CN110447457B - Haematococcus strain and artificial cultivation method and application thereof - Google Patents
Haematococcus strain and artificial cultivation method and application thereof Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
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- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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- A—HUMAN NECESSITIES
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- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention relates to a strain and an artificial cultivation method and application thereof, in particular to a pycnoporus sanguineus strain and an artificial cultivation method and application thereof. The Pycnoporus sanguineus strain is collected from Fujian Wuyi mountain, is identified as Pycnoporus sanguineus, is subjected to tissue separation to obtain an original strain, is named as Pycnoporus sanguineus (HMGIM-140415), is preserved in the China center for type culture collection (CCTCC NO) in 7-3.2019, and has the preservation number of CCTCC NO: m2019514. The strain of the invention has been artificially domesticated and cultivated, and compared with other strains of the same species, the strain shows stronger and more remarkable inhibition rate to tumors and staphylococcus.
Description
Technical Field
The invention relates to a strain and an artificial cultivation method and application thereof, in particular to a pycnoporus sanguineus strain and an artificial cultivation method and application thereof.
Background
At present, the industry of edible and medicinal fungi is rapidly developed, according to statistics of the edible fungi association in China, the yield of the edible and medicinal fungi in China in 2017 reaches 3712 ten thousand tons, the yield is increased by 3.21% compared with 2016, the yield is 2721.92 hundred million yuan, China accounts for more than 75% of the world, practitioners exceed 2000 thousand people, and the fifth place of the edible fungi industry excluding grains, vegetables, fruits and oil in the planting industry exceeds tea leaves and silkworm.
Nowadays, as the edible and medicinal fungi industry develops vigorously, more and more rare edible and medicinal fungi varieties gradually enter the visual field of people, and a plurality of original rare varieties are gradually domesticated, such as dictyophora phalloidea, agrocybe chaxingu, Lyophyllum and morel. However, there are also a large number of wild edible and medicinal fungi which have not been studied because they have not been recognized by human beings. According to research, about more than 300 ten thousand fungus species are existed in the world at present, only 1% of the species are known, wherein about 14000 large fungi are known, 1789 edible fungi and 798 medicinal fungi are recognized in China, and only less than 100 wild edible and medicinal fungi are domesticated by human beings, and the variety of large-scale cultivation is more than 30. There is a considerable road for the study and utilization of human beings from large fungi. With the gradual rise of living standard of people, the requirements on the quality of life are higher, and the macrofungi have very good effects on human health due to the fact that the macrofungi are rich in various components with nutrition and functional effects, including fungal polysaccharides, triterpenes, sterols and the like, and are increasingly paid attention to by people.
The dense red fungus is widely distributed, and is usually collected in summer and autumn, and then dried. The fungus has high medicinal value, and can relieve itching, stop bleeding, promote granulation, and promote qi circulation, and its fruiting body contains polyporin which has inhibitory effect on gram negative and positive bacteria, and can also be used for relieving inflammation.
Since trametes sanguinea has wide economic application and obvious medicinal value, more and more people develop and utilize the trametes sanguinea, but no report of large-scale cultivation and application exists so far.
Disclosure of Invention
In view of the above disadvantages, the invention provides a wild rare medicinal fungus strain Pycnoporus sanguineus strain with in vitro anti-tumor activity and bacteriostatic action, and an artificial domestication cultivation method and application thereof.
The invention achieves the above purposes through the following scheme:
in a first aspect, the Pycnoporus sanguineus strain is collected from Wuyi mountain in Fujian province, is identified as Pycnoporus sanguineus, is subjected to tissue separation to obtain an original strain, is named as Pycnoporus sanguineus (HMGIM-140415), is preserved in China center for type culture Collection (CCTCC for short, with the address: Wuhan, China) in 7-3 months in 2019, and has the preservation number of CCTCC NO: m2019514.
In a second aspect, the invention provides an artificial cultivation method of Pycnoporus sanguineus (CCTCC NO: M2019514), which comprises the steps of preparing a mother strain, preparing a production strain, cultivating, culturing and managing, wherein the cultivation material comprises, by weight, 37-41% of mixed wood chips, 18-20% of cottonseed hulls, 18-20% of corncobs, 16-18% of wheat bran, 2-3% of corn flour and 1-2% of lime.
