CN112143656B - Preparation method of yellow tumor spore powder - Google Patents
Preparation method of yellow tumor spore powder Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, relates to a preparation method of fungus traditional Chinese medicine raw materials, and particularly relates to a preparation method of a yellow tumor spore powder. The myxosporidia powder is produced from myxosporidia HLB-01 with a preservation number of CGMCC 20259. The preparation method of the yellow tumor spore powder comprises the steps of strain breeding, seed preparation, liquid strain preparation, strain spore production culture and spore powder collection and preparation. The invention utilizes the ultraviolet mutagenesis method to separate and select and breed the strain to obtain the yellow tumor spore mold, which has stronger stress resistance, quick hypha growth, strong spore production capability and the spore yield of 21.15mg/cm 2 Laying a good technical foundation for preparing the yellow tumor spore powder product. The preparation method of the yellow tumor spore powder provides new resources for expanding the development and utilization of medicinal fungi, solves the problem of large-scale preparation of yellow tumor spore powder, and provides a technical foundation for subsequent development and utilization; and provides a formulation of the xanthomonas sp and a spore production culture technology, and can flexibly shorten the production period according to the production conditions.
Description
Technical Field
The invention belongs to the technical field of bioengineering, relates to a preparation method of fungus traditional Chinese medicine raw materials, and particularly relates to a preparation method of a yellow tumor spore powder.
Background
Huang Liubao mould is often parasitic on the fruiting body of wild bolete, agaricus, producing a large number of golden yellow spores. The fungus is called as the jute coccidiosis on the traditional Chinese medicine, the sexual generation of the fungus is the golden spore parasitic fungus Hypomyces chrysospermus, the Chinese herbal medicine records that the spore powder of the jute coccidiosis is warm and slightly bitter, enters the lung channel, can be externally applied in proper amount, is mainly used for treating traumatic hemorrhage, has similar functions as the puffball spores, and is a good hemostatic. However, the difficulty of collecting spore powder in the field is high, the practical application progress is slow, few reports about the yellow tumor spore mold are in China, and only 2 spore powder are searched on a universal database, which is a resource introduction. The report on the bacteria is not more at home and abroad, mainly focuses on the research on extracting Huang Baola (chrysodin) and Huang Liubao (sepedonin) antibacterial peptide functional substances from the bacteria, and the bacteria have not been effectively developed and utilized at home and abroad. With the development of biotechnology, the development and utilization of fungal medicaments are more and more emphasized, compared with Ma Boxiang, the spore powder of the yellow tumor spore mould is prepared as a traditional Chinese medicine raw material, the preparation cycle of the spore powder is obviously shortened without going through sexual generation, and in addition, the yellow tumor spore mould also contains novel antibiotic substances such as yellow tumor spore extract (chrysodin), huang Liubao element (sepedonin) and the like, so that the development and utilization prospect is wide. Therefore, research and development of a preparation method of the yellow tumor spore powder is a problem to be solved urgently.
Disclosure of Invention
In view of the problems existing in the prior art, the invention aims to provide a preparation method of a yellow tumor spore powder. The spore powder of the yellow tumor spore is produced from yellow tumor spore HLB-01 with the preservation number of CGMCC No.20259. The preparation method of the yellow tumor spore powder comprises yellow strain breeding, seed preparation, liquid strain preparation, strain spore production culture and spore powder collection and preparation. The invention utilizes the ultraviolet mutagenesis method to separate and select and breed the strain to obtain the yellow tumor spore mold, the stress resistance is stronger, the hypha grows fast, the spore production capability is strong, and the spore yield can reach 21.15mg/cm 2 Lays a good technical foundation for the preparation of the yellow tumor spore powder product. The preparation method of the yellow tumor spore powder provides new resources for expanding the development and utilization of medicinal fungi, solves the problem of large-scale preparation of yellow tumor spore powder, and provides a technical foundation for subsequent development and utilization.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
A species of a genus of a fungus, which is a parasitic fungus of the genus chrysosporium (Hypomyces chrysospermus), and which is deposited in the China general microbiological culture Collection center (cmcs) at a deposit address of 9/11/2020: the collection number of the microbiological institute of China is CGMCC No.20259, and the collection number of the microbiological institute of China is China, national institute of sciences, no. 1, no. 3, north Chen West Lu, the Korean region of Beijing city.
