CN114480136B - Preparation method and application of haemoglobin bacterial strain and mycelium polysaccharide - Google Patents
Preparation method and application of haemoglobin bacterial strain and mycelium polysaccharide Download PDFInfo
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- 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
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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Abstract
The invention belongs to the field of microbiology, and in particular relates to a preparation method and application of a haemoglobin bacterial strain and mycelium polysaccharide. The haemoglobin bacterium strain HZXS-G1 (Trametes sanguinea HZXS-G1) is preserved in China center for type culture Collection (CCTCC M2020079) with the preservation number of China, and has the preservation address: the university of marchant collection in the marchand district of marchant, hubei province. The haemoglobin bacterium HZXS-G1 in the invention can be used for producing mycelium polysaccharide by liquid fermentation, so that the haemoglobin bacterium HZXS-G1 in the invention can be used for producing polysaccharide, and a novel method is provided for polysaccharide production. The mycelium polysaccharide preparation method is simple, convenient, quick, efficient and low in cost, and is suitable for mass production of the mycelium polysaccharide of the haemoglobus. The polysaccharide prepared by the invention has a protective effect on heart of mice with myocardial damage induced by DOX, and can be developed into auxiliary drugs for treating malignant tumors by DOX.
Description
Technical Field
The invention belongs to the field of microbiology, and in particular relates to a preparation method and application of a haemoglobin bacterial strain and mycelium polysaccharide.
Background
The haemoglobin (Trametes sanguinea) belongs to fungus of Polyporaceae (Polyporaceae) and Trametes (Trametes), and is a medicinal fungus. The fruiting body of the plant is commonly grown on broad-leaved trees such as oak, maple, poplar, willow and the like and conifer dried and inverted trees such as pine and fir and the like, and is widely distributed in China. The haemoglobin has the medical values of clearing heat and detoxicating, dispelling wind and removing dampness, diminishing inflammation, stopping bleeding, resisting tumor and the like. The research shows that the haemoglobin extract has the effect of inhibiting cancer cells, and the inhibition rate of the haemoglobin extract on an S-180 sarcoma mouse is 72.4%. The fruiting body also contains porous fungus with antibacterial effect, and can be used for relieving inflammation.
Polysaccharides are important and abundant active ingredients in medicinal fungi, and are attracting attention due to their unique medicinal value. Research has shown that the medicinal fungus polysaccharide has the effects of regulating immunity, resisting oxidation, resisting aging, resisting tumor, reducing blood lipid, reducing blood sugar and the like. Researches show that the haemoglobin polysaccharide has the effects of inhibiting tumor growth, regulating immunity and the like. Experiments have shown that the bioactive substances contained in the fermented mycelium are similar to or have the same therapeutic effect as the bioactive substances contained in the fruiting body. The natural haemoglobin liquid fermentation by biotechnology can shorten the culture time and reduce the production cost, and has great development potential.
Doxorubicin (DOX), one of the most widely used chemotherapeutic drugs that are well known, is used to treat a variety of cancers. However, patients have serious side effects such as dose-dependent cardiotoxicity in the body after prolonged use of DOX, and there is no clinically effective oral drug to alleviate this drug toxicity. Therefore, DOX-induced cardiotoxicity has resulted in a major limitation in its clinical application. In recent years, prevention of DOX-induced cardiotoxicity or alleviation of DOX-induced cardiotoxicity has become a focus of research. Research shows that the medicinal fungus polysaccharide has the function of resisting myocardial cell injury and protecting cardiac vessels, such as ganoderan, etc. The invention separates and obtains a haemoglobin bacterial strain, and the mycelium polysaccharide prepared by fermenting the bacterial strain is found to have a protective effect on heart of mice with DOX-induced myocardial damage.
Disclosure of Invention
The invention aims to provide a medicinal fungus strain HZXS-G1, which belongs to a haemophilus strain HZXS-G1 (Trametes sanguineaHZXS-G1) and is separated from haemophilus fruiting bodies on dead wood by adopting a tissue separation technology.
The invention also aims to provide mycelium polysaccharide produced by fermentation of the haemoglobin bacterial strain, which has a protective effect on heart of a DOX-induced myocardial injury mouse.
The invention also aims at providing a method for producing polysaccharide by fermenting the haemoglobin by using the HZXS-G1 liquid.