In a third aspect, the invention provides application of a pycnoporus sanguineus strain CCTCC NO: M2019514 or an extract thereof in resisting tumors or related diseases caused by bacteria.
In a fourth aspect, the invention provides an application of a haemophilus haemolyticus strain CCTCC NO: M2019514 or an extract thereof in preparing a medicament for resisting tumors or related diseases caused by bacteria.
In a fifth aspect, the invention provides a medicament for resisting tumors or related diseases caused by bacteria, which comprises the strain of densefruit pittosporum red bacterial strain CCTCC NO: m2019514 or an extract thereof and a carrier.
The strain of the invention has been artificially domesticated and cultivated, and compared with other strains of the same species, the strain shows stronger and more remarkable inhibition rate to tumors and staphylococcus.
Drawings
FIG. 1 is a diagram of a wild fruit body of Pycnoporus sanguineus of example 1.
FIG. 2 is a diagram showing the fruit body of artificially acclimated Pycnoporus sanguineus of example 2.
FIG. 3 is a diagram showing the fruit body of artificially acclimated Pycnoporus sanguineus of example 2.
Fig. 4 is the ITS sequence of example 1.
FIG. 5 is a schematic drawing of a streaking culture section of example 4.
FIG. 6 is a schematic diagram of Staphylococcus aureus-inhibiting bacteria of example 4.
Detailed Description
In a first aspect, the Pycnoporus sanguineus strain is collected from Wuyi mountain in Fujian province, is identified as Pycnoporus sanguineus, is subjected to tissue separation to obtain an original strain, is named as Pycnoporus sanguineus (HMGIM-140415), is preserved in China center for type culture Collection (CCTCC for short, with the address: Wuhan, China) in 7-3 months in 2019, and has the preservation number of CCTCC NO: m2019514.
The collected original strain is subjected to genome DNA extraction and ITS sequencing, the sequencing result is subjected to sequence Blast in GenBank, the similarity with the Pycnoporus sanguineus (L.) Murrill is found to be up to nearly 100%, and the macroscopic characteristics and the microscopic characteristics of the fungus specimen are consistent with the description of Pycnoporus sanguineus, and the identification result is Pycnoporus sanguineus combined with morphological identification.
The Pycnoporus sanguineus belongs to the order of Polyporales, the family of Polyporaceae, is the current name of Trametes sanguinea, the fruiting body is annual, the quality of leather, the pileus is fan-shaped, semicircular or kidney-shaped, the outward extension can reach 3cm, the width can reach 5cm, and the base part can reach 1.5 cm; the color of the material is changed from light reddish brown, rusty brown to yellow brown when the surface is fresh, and the color is faded in the later period, and is almost unchanged after the material is dried; sharp edges, lighter colors, and sometimes wavy. When the surface of the opening is fresh, the brick is red, and the color is almost unchanged after the opening is dried; the shape is approximate to a circle, and 5-6 pieces are used per millimeter; thin edge, full edge. The sterile edge is obvious, the apricot is yellow, and the width can reach 1 mm. The mushroom flesh is light red brown and the thickness can reach 13 mm. The fungus tube is reddish brown and can reach 2mm in length. Basidiospores are 3.6-4.4 multiplied by 1.7-2 mu m, are oblong to cylindrical, colorless, thin-walled, smooth, non-starchy and non-bluish. The single growth or the clustering growth in summer and autumn on the inverted wood, stump and rotten wood of various broad-leaved trees causes white rotten wood. Is used for medicine. All the zones are distributed.
In a second aspect, the invention provides an artificial cultivation method of Pycnoporus sanguineus (CCTCC NO: M2019514), which comprises the steps of preparing a mother strain, preparing a production strain, cultivating, culturing and managing, wherein the cultivation material comprises, by weight, 37-41% of mixed wood chips, 18-20% of cottonseed hulls, 18-20% of corncobs, 16-18% of wheat bran, 2-3% of corn flour and 1-2% of lime.
Preferably, the material-water ratio of the cultivation material is 1: 1.1-1.3.
Preferably, the cultivation material comprises 39% of mixed wood chips, 20% of cottonseed hulls, 20% of corncobs, 17% of wheat bran, 3% of corn flour and 1% of lime in percentage by weight, and the material-water ratio is as follows: 1:1.1-1.3.