The breeding method of the yellow tumor spore mold strain specifically comprises the following steps.
Step 1, separating strains: collecting fruiting body of Boletus edulis infected with Boletus edulis in the field, placing under 30W ultraviolet lamp, irradiating for 5min at a distance of 30cm, picking spore powder on fruiting body surface layer, placing into a test tube containing 10mL of sterile water, shaking uniformly, sucking spore liquid 100 μL onto comprehensive PDA culture medium with a pipette, scraping and smearing uniformly, culturing at 30deg.C for 4-7d, picking edge of target colony generating golden yellow spore, purifying for 2-3 times, and preserving for use.
Step 2, strain mutagenesis: under aseptic condition, 10mL of sterile water is slowly added into the strain in the inclined surface test tube, after gentle shaking, the spore suspension is poured into a sterile plate with the concentration of about 1 multiplied by 10 7 cfu/mL, placing under a 30W ultraviolet lamp, irradiating for 120s at a distance of 30 cm; under the aseptic condition, respectively sucking 100 mu L of spore suspension subjected to ultraviolet irradiation into a comprehensive PDA plate after proper dilution, uniformly smearing, culturing for 7d at 30 ℃, picking out the edge of a colony with the largest spore yield, purifying for 2 times, transferring to a PDA inclined-plane test tube for culturing to a full tube, and preserving for later use; and continuously passaging for 5 times, and investigating the spore yield of the strain.
A preparation method of the yellow tumor spore powder comprises the steps of strain breeding, seed preparation, liquid strain preparation, strain spore production culture and spore powder collection and preparation.
The preparation method of the yellow tumor spore powder specifically comprises the following steps.
Step one, strain breeding.
(1) Separating strains: collecting fruiting body of Boletus edulis infected with Boletus edulis in the field, placing under 30W ultraviolet lamp, irradiating for 5min at a distance of 30cm, picking spore powder on fruiting body surface layer, placing into a test tube containing 10mL of sterile water, shaking uniformly, sucking spore liquid 100 μL onto comprehensive PDA culture medium with a pipette, scraping and smearing uniformly, culturing at 30deg.C for 4-7d, picking edge of target colony generating golden yellow spore, purifying for 2-3 times, and preserving for use.
(2) Strain mutagenesis: under aseptic condition, 10mL of sterile water is slowly added into the strain in the inclined surface test tube, after gentle shaking, the spore suspension is poured into a sterile plate with the concentration of about 1 multiplied by 10 7 cfu/mL, placing under a 30W ultraviolet lamp, irradiating for 120s at a distance of 30 cm; under aseptic condition, the spore suspension irradiated by ultraviolet is diluted appropriately and respectively sucked into 100 mu LUniformly smearing on a comprehensive PDA plate, culturing for 7d at 30 ℃, picking the edge of a colony with the largest spore yield, purifying for 2 times, transferring to a PDA inclined-plane test tube for culturing until the tube is full, and preserving for later use; and continuously passaging for 5 times, and investigating the spore yield of the strain.
And step two, seed preparation.
Inoculating the yellow tumor spore mold to a mother strain eggplant bottle culture medium under the aseptic condition, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, and culturing in a constant temperature incubator at 30 ℃ for 7 days until the inclined surface of the eggplant bottle grows full to obtain the seeds.
And step three, preparing liquid strains.
And (3) the temperature of the liquid culture medium after sterilization is reduced to below 30 ℃ for inoculation, the inclined-plane seeds of the eggplant bottle are scratched into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by an inoculation shovel, and the inoculation blocks are transferred into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, and are cultured for 5 days at the temperature of 30 ℃ at the speed of 170rpm/min, wherein the proportion of the bacterial balls reaches more than 80% by volume, so that the liquid stock seed is obtained.