In order to solve the technical problems, the invention adopts the following technical scheme: a medicinal fungus strain, classified and named as haemoglobus HZXS-G1 (Trametes sanguinea HZXS-G1), is preserved in China center for type culture Collection (CCTCC M2020079) with a preservation number of China, and has a preservation address: the university of martial arts collection, postal code, in the marchand district of the city of marchand in Hubei province: 430072.
The haemoglobin bacterium HZXS-G1 of the invention belongs to Basidiomycotina (Basidiomycotina), agaricus (Agaricomycetes), aphyllophorales (Aphyllophorales), polyporaceae (Polyporaceae) and Trametes (Trametes).
The haemoglobin bacterial strain HZXS-G1 is cultured on a potato solid medium at 28 ℃, the bacterial colony grows faster, the initial stage of mycelium is white short villus, the bacterial colony is tightly combined with the medium, the bacterial colony diameter is 7.0-8.0cm after the bacterial colony grows for 3-4 days, and the mycelium is bright orange. When observed under a microscope, the mycelium is developed, transparent and smooth, and has a lock-like combination.
The invention successfully amplifies the ITS region sequence of the haemoglobus HZXS-G1 by extracting the total DNA of the haemoglobus, and using the universal primers ITS1 and ITS4 as primers, wherein the amplified sequence is about 616bp. They were submitted to Shanghai Bioengineering Limited for sequencing. The ITS sequence obtained by sequencing is subjected to Blast homology comparison, and the homology of the HZXS-G1 sequence of the strain disclosed by the invention and the Trametes sanguine [ MN 258679] sequence is 99%. Combining morphological observations with phylogenetic tree analysis, the fungus was identified as Trametes sanguinea. Wherein, the ITS sequence of the fungus is shown as SEQ ID NO: 1.
The invention also discloses application of the haemoglobin by HZXS-G1 in the aspect of producing polysaccharide by liquid fermentation.
Wherein, the specific formula of the liquid fermentation medium is as follows: 200+ -50 g of potato, 20+ -5 g of glucose, 20+ -0.2 g of KH 2PO41.5±0.5g,MgSO4·7H2 O1+ -50 mL of water, preferably 200g of potato, 20g of glucose, 1g of KH 2PO41.5g,MgSO4·7H2 O and 1000mL of water.
The invention also discloses a method for producing mycelium polysaccharide by adopting the haemoglobin bacterium HZXS-G1 liquid fermentation, which comprises the steps of inoculating the haemoglobin bacterium HZXS-G1 to a solid culture medium for activation, and then inoculating the haemoglobin bacterium to a liquid fermentation culture medium for 7-30 days.
Wherein the solid culture medium is a potato solid culture medium, and the liquid culture medium comprises the following specific formula: 200g of potato, 20g of glucose, 1g of KH 2PO4 1.5g,MgSO4·7H2 O and 1000mL of water. The fermentation conditions are as follows: the temperature is 24-32 ℃ and the rotating speed is 0-180r/min.
Wherein, the optimal fermentation conditions are as follows: shake culturing at 28deg.C under 150r/min for 7 days, and standing culturing at 28deg.C for 7 days.
The preparation method of the mycelium polysaccharide comprises the following steps: filtering the fermentation broth of the trametes heme, washing with distilled water for 2-3 times to obtain mycelium, drying the mycelium at 50-60 ℃, pulverizing, and sieving with a 50-60 mesh sieve for standby; placing the crushed mycelium into a beaker, adding distilled water, extracting with ultrasonic wave at 70-80deg.C for 1-2 times, 45 min/time, mixing the two filtrates, concentrating to 1/5 volume, adding 2-3 times of 70-80% ethanol, precipitating with ethanol for 18-24h, centrifuging at 2000-3000r/min for 10-15min, discarding supernatant, and oven drying the precipitate to obtain crude polysaccharide of mycelium of fermentation of Haemophilus. Wherein, the fermentation liquid of the haemoglobus is filtered and washed by distilled water for three times to obtain mycelium. The mycelium is dried in a 60 ℃ oven, crushed by a crusher and sieved by a 60-mesh sieve for standby. 20g of the crushed mycelium is taken and put into a 1000mL beaker, 400mL of distilled water is added, and ultrasonic extraction is carried out for 2 times at 80 ℃ for 45 min/time. Mixing the filtrates, concentrating to 1/5 volume in rotary evaporator, adding 3 times of 80% ethanol, precipitating with ethanol for 24 hr, centrifuging at 3000r/min for 10min, removing supernatant, and oven drying the precipitate in electric vacuum oven at 60deg.C to obtain crude polysaccharide (TSLFACP) of fermentation mycelium of Xylella.