Preferably, the cultivation comprises: transferring the production seeds into a cultivation material, culturing at a constant temperature of 25-26 ℃ in shade, and allowing mycelia to grow over the fungus bags with humidity of 60-70%, and performing cultivation management.
Preferably, the cultivation management comprises: continuously shading and post-ripening for 15 days after the hypha in the cultivation bag grows over the cultivation material in the bag, entering a fruiting stage, controlling the temperature at 23-27 ℃, increasing the ventilation volume, keeping the carbon dioxide content in the space below 1%, adjusting the relative air humidity to be above 90%, and removing the pileus after 5-7 days, so that the hypha is twisted and forms bright red rice-shaped primordium; after the primordium grows to 0.5cm, continuously controlling the temperature between 23 ℃ and 27 ℃ and the relative air humidity between 85% and 90%, illuminating for 9 hours every day with the illumination intensity of 300-.
Preferably, the preparing the mother seeds comprises: transferring the separated strain to a mother culture medium, performing dark culture at a constant temperature of 25 ℃, and picking tip hyphae when the hyphae grow but the bacteria do not grow to obtain a mother strain.
Preferably, the mother culture medium is a Bengal red culture medium.
Further preferably, the culture medium of Bengal red comprises, in weight percent: peptone 0.5%, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO4 & 7H2O) 0.05%, agar 2%, 1/3000 Bengal solution 10%, chloramphenicol 0.01%, and water in balance.
Preferably, the making and producing mother seeds comprises: transferring the mother strain to a culture medium for producing the mother strain, culturing at 25 deg.C in dark at constant temperature, and allowing mycelia to grow over the slant to obtain the production mother strain.
Preferably, the production mother culture medium is enriched comprehensive PDA.
Further preferably, the enriching comprehensive PDA comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1% of peptone, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate, trace vitamin B1 and the balance of water.
Preferably, the manufacturing process includes: inoculating production mother seed into the culture medium under sterile condition, embedding the material block of the production mother seed into the raw material, culturing at 25 deg.C under dark condition, and collecting the production seed after the hypha is full of the material.
Preferably, the production seed culture medium comprises, in weight percent: 98-99% of sorghum and 1-2% of calcium carbonate.
Further preferably, the production seed culture medium comprises, in weight percent: 98% sorghum and 2% calcium carbonate.
Preferably, the artificial cultivation method further comprises tissue isolation of the strain before the production of the mother strain.
An artificial culture method of dense red blood (Pycnoporus sanguineus) CCTCC NO: M2019514 comprises the steps of preparing a mother strain after strain tissue separation, preparing a production mother strain, preparing a production strain, and carrying out culture and culture management, wherein the culture material comprises, by weight, 37-41% of mixed wood chips, 18-20% of cottonseed hulls, 18-20% of corncobs, 16-18% of wheat bran, 2-3% of corn flour and 1-2% of lime.
Preferably, the tissue isolated species comprises: collecting the fruiting body of Fomitopsis sanguinea, wiping the surface with alcohol under aseptic condition, tearing, inoculating 0.2-0.5mm × 0.2-0.5mm inner mushroom flesh tissue in aseptic operation, culturing at 25 deg.C in dark at constant temperature, and collecting the separated strain after the mycelia grow over the inclined plane.
Preferably, the tissue isolation medium is an integrated PDA medium.
Further preferably, the comprehensive PDA culture medium comprises 20% of potatoes, 2% of glucose, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate and trace vitamin B1 in percentage by weight.
In a third aspect, the invention provides application of a pycnoporus sanguineus strain CCTCC NO: M2019514 or an extract thereof in resisting tumors or related diseases caused by bacteria.
Preferably, the extract is an ethyl acetate extract.
Preferably, the tumor is a tumor caused by HepG2 cells.
Preferably, the bacterium is staphylococcus, and more preferably staphylococcus aureus.
In a fourth aspect, the invention provides an application of a haemophilus haemolyticus strain CCTCC NO: M2019514 or an extract thereof in preparing a medicament for resisting tumors or related diseases caused by bacteria.
Preferably, the extract is an ethyl acetate extract.
Preferably, the tumor is a tumor caused by HepG2 cells.
Preferably, the bacterium is staphylococcus, and more preferably staphylococcus aureus.