And step four, cultivating the spawn sporulation.
(1) And (3) batching and sterilizing: preparing a culture medium from a culture medium formula A or a culture medium formula B, subpackaging and sterilizing, wherein the culture medium formula A is required to be filled into a high-temperature and high-pressure resistant glass container, the filling amount is 50% of the container volume, and sterilizing is carried out for 30 minutes at 121 ℃; the culture medium formula B can be filled into a polypropylene plastic bag with the size of 35cm multiplied by 17cm for producing edible fungi, the filling amount is about 200g, and the culture medium formula B is sterilized for 45 minutes at 121 ℃; and sterilizing the inoculation gun for standby.
(2) Inoculating and culturing: taking out the sterilized culture medium, inoculating the culture medium formula A to sterilized inoculating gun equipment, inoculating the culture medium to sterilized glass or plastic container, sealing with sterile plastic film, adding 20mL of liquid strain into the container with another inoculating gun after agar is solidified, and shaking uniformly; the culture medium formula B is cooled to about 30 ℃, 10mL of liquid strain is directly added into the culture medium under the aseptic condition, and the culture medium formula B is paved into a thickness of about 1.5cm after uniform mixing; after inoculation, the culture medium is placed in a 30 ℃ incubator or a culture room for culture management.
And fifthly, collecting and preparing spore powder.
Culture mediumFormula A is agar plate culture medium, yellow spores are distributed on the surface layer of the culture medium, and after scraping and collecting, the yellow spores are dried at 45 ℃ to obtain the yellow tumor spore powder, wherein the spore yield is 20.15mg/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the The culture medium formula B is a loose solid culture material, yellow spores are distributed on the surface layer of material particles, the material is taken out, dried at 45 ℃ and then screened by a 60-mesh standard sieve, and the undersize product is the yellow tumor spore powder, and the spore yield is 23.20mg/g of dry material. Packaging the obtained yellow tumor spore powder into plastic packaging bags, and vacuum packaging and preserving.
Further, the raw material composition of the comprehensive PDA culture medium in the first step comprises 200g of potato juice, 1.5 g of magnesium sulfate, 3 g of monopotassium phosphate, 20 g of glucose, 20 g of agar and the balance of water, wherein the sterilization condition of the culture medium is 121 ℃ for 30 minutes.
Further, the improved integrated PDA culture medium in the second step comprises the following raw materials: each liter of the culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of monopotassium phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water, wherein the sterilization condition of the culture medium is 121 ℃ for 30 minutes.
Further, the formula of the liquid strain culture medium in the third step comprises the following components in percentage by weight: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; the medium sterilization conditions were 21℃for 30 minutes.
Further, in the fourth step, the culture medium formula A comprises the following components in percentage by weight: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride, 2% of agar and the balance of water.
Further, the composition of the culture medium formula B in the step four is as follows in percentage by weight: 75.0% of bran, 15.0% of rice husk, 0.5% of glucose, 0.2% of brown sugar, 1.0% of peptone, 0.2% of boletus dry powder, 0.5% of dipotassium hydrogen phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and a feed-water ratio of 1:1.
And in the fourth step, the culture management comprises controlling the culture temperature between 25 ℃ and 30 ℃, the relative humidity of air is not required, white hypha grows in the initial stage, the culture is carried out until 3-5 days of visible yellow powdery spores are produced, and a large amount of yellow powdery spores are produced after 7-10 days of culture, so that spores can be collected.
The purity of the yellow tumor spore powder prepared by the preparation method of the yellow tumor spore powder is higher than 99 percent.
The preparation method of the yellow tumor spore powder can be used for preparing the medicament for stopping bleeding of the trauma.
Compared with the prior art, the invention has the following beneficial effects.
1) The invention utilizes the ultraviolet mutagenesis method to separate and select and breed the strain to obtain the yellow tumor spore mold, the stress resistance is stronger, the hypha grows fast, the spore production capability is strong, and the spore yield can reach 21.15mg/cm 2 Lays a good technical foundation for the preparation of the yellow tumor spore powder product.