Wherein, MTT method is used to detect TSLFACP effect on iHUVEC cell and CCC-HEH cell activity. Results iHUVEC cells treated for 24h with different concentrations TSLFACP showed a significant increase in viability; after 24h of 2.5 mu MDOX treatment, the activity of CCC-HEH cells is obviously reduced, and after 24h of 25 mu g/MLTSLFACP treatment, the activity of CCC-HEH cells is obviously increased.
Wherein, 18 male Balb/c mice were randomly divided into three groups: blank, DOX, DOX+ TSLFACP, in vivo mouse myocardial injury model was established, changes in body weight of mice before and after dosing were recorded, and three groups of heart tissue section morphological changes were observed by hematoxylin eosin staining. Results the body weight was significantly reduced in the DOX and DOX + TSLFACP groups compared to the Control group. Compared with the Control group, the DOX treatment group myocardial tissue has obvious unclear texture, shrinkage and plasma dissolution; and the DOX+ TSLFACP treatment group myocardial cells are orderly arranged, clear in texture and smaller in shrinkage range.
Wherein, the serum central muscle injury index myocardial troponin I (CTNI) content of mice in blank group, DOX group and DOX+ TSLFACP group is detected by adopting an ELISA method. Results the level of myocardial injury index CTNI was significantly reduced in the DOX + TSLFACP treated group compared to the DOX group.
The beneficial effects are that: compared with the prior art, the invention has the advantages that: the haemoglobin bacterium HZXS-G1 in the invention can be used for producing mycelium polysaccharide by liquid fermentation, so that the haemoglobin bacterium HZXS-G1 in the invention can be used for producing polysaccharide, and a novel method is provided for polysaccharide production. The mycelium polysaccharide preparation method is simple, convenient, quick, efficient and low in cost, and is suitable for mass production of the mycelium polysaccharide of the haemoglobus. The polysaccharide prepared by the invention has a protective effect on heart of mice with myocardial damage induced by DOX, and can be developed into auxiliary drugs for treating malignant tumors by DOX.
Drawings
FIG. 1 shows colony morphology and microscopic morphology of the haemoglobus HZXS-G1 (a: plate colony morphology; b: microscopic morphology)
FIG. 2 shows a phylogenetic tree (constructed by adopting the adjacent method) of the haemoglobin string HZXS-G1 based on the ITS sequence
FIG. 3 shows the preparation of a fermentation broth of Xemollients HZXS-G1 (a: fermentation broth in a shake flask; b: mycelium obtained by filtration)
FIG. 4 is a graph showing the effect of Xemollients HZXS-G1 mycelium polysaccharides on iHUVEC and CCC-HEH cell viability (A: effect of different concentrations TSLFACP on CCC-HEH cell viability; B: TSLFACP on CCC-HEH cell viability after DOX pretreatment .*P or#P<0.05,**P or##P<0.01,***Por###P<0.001,*vs.Control group, # vs. DOX group.)
FIG. 5 shows the effect of trametes rubra HZXS-G1 mycelium polysaccharide on myocardial injury in mice (A: weight change in three groups of mice before and after administration; B: HE staining pattern of heart tissue sections of three groups of mice. *P<0.05,**P<0.01,***P<0.001,* vs. control group.)