In a fifth aspect, the invention provides a medicament for resisting tumors or related diseases caused by bacteria, which comprises a haemophilus haemolyticus strain CCTCC NO: M2019514 or an extract and a carrier.
Preferably, the extract is an ethyl acetate extract.
Preferably, the tumor is a tumor caused by HepG2 cells.
Preferably, the bacterium is staphylococcus, and more preferably staphylococcus aureus.
The present invention is further illustrated by the following specific examples.
Example 1:
identification of Pycnoporus sanguineus (L.) Murrill
A sample of dense red fungus (preliminary identification) was collected from Liu Yuan super and Huang Shi Yong in Fujian Wu mountain, as shown in FIG. 1. Obtaining a PDA pure culture by a tissue isolation method, collecting hyphae by liquid culture, drying at low temperature (40 ℃), grinding by using liquid nitrogen, extracting DNA genome by using an Ezup column type fungus genome DNA extraction kit, and refrigerating the obtained DNA solution at-20 ℃ for later use.
The ITS-PCR experiment of the material was performed by using fungal ribosomal intergenic region universal primer ITS1/ITS4(ITS1: TCCGTAGGTGAACCTGCGG, ITS4: TCCTCCGCTTATTGATATGC, synthesized by Biotechnology, Inc., Shanghai), amplification was performed on a Biometra PCR instrument, and the composition of the PCR reaction solution (50. mu.l in total) was:
TaKaRaTaq(5units/μl) 0.25μl
10×PCR Buffer 5μl
dNTP mix (2.5 mM each) 4. mu.l
DNA template 2. mu.l
Primer 1 (10. mu. mol. L-1) 5. mu.l
Primer 2 (10. mu. mol. L-1) 5. mu.l
Sterilized distilled water 28.75. mu.l
The reaction conditions are that the reaction is carried out for 5min at 94 ℃; reacting at 94 ℃ for 1min, at 55 ℃ for 1min, at 72 ℃ for 1min, and performing 30 cycles; and (3) reacting at 72 ℃ for 10min, and directly detecting a PCR product to perform bidirectional sequencing, wherein the bidirectional sequencing is completed by the Huada gene.
The ITS sequences obtained by sequencing are shown in FIG. 4.
Sequence Blast was performed on GenBank as a result of sequencing of fig. 4, and it was found that the similarity to the Pycnoporus sanguineus (L.) Murrill was as high as nearly 100%, and the macroscopic and microscopic characteristics of the fungal specimen were consistent with those described for Pycnoporus sanguineus, in combination with morphological identification, and the result was Pycnoporus sanguineus. The strain E140415 is preserved in the China center for type culture Collection (address: Wuhan, China) in 2019 and 7 months, and the preservation number is CCTCC NO: m2019514.
Example 2
Firstly, a culture medium (in percentage by weight):
1. tissue isolation medium (comprehensive PDA):
20% of potato, 2% of glucose, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate, a trace amount of vitamin B1 and the balance of water.
2. Purified stock culture medium (menglar red medium):
peptone 0.5%, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO4 & 7H2O) 0.05%, agar 2%, 1/3000 Bengal solution 10%, chloramphenicol 0.01%, and water in balance.
3. Production mother culture medium (enriched integrated PDA):
20% of potato, 2% of glucose, 1% of peptone, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate, trace vitamin B1 and the balance of water.
4. Production of seed culture medium:
98-99% of sorghum and 1-2% of calcium carbonate.
5. Cultivation material:
39% of mixed wood dust, 20% of cottonseed hulls, 20% of corncobs, 17% of wheat bran, 3% of corn flour and 1% of lime, wherein the material-water ratio is as follows: 1:1.1-1.3.
Secondly, the method comprises the following steps:
1. tissue isolation of strains:
preparing tissue isolation culture medium, subpackaging test tubes, performing moist heat sterilization at 0.11MPa atmospheric pressure and 121 deg.C high temperature and high pressure for 30min, taking out, cooling, and placing into inclined plane. Collecting wild fasciculate sporophore, wiping surface with 75% alcohol under aseptic condition, tearing, and aseptically inoculating 0.2-0.5mm × 0.2-0.5mm inner mushroom flesh tissue. Culturing in 25 deg.C incubator at constant temperature in dark condition, and transferring after mycelia grow over the inclined plane for 10-15 days.