2) The preparation method of the yellow tumor spore powder provides new resources for expanding the development and utilization of medicinal fungi, solves the problem of large-scale preparation of yellow tumor spore powder, and provides a technical foundation for subsequent development and utilization.
3) The preparation method of the yellow tumor spore powder provides a yellow tumor spore mold seed formula and a spore production culture technology, and can flexibly shorten the production period according to the production conditions.
Drawings
FIG. 1 is the rDNA-ITS sequence of F.chrysosporium of example 1.
Detailed Description
The invention is further described below with reference to the drawings and specific examples.
Example 1 isolation and identification of the parasitic fungus Hypomyces chrysospermus of the fungus and study of its biological properties.
In 2012, in research on large-scale fungus resources in Liaoxi, a strain of bolete pathogenic bacteria is separated, hyphae grow flatly on bolete fruiting bodies, the initial infection period is white, and the hyphae stage is mainly adopted at the moment; the fruiting body gradually decays to generate a large amount of yellow powder in the later stage of infection, which is the spore-producing stage. The strain separation is carried out by adopting a dilution method, and the high-yield spore strain breeding is carried out by adopting an ultraviolet mutagenesis method.
1. A culture medium.
Comprehensive PDA medium: the culture medium contains 200g of potato juice, 1.5 g of magnesium sulfate, 3 g of monopotassium phosphate, 20 g of glucose, 20 g of agar and the balance of water in percentage by mass, after the triangular flask is split, 7 bottles of the culture medium are respectively sterilized with 40% NaOH and 0.1mol/LHCl to adjust the pH of the culture medium to 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5, and the culture medium is sterilized at 121 ℃ for 30 minutes.
10 sterile water test tubes of 10mL are filled for later use.
2. And (5) separating and identifying the strain.
(1) Separating strains: collecting fruiting body of Boletus edulis infected with Boletus edulis in the field, placing under 30W ultraviolet lamp, irradiating for 5min at a distance of 30cm, picking spore powder on fruiting body surface layer, placing into a test tube containing 10mL of sterile water, shaking uniformly, sucking spore liquid 100 μL onto comprehensive PDA culture medium with a pipette, scraping and smearing uniformly, culturing at 30deg.C for 4-7d, picking edge of target colony generating golden yellow spore, purifying for 2-3 times, and preserving for use.
(2) Strain mutagenesis: under aseptic conditions, 10mL of sterile water was slowly added to the slant tube strain, and after gentle shaking, the spore suspension was poured into a sterile dish (concentration about 1X 10) 7 cfu/g), placed under a 30W ultraviolet lamp, irradiated for 120s at a distance of 30 cm. Under the aseptic condition, respectively sucking 100 mu L of spore suspension irradiated by ultraviolet into a comprehensive PDA flat plate, uniformly smearing, culturing for 7d at 30 ℃, picking the colony edge with the largest spore yield, purifying for 2 times, transferring to a PDA inclined plane test tube, culturing to a full tube, and preserving for later use. And sending the slant strain to Shanghai worker for ITS sequence analysis.
The bacterial colony is white at the initial stage and neat in edge, yellow spores are generated after culturing on PDA for 4-5 days, and yellow spores are fully distributed on the surface layer of the bacterial colony at the later stage; the microscopic morphological characteristics are that hyphae have diaphragms, branches are developed, creeping growth is carried out, and the hyphae are nearly colorless. Conidiophores are short and multi-branched. The conidiophore is on the top of the peduncles, and the conidiophore is cylindrical or elliptic, and 3.1-5.4X4.5-7.3 μm; chlamydospores are single-born and bear on hypha side branches, are spherical, have tumor-shaped protrusions on the surface layer, are colorless at first, and are yellow to golden yellow at first, extremely large in quantity and 12.2-18.5 mu m in diameter. Based on the morphological characterization of the fungus in combination with the analysis of the rDNA-ITS sequence (shown in FIG. 1), the fungus was identified as a fungus parasitic fungus (Hypomyces chrysospermus), the sexual generation of which was a fungus parasitic fungus (Hypomyces chrysospermus Tul. & C.Tul.).