FIG. 6 shows the CTNI levels (*P or#P<0.05,**P or##P<0.01,***P or###P<0.001,*vs.Control, # vs. DOX in the serum of each group of mice. )
Detailed Description
Example 1: separation of haemoglobus HZXS-G1
In this example, the haemoglobin fungus HZXS-G1 was isolated from healthy haemoglobin fungus fruiting bodies. Experimental materials were collected from a rotten wood of a forest park in the western mountain country of hangzhou in 3 months of 2019. The separation is carried out according to the following steps:
Healthy and fresh wild hemoglobus fruit bodies are taken and washed clean by distilled water. Sterilizing with 75% alcohol for 1min, cleaning with sterile water for 3 times, sterilizing with 3% sodium hypochlorite solution for 1min, and washing with sterile water for 3 times. Cutting fruiting body into small pieces under aseptic condition, implanting onto PDA plate, and culturing in incubator at 28deg.C for 3-7d. Sterile water for the last washing of the samples was applied to the PDA plates as a control, incubated under the same conditions, and observed to ensure thorough surface disinfection. And (3) observing the growing condition of fungi at regular time every day, after hyphae grow out, picking the tip part of the hyphae, transferring the tip part to a fresh PDA plate, and purifying and culturing for multiple times until pure strains are obtained.
Potato solid medium (PDA): 200g of peeled potato pieces were weighed, 500mL of water was added thereto, and after boiling for 30 minutes, the potato pieces were filtered with 4 layers of gauze. 20g of glucose and 20g of agar are added into the filtrate, water is added to 1000mL after heating and dissolving, and the pH is natural. Sterilizing with steam at 121deg.C for 20min.
Example 2: identification of the haemoglobin HZXS-G1 Strain
1. Strain plate morphology observation
The strain of the present invention was inoculated onto a PDA plate, cultured in an incubator at 28℃and the morphology of colonies was observed.
Referring to FIG. 1, a colony morphology diagram of the haemoglobin of the invention HZXS-G1 is shown, wherein the colony morphology of the haemoglobin HZXS-G1 is: the strain grows faster on the PDA plate, the mycelium is white short villus at the initial stage, the mycelium is tightly combined with the culture medium, the colony diameter is 7.0-8.0cm after the strain grows for 3-4 days, and the mycelium shows bright orange. When observed under a microscope, the mycelium is developed, transparent and smooth, and has a lock-like combination.
2. Microscopic morphology observation of strains
The microscopic morphology of the strain of the invention, xylella HZXS-G1, was observed by the insert method.
Referring to FIG. 3, a microscopic morphology of the haemoglobus HZXS-G1 of the present invention is shown in FIG. 3, wherein the microscopic morphology (10X 40 high-power microscope) of the haemoglobus HZXS-G1 is: the mycelium is colorless and transparent, branches, developed, and has a lock-shaped combination.
3. Molecular biological identification of strains
(1) Extraction of DNA
Mycelium was collected and fermented in liquid medium for 7 days. After the mycelium was ground with liquid nitrogen, total DNA was extracted with a fungal genome DNA extraction kit (Shanghai Biotechnology Co., ltd.).
(2) PCR amplification of ITS region sequences
The primer sequences are as follows: ITS1 (5'-TCCGTAGGTGAACCTGCGC-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3').
The PCR reaction conditions were: preheating for 5min at 94 ℃, denaturing for 1min at 94 ℃, annealing for 40s at 55 ℃ and extending for 50s at 72 ℃ for 30 cycles; extending at 72℃for 10min. The PCR amplification products were sequenced by Biotechnology (Shanghai) Inc.
The base sequence of the haemoglobin ITS is shown in the appendix (SEQ ID NO: 1).
(3) Data processing
Sequence data were aligned for homology searches in Genbank of NCBI and analyzed for homology. The results show that: the homology of the HZXS-G1 sequence of the strain of the invention with Trametes sanguine [ MN 258679] sequence is 99%. Combining morphological observations with phylogenetic tree analysis, the fungus was identified as Trametes sanguinea. Please refer to fig. 2, which constructs phylogenetic tree of strain HZXS-G1 using MEGA11.0 software.
And combining the morphological classification identification result and the molecular biological classification identification result to finally identify the strain as the haemoglobus HZXS-G1 (Trametes sanguine HZXS-G1).
Example 3: preparation of haemoglobin by means of HZXS-G1 fermentation
The strain of the present invention, xemollients HZXS-G1, was inoculated onto PDA plates and cultured at 28℃for 5d to activate the strain. 1 bacterial block with the diameter of 6mm is inoculated into a triangular flask (500 mL) containing 150mL of liquid fermentation medium, shake fermentation culture is carried out for 7d at the temperature of 28 ℃ and the speed of 150r/min, and then standing fermentation culture is carried out for 7d at the temperature of 28 ℃. Filtering the fermentation liquor with four layers of gauze, and obtaining filter residues which are the fermentation mycelium of the haemoglobus hystericus HZXS-G1. FIG. 3 shows the results of shake flask fermentation of the present invention with the trametes at HZXS-G1 and the mycelium obtained.