2. Preparing a purified mother strain:
preparing a purified culture medium according to the formula, subpackaging test tubes, performing moist heat sterilization at the atmospheric pressure of 0.11MPa and the high temperature of 121 ℃ for 30min, and transferring the separated bacteria infected strains. Placing in an incubator at 25 deg.C, dark culturing at constant temperature, picking and transferring tip mycelium when mycelium grows and bacteria do not grow to obtain purified mother strain.
3. Preparing a production mother strain:
preparing a culture medium for producing mother seeds, subpackaging test tubes, performing moist heat sterilization at the atmospheric pressure of 0.11MPa and the high temperature of 121 ℃ for 30min, taking out, cooling and performing aseptic operation, and inoculating the purified mother seeds which are successfully separated. Culturing in 25 deg.C incubator at constant temperature in dark condition, and inoculating the strain after mycelia grow on the slant. The time for the production mother seed to grow is between 15 days and 20 days.
4. Production of seeds
Weighing sorghum according to a required proportion, soaking the sorghum overnight in water, mixing the sorghum with calcium carbonate according to a proportion, putting the mixture into a 250ml conical flask, converting the content of dry materials in each flask into 150g of dry materials, obtaining a production seed culture medium, sealing the culture medium by using a silica gel plug, carrying out moist heat sterilization at the atmospheric pressure of 0.147MPa and the high temperature of 128 ℃ for 90min, taking out the culture medium, cooling, shaking the culture medium, and inoculating the culture medium into a production mother seed in an aseptic operation. And ensuring that a production mother material block is buried in the production seed material during inoculation. Culturing at 25 deg.C in incubator at constant temperature, and inoculating into cultivation bag after mycelium is full of material (about 20 days).
5. Cultivation of plants
Weighing the artificial domestication culture medium, taking the culture material in required proportion, fully mixing, adding water (the water content is 55-65%), and filling into a 17cm × 35cm high-temperature-resistant transparent polypropylene strain bag. 400-420g of dry materials in each bag. After the materials are filled, a small wood bar is used for punching a hole in the bag materials, the hole is deep to the bottom of the bag, then a plastic ring is sleeved on the bag opening, and a matched cover is buckled, so that the finished cultivation material bag is obtained. Performing moist heat sterilization at the atmospheric pressure of 0.147MPa and the high temperature and the high pressure of 128 ℃ for 90min, taking out, cooling, performing sterile operation, and inoculating the seeds. When in inoculation, the material block is ensured to be embedded in the cultivation material. Culturing at 25 + -1 deg.C and air relative humidity of 60-70% in dark place. After the hypha is full of the material (about 18 days), the cultivation management can be carried out.
6. Cultivation management (including after ripening management, primordium formation, fruiting body growth)
(1) Management of after ripening
After the cultivation material in the cultivation bag is fully grown by hypha in the cultivation bag, continuously shading and post-ripening for 15 days, and entering a fruiting stage.
(2) Formation of primordia
Controlling the temperature at 23-27 deg.C, increasing ventilation rate, keeping carbon dioxide content in space below 1%, adjusting air relative humidity to above 90%, removing pileus after 5-7 days, and arranging the cultivation bags horizontally (gaps should be left between the bags), at which time the hypha starts to kink and form bright red rice-shaped primordium. The relative humidity is maintained at this time without spraying water directly onto the original substrate.
(3) Growth period of fruiting body
After the primordium grows to 0.5cm, continuously controlling the temperature between 23 ℃ and 27 ℃ and the relative air humidity between 85% and 90%, and illuminating for 9 hours every day with the illumination intensity of 300-. After about 30 days, the fruit body is completely mature. During the period, spraying water mist to the young mushroom for 1-2 times every day until the size of the fruiting body is basically unchanged, indicating that the fruiting body is mature, and then harvesting. About 35 days passes from the growth of primordia to the maturation of fruiting bodies. As shown in fig. 2 and 3.
Third, fruiting status
1. And (3) fruiting period: the fruiting period of the variety is 81 days, and the variety is picked as a tide.
2. Yield: each bag has fruiting rate of 9.87-13.69 g, average bag yield of 11.99 g, and biotransformation rate of about 3.43%.
3. And (3) fruiting body properties: the fruit body is in fan shape or Ruyi shape, and is bright red after stacking to single growth.