3. Strain biological property study.
The biological characteristics of the strain are developed and researched, and test results show that the optimal growth temperature of the yellow tumor spore mould is 25-30 ℃, the optimal pH value is 6.5, and the yellow tumor spore mould is cultured for 96 hours on a PDA culture medium, and the colony diameter can reach 69.18mm, so that a large number of yellow spores are generated. Above 35 ℃, the strain is limited in growth, does not grow at 40 ℃, but can recover growth after recovering to the culture temperature below 30 ℃, and the growth is recovered, as shown in tables 1-2.
Table 1. The effect of different culture temperatures on the growth of F.xanthomatosis.
Note that: "-" means not grown; "+" indicates a small amount of sporogenes, and "++ +" indicates a large amount of sporogenes.
Table 2. Effects of different pH on growth of F.flavum.
Note that: "-" means not grown; "+" indicates a small amount of produced spore, "++" indicates a medium amount of produced spore, and "++ +" indicates a large amount of produced spore.
Example 2 influence of different species types on spore production.
1. A culture medium.
The improved comprehensive PDA culture medium comprises the following raw materials in percentage by mass: each liter of culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of monopotassium phosphate, 1.4 g of magnesium sulfate, 2.0 g of boletus dry powder, 20 g of agar and the balance of water, and after the culture medium is prepared, the culture medium is filled into eggplant bottles respectively with 70mL of culture medium per bottle.
The formula of the liquid strain culture medium comprises the following components: the potato juice comprises, by weight, 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.1% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water, wherein each bottle contains 50mL of water.
Spore-forming medium formulation a: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride, 2% of agar and the balance of water.
Medium sterilization conditions: 121 ℃,30 minutes.
2. And (5) seed preparation.
(1) The preparation method of the eggplant bottle seeds comprises the following steps: inoculating the yellow tumor spore mold to a mother strain eggplant bottle culture medium under a sterile condition, and culturing in a constant temperature incubator at 30 ℃ for 7 days until the inclined surface of the eggplant bottle grows to be full to obtain seeds; the preparation method of the liquid strain comprises the following steps: and (3) the temperature of the liquid culture medium after sterilization is reduced to below 30 ℃ for inoculation, the inclined-plane seeds of the eggplant bottle are scratched into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by an inoculation shovel, and the inoculation blocks are transferred into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, and are cultured for 5 days at the temperature of 30 ℃ at the speed of 170rpm/min, wherein the proportion of the bacterial balls reaches more than 80% by volume, so that the liquid stock seed is obtained.
3. And (5) spore production culture.
Pouring the spore-producing culture medium into a sterilization plate with the diameter of 90mm, and cooling the plate for later use. Adding 100mL sterile water into eggplant bottle strain, gently scraping off surface spore with inoculating needle to obtain spore suspension with concentration of 1×10 6 cfu/mL, and then sequentially diluted to 1X 10 2 cfu/mL. Adding 100mL sterile water into the liquid strain for dilution, and secondarily diluting to 1×10 2 Individual pellet/mL. 0.1mL of bacterial suspension or 0.1mL of liquid bacterial strain dilution is added into each dish, and the mixture is uniformly smeared by a spatula. After inoculation, the cells were incubated at a constant temperature in an incubator at 30 ℃. The test result shows that the liquid strain is obviously superior to spore suspension, has quick germination, quick growth, large spore yield and obvious reduction, and is shown in Table 3。
Table 3. Influence of different strains on growth and production of Mortierella verticillata.
Note that: "-" means not grown; "+" indicates a small amount of produced spore, "++" indicates a medium amount of produced spore, and "++ +" indicates a large amount of produced spore.
Example 3 influence of different culture modes and culture media on spore production.
1. And (5) preparing a culture medium.
(1) Mother culture medium composition: each liter of the culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of monopotassium phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water. Medium sterilization conditions: 121 ℃,30 minutes.