The formula of the liquid fermentation medium is as follows: 200g of potato, 20g of glucose, 1g of KH 2PO4 1.5g,MgSO4·7H2 O and 1000mL of water.
Example 4: preparation of polysaccharide from mycelium of haemoglobin
Filtering the fermentation liquid of the trametes, and washing with distilled water for three times to obtain mycelium. The mycelium is dried in a 60 ℃ oven, crushed by a crusher and sieved by a 60-mesh sieve for standby. 20g of the crushed mycelium is taken and put into a 1000mL beaker, 400mL of distilled water is added, and ultrasonic extraction is carried out for 2 times at 80 ℃ for 45 min/time. Mixing the filtrates, concentrating to 1/5 volume in rotary evaporator, adding 3 times of 80% ethanol, precipitating with ethanol for 24 hr, centrifuging at 3000r/min for 10min, removing supernatant, and oven drying at 60deg.C in electric vacuum oven to obtain TSLFACP.
Example 5: TSLFACP Effect on endothelial cell proliferation Activity
IHUVEC and CCC-HEH cells were resuscitated, inoculated into DMEM medium containing 10% FBS and 0.1% diabody, cultured to logarithmic phase at 37deg.C in 5% CO 2 incubator, collected by digestion centrifugation, inoculated into 96 well plates, and cultured in 5% CO 2 incubator at 37deg.C. iHUVEC cells set up control and dosing groups (TSLFACP, 20, 50, 100, 200 μg/ml), 4 duplicate wells; CCC-HEH cells were set up in control, model (2.5. Mu.M DOX pretreatment for 4 h), dosing (25. Mu.g/ML TSLFACP) and 4 replicate wells. After 24h of incubation, 20. Mu.L of MTT solution with a concentration of 5 g.L －1 was added to each well, incubation was terminated for 4h in an incubator, 200. Mu.L of the triple solution was added to each well, and the wells were incubated at 37℃overnight, and absorbance values at 570nm were measured for each well using an ELISA reader.
Experimental detection shows that iHUVEC cells treated for 24h with different concentrations TSLFACP have significantly increased viability (P < 0.001), and the results show that TSLFACP has the effect of promoting endothelial cell growth, and that CCC-HEH cell viability is significantly reduced after 24h treatment with 2.5 mu M DOX, and CCC-HEH cell viability is significantly increased after 24h treatment with 25 mu g/ML TSLFACP, as shown in FIG. 4A.
Example 6: TSLFACP protective action on myocardial injury of mice
1. Model construction and drug treatment
The 18 male Balb/c mice were randomly divided into three groups: control group, DOX group, DOX+ TSLFACP group. Control group animals were intraperitoneally injected with 100. Mu.L of 0.9% saline and perfused with 200. Mu.L of ddH 2 O; the DOX group animals were intraperitoneally injected with 100. Mu.L DOX (15 mg/kg, formulated with 0.9% saline) and lavaged with 200. Mu LddH 2 O; animals in the DOX+ TSLFACP group were intraperitoneally injected with 100. Mu. LDOX and lavaged with 200. Mu. L TSLFACP (200 mg/kg, formulated with ddH 2 O). Three groups were treated continuously for 8 days. After 8 days, blood samples were collected from all animals rapidly and the animals were sacrificed to obtain heart samples rapidly and centrifuged to obtain serum samples.
2. Heart histopathology
Taking mouse heart tissue fixed in formalin, dehydrating with gradient ethanol, adding dimethylbenzene for treatment for 60min, immersing in paraffin for 3h, cutting into slices with the thickness of 3-5 mu m, pasting the slices on a glass slide, baking the slices at 62 ℃ for 120min, dewaxing and hydrating with the dimethylbenzene and the gradient ethanol, dyeing with an HE kit, dehydrating the slices in the gradient ethanol after dyeing, placing the slices in the dimethylbenzene for transparentization, sealing the slices with neutral resin, and observing the heart histopathological change of the mouse under a microscope.