4. Compared with the wild state, the fruiting body individuals of the variety are increased by 2-3 times after artificial domestication, the appearance is round, and the yield is higher.
Example 3 inhibition of tumor cell function
First, experiment method
1. Preparation of ethyl acetate extract
The trametes sanguinea strain is subjected to solid fermentation, and the culture medium is rice and water. Soaking rice culture medium full of mycelia in ethyl acetate for 10 hr after culturing for 45 days, ultrasonically extracting for 100min twice, and mixing the two organic phases. And (3) after the collected organic phase is subjected to suction filtration, carrying out rotary evaporation on the liquid obtained by suction filtration by using a rotary evaporator until no liquid drops exist, and preserving at a low temperature (4 ℃) for later use. The same procedure was performed for simultaneous fermentation of other wild strains as controls. Paclitaxel was used as a positive control at a concentration of 0.5 mg/ml.
2. Tumor cell culture
Tumor cells HepG2 (human liver cancer) were cultured in DMEM medium containing penicillin, streptomycin and 10% Fetal Bovine Serum (FBS). DMEM cell suspension containing 10% FBS was seeded at a cell concentration of 2X 10 onto 96 well tissue culture plates4cells/ml. The culture was incubated at 37 ℃ with 5% CO2Cultured in a tissue culture box for 4 hours for later use.
3. Tumor cell inhibition assay
(1) Sample preparation
The ethyl acetate extract was dissolved in DMSO to prepare a mother liquor having a concentration of 20mg/ml, and the mother liquor was diluted to a sample solution of 100ug/ml with a DMEM culture solution containing 10% Fetal Bovine Serum (FBS).
(2) Sample application
The cell culture medium was carefully aspirated from the 96-well plate and a final concentration of 100ug/ml of sample solution was added. Triplicate wells per sample. At the same time, a zero setting well (culture medium) and a control well (cells, culture solution) are arranged.
(3) Culturing
Placing at 37 ℃ and 5% CO2Cultured in a tissue culture box for 44 hours.
(4) Observation of tumor cell inhibiting ability
The observation was performed by the MTT method. After 44 hours of incubation, 20ul MTT per well was added and incubation continued for 4 h. After uniform shaking, the inhibition was calculated by reading at 490nm with a microplate reader. The sample was run in duplicate.
(5) IC50 calculation
Repeating (1) to (4), and diluting the mother liquor to 15.625, 31.25, 62.5, 125, 250ug/ml gradient sample solution by using DMEM culture solution containing 10% Fetal Bovine Serum (FBS) when the concentration of (1) is prepared. The half fatality rate is calculated after experiments.
4. Data analysis
Statistics were performed using spss.21 proprietary statistical software, with P <0.05 significantly different using paired t-test.
Second, experimental results
Statistics were performed by SPSS.21 proprietary statistical software, with the results shown in Table 1.
Table 1:
the results in Table 1 show that at a concentration of 100. mu.g/ml, the inhibition rate of trametes sanguineus on HepG2 is 96.70%, and the IC50 value for HepG2 is 0.34ug, indicating that it has a strong tumor cell inhibiting effect in vitro. In the control experiment, most of the other wild bacteria had no effect of inhibiting tumor cells. In other parallel experiments, the inhibition rate of the ethyl acetate extract of the rice fermentation product of the strain A140247 of the Pycnoporus sanguineus to the HepG2 at the concentration of 100 mu g/ml is 46.9 percent, which proves that the in vitro tumor cell inhibition HepG2 effect of the strain of the invention is more remarkable.
Example 4 fermentation broth bacteriostatic activity assay
First, experiment method
1. Preparation of the culture Medium
Culture medium for bacteria: nutrient agar/broth medium;
nutrient agar/broth medium: 3g of beef extract, 10g of peptone, NaCL5g and 15g of agar, and adding water to adjust the volume to 1000ml and adjust the pH to 7.4 (the nutrient broth does not contain agar).
2. Preparation of samples
Preparation of control: taking ampicillin frozen stock solution, diluting with sterile water to obtain control solution with concentration of 5 μ g/mL, and ultraviolet sterilizing for 30 min.