(2) The formula of the liquid strain culture medium comprises the following components: the potato juice comprises, by weight, 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; medium sterilization conditions: 121 ℃,30 minutes.
Spore-forming medium formulation a: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride, 2% of agar and the balance of water. Packaging into high temperature and high pressure resistant glass container, sterilizing at 121deg.C for 30 min.
Spore-forming medium formulation B: 75.0% of bran, 15.0% of rice hull, 0.5% of glucose, 0.2% of brown sugar, 1.0% of peptone, 0.2% of boletus dry powder, 0.5% of dipotassium hydrogen phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and a feed water ratio of 1:1. packaging into polypropylene plastic bag with size of 35cm×17cm for edible fungus production, packaging amount of about 200g, and sterilizing at 121deg.C for 45 min.
And sterilizing the inoculation gun for standby.
2. And (5) preparing liquid strains.
Inoculating the yellow tumor spore mold to a mother strain eggplant bottle culture medium under a sterile condition, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, and culturing in a constant temperature incubator at 30 ℃ for 7 days until the inclined surface of the eggplant bottle grows full to obtain seeds; and (3) the temperature of the liquid culture medium after sterilization is reduced to below 30 ℃ for inoculation, the inclined-plane seeds of the eggplant bottle are scratched into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by an inoculation shovel, and the inoculation blocks are transferred into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, and are cultured for 5 days at the temperature of 30 ℃ at the speed of 170rpm/min, wherein the proportion of the bacterial balls reaches more than 80% by volume, so that the liquid stock seed is obtained.
3. And (5) cultivating the strain to produce spores.
Spore-forming medium formulation a: taking out the sterilized culture medium, connecting sterilized inoculating gun equipment, inoculating the culture medium into sterilized glass or plastic container, sealing with sterile plastic film, adding 20mL liquid strain into the container with another inoculating gun after agar is solidified, and shaking uniformly.
Spore-forming medium formulation B: cooling to about 30deg.C, directly adding 10mL of liquid strain into the culture material under aseptic condition, mixing, and spreading to a thickness of about 1.5 cm.
After inoculation, the cells were placed in an incubator at 30 ℃. Periodically sampling in the peak period of spore production, and reserving for use.
The sampling method comprises the following steps: the culture medium formula A is an agar plate culture medium, yellow spores are distributed on the surface layer of the culture medium, and the yellow spores are dried at 45 ℃ after being scraped and collected by a spatula to obtain a yellow tumor spore powder sample.
The culture medium formula B is a loose solid culture material, yellow spores are distributed on the surface layer of material particles, the material is taken out, dried at 45 ℃ and then screened by a 60-mesh standard sieve, and the undersize is collected to obtain a sample of the yellow tumor spore powder.
The obtained myxoma xanthomonas spore powder samples sampled in different culture periods are weighed, and the myxoma xanthomonas yield is calculated according to the following formula.
The spore production efficiency of the culture medium formula A is closely related to the surface area of the culture medium, and the spore production amount is measured by the change of the spore production amount per unit area.
The spore production efficiency of the culture medium formula B is closely related to the weight of the materials, and the spore production amount is mainly measured by the change of the spore production amount per unit weight.
Test results show that the two culture modes can obtain better yield of the yellow tumor spore powder, wherein the culture medium A is adopted to grow and produce spores on the surface layer of the culture medium, so that the yellow tumor spore powder with higher purity can be obtained, the production cost is considered, the spore production period is proper for 7 days, and the spore yield is 20.15mg/cm 2 The production period is short; the culture medium formula B has the advantages of simple operation, wide applicability and the like, and the production period is 10 days, and the spore yield is 23.20mg/g of dry material, which is shown in Table 4, by combining the production cost.
Table 4. Influence of different strains on growth and production of Mortierella verticillata.
Note that: "-" means not grown; "+" indicates a small amount of produced spore, "++" indicates a medium amount of produced spore, and "++ +" indicates a large amount of produced spore.
