract is obtained. Half-maximal toxicity concentration (CC) was calculated using SPSS regression analysis50）。
1.6 Activity test of Phellinus Linteus extract against avian influenza at MDCK cell level
In the in vitro AIV cell resisting model, a normal cell control group (only adding cell maintenance liquid), a virus control group (only adding virus liquid) and a blank zero-adjusting group without cells are arranged, the maximum mass concentration is selected to be 2 times, the dilution is carried out for 4 concentrations, and 3 holes are arranged for repeating. Abandoning supernatant in 96-well cell culture plate, adding 10000TCID50Virus liquid 100 muL/well, 3 replicates were set. Adsorbing at 37 deg.C for 1.5h, discarding virus liquid supernatant, and adding sample solution of different concentrations at 100 μ L/well. Standing at 37 deg.C for 5% CO2The cultivation was continued in the incubator for 48 h. After culturing for 48h, terminating the test by adopting an MTT method, measuring the OD value, and calculating the virus inhibition rate according to a virus inhibition rate formula. When the OD value of the sample adding group is obviously greater than that of the virus control group, the liquid can obviously inhibit virus infection MDCK and has certain antiviral activity. Half Effective Concentration (EC) was calculated using SPSS regression analysis50）。
1.3 data analysis
Calculation of sample CC50And EC50SI = CC is obtained50／EC50The efficacy of each extract on AIV inhibition was measured using the Selection Index (SI) as an evaluation index. Duncan' S and T-test multiplex analysis with statistical software SPSS17.0, comparison of the difference in cytopathic effects between the test and virus control groups, P<0.05 shows that the data have significant difference, and the mean value plus or minus standard deviation (is adopted for the measurement data)
SD).
2 results
TABLE 1 Phellinus linteus fermentation broth polysaccharide EPS1 and EPS2 cytotoxicity and in vitro anti-avian influenza virus H5N1 strain pharmacodynamic results
The cell-level H5N1 avian influenza virus activity detection method is adopted to test the inhibitory activity of phellinus igniarius fermentation broth polysaccharides EPS1 and EPS2 on H5N1 avian influenza virus, and the EC50 of the phellinus igniarius fermentation broth polysaccharides is 10.578 ug/mL and 0.935 ug/mL respectively (Table 1).
The 2 samples showed different levels of cytotoxicity on canine kidney MDCK cells with half-cell death concentrations CC50 of 17.324ug/mL and 2.511ug/mL, respectively, and maximum safe concentrations of 7.6ug/mL and 2.0ug/mL, respectively (Table 1).
Experimental results show that the selection coefficients SI of the phellinus igniarius fermentation broth polysaccharides EPS1 and EPS2 are 1.64 and 2.686 respectively, and the phellinus igniarius fermentation broth polysaccharides have good in-vitro anti-H5N 1 avian influenza virus activity and have the potential of further developing novel anti-avian influenza virus medicines.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> Qingdao agricultural university
Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 764
<212> DNA
<213> Phellinus linteus (Sanghuangpolus sanghuang)
<400> 1
cgatgggtac tgcggaggtc attatcgagt tttgaaagcg agacctgctg ctggtgcgaa 60
atcgcgcatg tgcacggtct tcgcgctcaa atccaactca aacccctgtg caccttatat 120
atcgcgagtc gaagttagta gcctgaggtc ttgtaagtaa ttagtagaag ggcgaaagcg 180
agtcttgctc gttaggtagc ctttcgaaaa tgaaagcgag tgcgtcgggt gaagacttcg 240
gcttgtcgtt acaaaacacc ttatattgtc tttgtgaatg taatgctcct tgtgggcgaa 300
aataaataca actttcaaca acggatctct tggctctcgc atcgatgaag aacgcagcga 360
aatgcgataa gtaatgtgaa ttgcagaatt cagtgaatca tcgaatcttt gaacgcacct 420
tgcgcccctt ggtattccga ggggcatgcc tgtttgagtg tcatgtttat ctcaaaccgc 480
tcgtctttct taattgaagg gcttgaggtt tggacttgga ggtttactgc tggcgccttt 540
cgaggggtcg gctcctctta aatacattag ctgggctttg gctcgcgttt acggtgtaat 600
agttgattcc attcaccaac gagcgcttgc ctgacgagct tgcttctagc cgtccgcgtc 660
gtcggacaag gagtcacctc cttcttgaca cctttgacct caaatcaggt aggattaccc 720
gccgacttac taattaaagg gggggggggg gggaagaaat tgaa 764
Claims (2)
1. Phellinus igniarius with preservation number of CGMCC NO.21068Sanghuangporus sanghuang) The application of fermentation broth polysaccharide in preparing anti-avian influenza virus medicine;
the phellinus igniarius fermentation broth polysaccharide is extracted by the following method:
(1) extraction: concentrating the Phellinus linteus fermentation broth obtained after shaking culture for 8-10d to 1/20-1/10 of the original volume, adding 2-5 times of 75-95% ethanol, standing for precipitating with ethanol for 12-48h, centrifuging at 5000 rpm for 10-30 min to obtain an ethanol precipitate, freeze-drying, pulverizing, and sieving with 40-80 mesh sieve;
(2) and (3) filtering: dissolving appropriate amount of above dry powder of alcohol precipitate with 50-100 times volume of distilled water, dissolving with ultrasonic oscillation at 30-60 deg.C for 1-3 hr, dissolving for 15-30 min under magnetic stirring, and centrifuging at 5000 rpm for 10-30 min; dissolving the filter residue with 10-50 times of distilled water for two times, centrifuging, mixing the supernatant for 3 times, concentrating, and freeze drying to obtain polysaccharide crude extract of Phellinus linteus fermentation liquid, and labeling as EPS;
(3) and (3) purification: dissolving EPS sample with 100 times of distilled water, centrifuging at 8000 rpm for 10-30 min to remove insoluble substances, and purifying supernatant by Sephadex G-100;
(4) collecting: collecting the purified sample, and drying to obtain phellinus igniarius polysaccharides;
the phellinus igniarius fermentation liquor is obtained by the following method:
(1) the plate strain culture method comprises the following steps: sterile operation, taking out the area of 0.25cm from the slant mother seeds2Transferring the strain block to the center of a plate culture medium cooled to below 28 ℃, putting the plate culture medium in a constant-temperature incubator at 25-28 ℃, keeping out of the sun, inverting, and performing standing culture for 6-10 days to obtain plate strains;
(2) liquid spawnThe culture method comprises the following steps: sterile operation, 500 mL triangular flask, liquid loading amount of 200mL, inoculating 6-8 blocks of uniform age, and area of 0.25cm2Performing shake culture on activated plate strain at 28 deg.C and 150 rpm in dark for 6 d to obtain liquid strain;
(3) the liquid fermentation culture method comprises the following steps: and (3) performing aseptic operation, namely inoculating the liquid strain into a liquid fermentation tank, wherein the inoculation amount is 5-15%, the temperature is 25-30 ℃, the rotation speed is 150 rpm, performing vibration culture in a dark place for 6-10 days, stopping fermentation, and filtering by using a gauze with 60-200 meshes to obtain the phellinus igniarius fermentation broth.
2. The application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus drugs as claimed in claim 1, wherein the liquid strain culture medium used in the liquid strain culture method comprises the following components in percentage: 1-2% of glucose, 0.2-0.5% of yeast extract and KH2PO4 0.05-0.1%，MgSO4·7H2O0.02-0.05%, pH is natural;
the liquid fermentation culture method comprises the following steps of: corn flour 0.3-0.4%, glucose 1-2%, peptone 0.1-0.3%, yeast extract 0.2-0.5%, KH2PO4 0.05-0.1%，MgSO4·7H20.02-0.08% of O, 1-3% of bran and the pH value of 5-7.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110174398.0A CN112626149B (en) | 2021-02-07 | 2021-02-07 | Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine |
| AU2021104232A AU2021104232A4 (en) | 2021-02-07 | 2021-07-16 | Application of sanghuangporus sanghuang fermentation broth polysaccharides in medicaments of anti-avian influenza virus |
| ZA2021/06499A ZA202106499B (en) | 2021-02-07 | 2021-09-06 | Application of sanghuangporus sanghuang fermentation broth polysaccharides in medicaments of anti-avian influenza virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110174398.0A CN112626149B (en) | 2021-02-07 | 2021-02-07 | Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112626149A CN112626149A (en) | 2021-04-09 |
| CN112626149B true CN112626149B (en) | 2022-04-12 |
Family
ID=75295339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110174398.0A Active CN112626149B (en) | 2021-02-07 | 2021-02-07 | Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine |
Country Status (3)
| Country | Link |
|---|---|
| CN (1) | CN112626149B (en) |
| AU (1) | AU2021104232A4 (en) |
| ZA (1) | ZA202106499B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103816176A (en) * | 2013-09-06 | 2014-05-28 | 青岛农业大学 | Application of maitake 253 strain crude polysaccharide to resisting H5N1 avian influenza virus |
| CN104087632A (en) * | 2014-07-15 | 2014-10-08 | 江苏神华药业有限公司 | Method for producing phellinus igniarius extracellular polysaccharides by deep liquid fermentation |