Preparing a test sample: the trametes sanguinea strain is subjected to solid fermentation, and the culture medium is rice and water. Soaking the rice culture medium with ethyl acetate for 10 hr after culturing for 45d, ultrasonically extracting for 100min twice, and mixing the two organic phases. And (3) after the collected organic phase is subjected to suction filtration, carrying out rotary evaporation on the liquid obtained by suction filtration by using a rotary evaporator until no liquid drops exist, and preserving at a low temperature (4 ℃) for later use. For the experiment, 10mg/mL of ethyl acetate solution containing 1% DMSO was prepared, and sterilized by filtration using a sterile syringe and a filter membrane.
3. Preparation of the bacterial suspension
(1) Taking out the staphylococcus aureus frozen stock solution from a refrigerator at the temperature of-80 ℃, and carrying out plate streaking inoculation to obtain a single colony;
note: selecting a smooth inoculating loop, and picking a small amount of bacteria-containing samples according to an aseptic operation method. As shown in fig. 5. 1) Marking an area A: placing the flat plate beside the flame of an alcohol burner, grasping a culture dish cover by using a left index finger and a thumb, supporting the bottom of the culture dish by using other three fingers, opening the culture dish facing the flame, holding an inoculating loop containing bacteria by using a right hand, lightly scribing 3-4 continuous parallel lines in an area A as a bacteria source for primary dilution, and burning out residual bacteria samples on the inoculating loop; 2) dividing other areas: cooling the burned inoculating loop at the edge of a plate culture medium, transferring a B area to a scribing position, moving the inoculating loop to the B area through the A area (a bacteria source area), scribing 6-7 compact row lines on the B area lightly, and scribing more parallel lines on the C area and the D area by the same operation, wherein the lines of the D area are parallel to the A area (but cannot be in contact with the lines of the A area or the B area); 3) constant-temperature culture: culturing the streaked plate at 37 ℃ for 2-3 days; 4) selecting a small amount of thallus from a typical single colony to a test tube inclined plane, and obtaining a primary separated pure strain after culturing.
(2) And (3) picking a single colony by using a sterile inoculating loop, inoculating the single colony in a nutrient broth culture medium, and performing shaking culture at 37 ℃ and 220r/min for 24h to obtain a bacterial suspension of the test bacteria.
(3) Sucking bacterial suspension of test bacteria, and diluting with sterile distilled water to obtain 10-1～10-8Bacterial liquid with the original concentration;
(4) respectively sucking 200 μ L of bacterial liquid with each concentration, uniformly coating on nutrient agar plates, culturing at 37 deg.C for 24h, and repeating each concentration for 3 times;
(5) and determining the concentration of the bacterial liquid for later use by a plate counting method.
4. Determination of zone of inhibition
Injecting a certain amount of bacteria liquid into a plate culture medium which is cooled to about 50 ℃ by adopting a pre-bacteria liquid pouring plate method to ensure that the bacteria concentration in the culture medium is 105～106cfu/mL, shaking up, pouring the plate (about 30 mL/plate), horizontally standing for later use after solidification, uniformly punching holes on the plate containing bacteria by using a sterilized puncher, carefully selecting the culture medium in the holes, sucking 200 mu L of a reference product/test product under an aseptic condition, punching the hole into the hole, placing the hole for standing and culturing for 1 day under a condition of 37 ℃, and measuring the size of a bacteriostatic circle. The positive control was 10. mu.g/mL ampicillin.
II, experimental results:
the results are shown in Table 2.
Table 2E140415 zone diameter for inhibition of staphylococcus aureus (x ± s, n ═ 3)
| Sample (I) | Diameter of bacteriostatic circle (mm) |
| Positive control | 19.79±1.7 |
| Pycnoporus sanguineus E140415 | 19.39±0.86 |
As shown in FIG. 6, the upper left corner is blank, the upper right corner is a positive control, the lower left corner is Pycnoporus sanguineus E140415, and the lower right corner is another control variety Lepidium W140054.
In more than 600 wild edible and medicinal fungus variety ethyl acetate extracts which are detected by adopting the concentration of 10mg/mL, the strain has an obvious inhibition zone, is one of 10 samples with the most obvious effect, and has strong antibacterial activity on staphylococcus aureus.
In conclusion, the trametes sanguinea strain is a new variety which is not developed and researched, pure strains are separated through field collection and artificial cultivation, and from the view of functional experiments, the ethyl acetate extract of the trametes sanguinea strain has the characteristics of obvious in-vitro tumor inhibition effect, obvious staphylococcus aureus inhibition effect, good agronomic characters after cultivation and the like, and is a strain with development prospect.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.