Example 4 use of a spore powder of Paeonia flavescens.
The yellow tumor spore powder obtained according to example 3 can be used for hemostasis of wounds.
Can provide raw materials with purity higher than 99% for the medicinal development of the yellow tumor spore powder.
Sequence listing
<110> Liaoning national institute of microbiology
<120> preparation method of yellow tumor spore powder
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 568
<212> DNA
<213> artificial sequence
<400> 1
TGCGGAGGGA TCATTACCGA GTTTACAACT CCCAAACCCC ATGTGAACCT ACCACTACGT 60
TGCTTCGGCG GGATGCCCCG GGCGCGGGCC GCAAGGCCTC AGCCCCGGAA CCAGGCGCCC 120
GCCGGAGGCC CAAACCAAAC TCTTGTTTTT ATAGAATCTT CTGAGTGGCC TTTTAGGCAA 180
CAAATGAATC AAAACTTTCA ACAACGGATC TCTTGGTTCT GGCATCGATG AAGAACGCAG 240
CGAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA TCATCGAATC TTTGAACGCA 300
CATTGCGCCC GCCAGCATTC TGGCGGGCAT GCCTGTTCGA GCGTCATTTC AACCCTCAAG 360
CCCCCCCGGG GGCCTGGTGT TGGGGACCGG CCCCGCCCCG TCGCGGGTCG CCGCCCCCTA 420
AATTCAGTGG CGGTCCTGCC GCAGCCTCCT CTGCGTAGTA GCTTTTGAAA CCTCGCACCG 480
GAGAGCGGCT CGGCCACGCC GTTAAACCCC AACTTTCTAG AGTTTGACCT CGGATCAGGT 540
AGGAATACCC GCTGAACTTA AGCATATC 568
Claims (8)
1. The method is characterized in that the yellow tumor spore mold is yellow tumor spore mold HLB01, and the strain is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) on 9 and 11 days of 2020, and the preservation address is: the collection number of the microbiological institute of China is CGMCC No.20259, and the national institute of sciences of China is No. 1, no. 3, north Chen West Lu, the Korean region of Beijing city.
2. The preparation method of the yellow tumor spore powder is characterized by comprising the following steps of:
step one, seed preparation
Inoculating the xanthoma fungus strain in claim 1 to a mother strain eggplant bottle culture medium under aseptic condition, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, culturing in a constant temperature incubator at 30deg.C for 7 days, and obtaining seeds after the inclined surface of eggplant bottle is full;
step two, liquid strain preparation
Inoculating the sterilized liquid culture medium at a temperature below 30deg.C, dividing the slant seed of the eggplant bottle into inoculating pieces with a size of about 0.5cm×0.5cm by using an inoculating shovel, transferring 5-6 pieces to 150mL liquid strain culture medium under aseptic condition, culturing at 30deg.C at 170rpm/min for 5 days until the ratio of the bacterial balls reaches above 80% by volume, and obtaining liquid stock seed;
step three, spawn spore production culture
(1) And (3) batching and sterilizing: preparing a culture medium from a culture medium formula A or a culture medium formula B, subpackaging and sterilizing, wherein the culture medium formula A is required to be filled into a high-temperature and high-pressure resistant glass container, the filling amount is 50% of the container volume, and sterilizing is carried out for 30 minutes at 121 ℃; the culture medium formula B can be filled into a polypropylene plastic bag with the size of 35cm multiplied by 17cm for producing edible fungi, the filling amount is about 200g, and the culture medium formula B is sterilized for 45 minutes at 121 ℃; simultaneously sterilizing the inoculation gun for standby;
(2) Inoculating and culturing: taking out the sterilized culture medium, inoculating the culture medium formula A to sterilized inoculating gun equipment, inoculating the culture medium to sterilized glass or plastic container, sealing with sterile plastic film, adding 20mL of liquid strain into the container with another inoculating gun after agar is solidified, and shaking uniformly; the culture medium formula B is cooled to about 30 ℃, 10mL of liquid strain is directly added into the culture medium under the aseptic condition, and the culture medium formula B is paved into a thickness of about 1.5cm after uniform mixing; after inoculation, placing the strain in a 30 ℃ incubator or a culture room for culture management;
step four, spore powder collection and preparation
The culture medium formula A is an agar plate culture medium, yellow spores are distributed on the surface layer of the culture medium, and the yellow spores are dried at 45 ℃ after being scraped and collected by a spatula to obtain the yellow tumor spore powder; the culture medium formula B is a loose solid culture material, yellow spores are distributed on the surface layer of material particles, the material is taken out, dried at 45 ℃ and then screened by a 60-mesh standard sieve, and the undersize product is collected to obtain the yellow tumor spore powder;
packaging the obtained yellow tumor spore powder into plastic packaging bags, and vacuum packaging and preserving.
3. The method for preparing the yellow tumor spore powder according to claim 2, wherein the modified integrated PDA culture medium in the first step comprises the following raw materials: each liter of the culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of monopotassium phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water, wherein the sterilization condition of the culture medium is 121 ℃ for 30 minutes.
4. The method for preparing the yellow tumor spore powder as claimed in claim 2, wherein the liquid strain culture medium formula in the second step comprises the following components in percentage by weight: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; the medium sterilization conditions were 21℃for 30 minutes.
5. The method for preparing the yellow tumor spore powder according to claim 2, wherein the composition of the culture medium formula A in the step three is as follows in percentage by weight: 20% of potato juice, 0.3% of brown sugar, 0.5% of glucose, 1.5% of dipotassium hydrogen phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride, 2% of agar and the balance of water.
6. The method for preparing the yellow tumor spore powder according to claim 2, wherein the composition of the culture medium formula B in the step three is as follows in percentage by weight: 75.0% of bran, 15.0% of rice husk, 0.5% of glucose, 0.2% of brown sugar, 1.0% of peptone, 0.2% of boletus dry powder, 0.5% of dipotassium hydrogen phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and a feed-water ratio of 1:1.
7. The method for preparing the yellow tumor spore powder as defined in claim 2, wherein the culturing in the third step comprises controlling the culturing temperature between 25 ℃ and 30 ℃, the relative humidity of air is not particularly required, white hypha grows in the initial stage, yellow powdery spores are produced after culturing for 3-5 days, a large amount of yellow powdery spores are produced after culturing for 7-10 days, and spores can be collected.
8. The process for preparing a yellow tumor spore powder according to claims 2-7, wherein the yellow tumor spore powder has a purity of more than 99%.
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| JP2004215586A (en) * | 2003-01-15 | 2004-08-05 | Daicel Chem Ind Ltd | Method for producing 4-hydroxybenzoic acid |
| CN103451113A (en) * | 2013-09-02 | 2013-12-18 | 山西农业大学 | Separation and cultivation method for isaria fumosorosea |
| CN103555821A (en) * | 2013-08-17 | 2014-02-05 | 中国计量学院 | Detection method for yeast-like symbiotes in brown planthopper on rice varieties with different resistance |
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| JP2004215586A (en) * | 2003-01-15 | 2004-08-05 | Daicel Chem Ind Ltd | Method for producing 4-hydroxybenzoic acid |
| CN103555821A (en) * | 2013-08-17 | 2014-02-05 | 中国计量学院 | Detection method for yeast-like symbiotes in brown planthopper on rice varieties with different resistance |
| CN103451113A (en) * | 2013-09-02 | 2013-12-18 | 山西农业大学 | Separation and cultivation method for isaria fumosorosea |
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Effective date of registration: 20240417 Address after: 122000 No.8, Section 2, Longquan street, Chaoyang Development Zone, Chaoyang City, Liaoning Province Patentee after: LIAONING WKIOC BIOENGINEERING Co.,Ltd. Country or region after: China Address before: 122000 No. four, Longshan street, Twin Towers District, Liaoning, Chaoyang City province 820 Patentee before: LIAONING SCIENTIFIC ACADEMY OF MICROBIOLOGY Country or region before: China |