3. Detection of CTNI levels in mouse serum
Mouse serum is taken, the operation is carried out according to the instruction of an ELISA kit, the absorbance is measured at 450nm, and the CTNI content in the mouse serum is calculated.
Referring to fig. 5A, the DOX and dox+ TSLFACP groups had significantly reduced body weight compared to the Control group. As shown in fig. 5B, the DOX treated myocardial tissue showed significant texture, shrinkage and plasmatic dissolution compared to the Control group; and the DOX+ TSLFACP treatment group myocardial cells are orderly arranged, clear in texture and smaller in shrinkage range.
Referring to fig. 6, the DOX treatment group showed significantly increased level of myocardial injury index CTNI compared to Control group; the level of myocardial injury index CTNI was significantly reduced in the DOX + TSLFACP treated group compared to the DOX group.
Sequence listing
<110> Zhejiang university of Tree people (Zhejiang college of Tree people)
<120> Preparation method and application of haemoglobin bacterial strain and mycelium polysaccharide
<130> 2021
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 616
<212> DNA
<213> Hemoglobinula (Trametes sanguinea)
<400> 1
aaaccaaaat cggaaacgag tgatttgagg tcatatgtgg gaggttgtcc catacgggac 60
ggttagaagc tcgccaaacg cttcagggtc acagcgtaga caattatcac actgagagcc 120
gatccgcacg gaatcaagct aatgcattca agaggagccg accgacgagg gccagcaagc 180
ctccaagtcc aagcccacag catcacaagg acgtgtgggt tgagaattcc atgacactca 240
aacaggcatg ctcctcggaa taccaaggag cgcaaggtgc gttcaaagat tcgatgattc 300
actgaattct gcaattcaca ttacttatcg catttcactg cgttcttcat cgatgcgaga 360
gccaagagat ccgttgctga aagttgtatt tagatgcgtt agacgctaat acattctgtt 420
actttatgtg tttgtagtga tacataggcc ggcagaatgc ctcaaagacc cggaggcccc 480
gaagcccacg ccaaacctac agtaagtgca caggtgtaga gtggatgagc agggtgtgca 540
catgccccgg aaggccagct acaacccctt tcagaactcg ttaatgatcc ttccgcaggt 600
tcacctacgg aagaag 616
Claims (2)
1. An application of a haemoglobin bacterial strain HZXS-G1 in the aspect of liquid fermentation production of mycelium polysaccharide, wherein the bacterial strain has a preservation number of CCTCC M2020079, a preservation date of 2020, 5 months and 9 days, a preservation unit of China center for type culture collection, and a preservation address of China center for type culture Collection in Wuhan, china center for type culture collection, and the method for liquid fermentation production of mycelium polysaccharide is characterized by comprising the following steps: inoculating the strain to a solid culture medium for activation, then inoculating to a liquid fermentation culture medium for culturing for 7-30 days, collecting mycelia, and extracting and separating the mycelia to obtain mycelia polysaccharide; the solid culture medium is a potato solid culture medium, and the specific formula of the liquid culture medium is as follows: 200+/-50 g of potato, 20+/-5 g of glucose, 20+/-5 g of KH 2PO4 1.5±0.5 g,MgSO4·7H2 O1+/-0.2 g and 1000+/-50 mL of water, wherein the liquid fermentation conditions are as follows: shake culturing at 28deg.C and 150 r/min for 7 days, standing at 28deg.C for 7 days, filtering the fermentation broth, washing with distilled water for 2-3 times to obtain mycelium, oven drying at 50-60deg.C, pulverizing, and sieving with 50-60 mesh sieve; placing the crushed bacterial body into a beaker, adding distilled water, extracting for 1-2 times at 70-80 ℃ by ultrasonic wave, 45-min times, combining the two filtrates, concentrating to 1/5 volume, adding 2-3 times of 70-80% ethanol, precipitating with ethanol for 18-24 h, centrifuging for 10-15 min at 2000-3000 r/min, discarding supernatant, and drying the obtained precipitate to obtain the crude polysaccharide of the fermentation mycelium of the trametes hemsleyanum.
2. Use of the mycelium polysaccharide produced by liquid fermentation of the strain of claim 1 in the preparation of a heart medicament for protecting against DOX-induced myocardial damage.
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