Claims (12)
1. The Pycnoporus sanguineus strain is Pycnoporus sanguineus (HMGIM-140415) with the preservation number of CCTCC NO: m2019514.
2. An artificial cultivation method of a pycnoporus sanguineus strain CCTCC NO: M2019514 is characterized by comprising the steps of preparing a mother strain, preparing a production seed, cultivating, culturing and managing, wherein the cultivation material comprises 37-41% of mixed wood chips, 18-20% of cottonseed hulls, 18-20% of corncobs, 16-18% of wheat bran, 2-3% of corn flour and 1-2% of lime in percentage by weight.
3. The artificial cultivation method according to claim 2, wherein the cultivation material comprises 39% of miscellaneous wood chips, 20% of cotton seed hulls, 20% of corn cobs, 17% of wheat bran, 3% of corn flour, 1% of lime, and the ratio of material to water is as follows: 1:1.1-1.3.
4. The artificial cultivation method as claimed in claim 2, wherein the cultivation culture comprises: transferring the production seeds into a cultivation material, culturing at a constant temperature of 25-26 ℃ in shade, and allowing mycelia to grow over the fungus bags with humidity of 60-70%, and performing cultivation management.
5. The artificial cultivation method as claimed in claim 4, wherein the cultivation management includes: continuously shading and post-ripening for 15 days after the hypha in the cultivation bag grows over the cultivation material in the bag, entering a fruiting stage, controlling the temperature at 23-27 ℃, increasing the ventilation volume, keeping the carbon dioxide content in the space below 1%, adjusting the relative air humidity to be above 90%, and removing the pileus after 5-7 days, so that the hypha is twisted and forms bright red rice-shaped primordium; after the primordium grows to 0.5cm, continuously controlling the temperature between 23 ℃ and 27 ℃ and the relative air humidity between 85% and 90%, illuminating for 9 hours every day with the illumination intensity of 300-.
6. The artificial cultivation method as claimed in any one of claims 2 to 5, wherein the preparing of the mother seeds comprises: transferring the separated strains to a mother strain culture medium, performing constant-temperature dark culture at 25 ℃, and picking tip hyphae when the hyphae grow but the bacteria do not grow to obtain a mother strain; and/or the presence of a gas in the gas,
the production mother seed comprises the following steps: transferring the mother strain to a culture medium for producing the mother strain, carrying out dark culture at a constant temperature of 25 ℃, and obtaining the production mother strain when hyphae grow over an inclined plane; and/or the presence of a gas in the gas,
the manufacturing production method comprises the following steps: inoculating production mother seed into the culture medium under sterile condition, embedding the material block of the production mother seed into the raw material, culturing at 25 deg.C under dark condition, and collecting the production seed after the hypha is full of the material.
7. The artificial cultivation method according to claim 6, wherein the mother culture medium is a Bengal culture medium, and the Bengal culture medium comprises, in weight percent: peptone 0.5%, glucose 1%, potassium dihydrogen phosphate 0.1%, magnesium sulfate (MgSO4 & 7H2O) 0.05%, agar 2%, 1/3000 Bengal solution 10%, chloramphenicol 0.01%, and water in balance; and/or the presence of a gas in the gas,
the production mother culture medium is enriched comprehensive PDA, and the enriched comprehensive PDA comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1% of peptone, 2% of agar, 0.3% of monopotassium phosphate, 0.15% of magnesium sulfate, trace vitamin B1 and the balance of water;
the production seed culture medium comprises the following components in percentage by weight: 98-99% of sorghum and 1-2% of calcium carbonate.
8. The application of the dense red blood fungus strain CCTCC NO: M2019514 or the extract thereof is characterized in that the strain is used for preparing the medicine for resisting tumor or relevant diseases caused by bacteria.
9. The use according to claim 8, wherein the extract is an ethyl acetate extract.
10. The use of claim 8, wherein the tumor is a tumor caused by HepG2 cells; and/or, the bacterium is a staphylococcus.
11. The use of claim 10, wherein the staphylococcus is staphylococcus aureus.
12. A medicine for treating tumor and the diseases caused by bacteria is prepared from the strain of red blood dense pore (CCTCC) M2019514 or its extract and carrier.
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