| CN105154338A (en) * | 2015-09-18 | 2015-12-16 | 南京师范大学 | Tabasheer separated fungus acremonium cavaraeanum and application thereof in ergot sterone production |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100663712B1 (en) * | 2005-05-24 | 2007-01-03 | (주)새롬바이오 | A crude exopolysaccharides produced from Phellinus baumii mycellium having hypoglycemic activity and preparation method thereof |
-
2021
- 2021-02-07 CN CN202110174398.0A patent/CN112626149B/en active Active
- 2021-07-16 AU AU2021104232A patent/AU2021104232A4/en not_active Ceased
- 2021-09-06 ZA ZA2021/06499A patent/ZA202106499B/en unknown
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103816176A (en) * | 2013-09-06 | 2014-05-28 | 青岛农业大学 | Application of maitake 253 strain crude polysaccharide to resisting H5N1 avian influenza virus |
| CN104087632A (en) * | 2014-07-15 | 2014-10-08 | 江苏神华药业有限公司 | Method for producing phellinus igniarius extracellular polysaccharides by deep liquid fermentation |
| CN105154338A (en) * | 2015-09-18 | 2015-12-16 | 南京师范大学 | Tabasheer separated fungus acremonium cavaraeanum and application thereof in ergot sterone production |
Non-Patent Citations (2)
| Title |
|---|
| A newly characterized exopolysaccharide from Sanghuangporus sanghuang;Xiao-Kui Ma等;《J Microbiol》;20190614;第57卷(第9期);第812-820页 * |
| 药用真菌桑黄液体培养过程中的抗氧化活性研究;郑飞等;《菌物学报》;20171231;第36卷(第1期);第98-111页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA202106499B (en) | 2021-10-27 |
| AU2021104232A4 (en) | 2021-09-09 |
| CN112626149A (en) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107501429B (en) | Method for extracting bioactive β -glucan from sparassis crispa liquid fermentation mycelium | |
| CN108330072A (en) | Inonotus obliquus liquid submerged fermentation culture composition and preparation method and application | |
| CN101492706A (en) | Method for improving cordyceps sinensis bacterium native volume of production with cordyceps militaris link liquid fermentation | |
| CN111500472A (en) | Corynebacteria mycelium rich in flavone and polyphenol and production method thereof | |
| CN113308378B (en) | Ganoderma lucidum strain for high-yield ergothioneine and application thereof | |
| CN110713550A (en) | Method for preparing refined polysaccharide with antibacterial activity by using cordyceps culture | |
| CN105713841B (en) | A kind of fusarium oxysporum and its application in the sprouting pretreatment of bitter buckwheat Wheat Seeds | |
| CN112626149B (en) | Application of phellinus igniarius fermentation broth polysaccharide in anti-avian influenza virus medicine | |
| CN103103127B (en) | Culture method for microalgae | |
| CN110711203B (en) | Application of cordycepin polysaccharide in preparation of medicament for resisting gram-negative plant pathogenic bacteria | |
| CN105779306A (en) | Peltate yam rhizome endophyte for generating saponinase and application | |
| CN108624509B (en) | Recycling method of solid culture medium in artificial culture process of cordyceps sobolifera | |
| CN104278070A (en) | Method for improving content of ergosterol in liquid fermentation products of phellinus igniarius | |
| CN1177925C (en) | Fermenting culture process of produicng Cordyceps extract | |
| CN109731015B (en) | Immunity enhancer based on hirsutella sinensis and preparation method | |
| CN109503613B (en) | Gryllus chinensis bipolaris staurosporine I and preparation method and application thereof, and Gryllus chinensis bipolaris staurosporine J and preparation method and application thereof | |
| CN110423788B (en) | Method for producing grifola frondosa polysaccharide by using grifola frondosa strains generated by mutagenesis | |
| CN110172411B (en) | Xylaria cruzi strain ZJ1811 and culture method and application thereof | |
| CN108934769B (en) | Submerged fermentation medium for fomes ruditapes, and preparation and application methods thereof | |
| CN113563488A (en) | Preparation method of pharmaceutical-grade micromolecular marine organism polysaccharide | |
| Zhu et al. | Screening of Cordyceps strains and optimization of its solid-state fermentation conditions on bioconversion of Astragalus residue | |
| CN101270337A (en) | Method for improving productivity of cultivation of cell dioscorea opposita sapogenin by using peltate yam endogenetic oligose | |
| KR20030015504A (en) | Cultivation Method of mushroom mycelium using citrus juice and mushroom mycelium thereof | |
| CN1294151C (en) | Marine fungi polysaccharide and its extraction method and use | |
| CN113444646B (en) | Mould for producing beta-caryophyllene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |