CN112143656A - Preparation method of xanthoma spore powder - Google Patents
Preparation method of xanthoma spore powder Download PDFInfo
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Abstract
The invention belongs to the technical field of bioengineering, relates to a preparation method of a fungus traditional Chinese medicine raw material, and particularly relates to a preparation method of xanthoma spore powder. The xanthomonas campestris spore powder is produced from the xanthomonas campestris HLB-01, and the preservation number is CGMCC 20259. The preparation method of the xanthomonas powder comprises strain breeding, seed preparation, liquid strain preparation and strain spore productionCulturing, collecting and preparing spore powder. The method utilizes an ultraviolet mutagenesis method to separate and breed strains to obtain the strain of the xanthomonas fulva, has stronger stress resistance, fast hypha growth and strong spore production capacity, and the spore production can reach 21.15mg/cm2And a good technical foundation is laid for the preparation of the xanthomonas campestris spore powder product. The preparation method of the xanthomonas campestris spore powder provides new resources for developing and utilizing medicinal fungi, solves the problem of large-scale preparation of the xanthomonas campestris spore powder, and provides a technical basis for the subsequent development and utilization of the xanthomonas campestris spore powder; and provides a formula of the xanthomonas campestris strain 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 a fungus traditional Chinese medicine raw material, and particularly relates to a preparation method of xanthoma spore powder.
Background
The fulvia fulva is often parasitic on fruiting bodies of wild bolete and agaricus to generate a large amount of golden yellow spores. In the traditional Chinese medicine, the fungus is named as the staphylotrichum marovii, the sexual generation of the staphylotrichum marovii is the staphylococcus chrysospermus, the spore powder of the staphylotrichum marovii is recorded in Chinese materia medica, the spore powder is warm and slightly bitter, the spore powder enters lung channels, the spore powder can be applied externally in a proper amount to treat traumatic hemorrhage, the function of the spore powder is similar to that of the spore of the puffball, and the spore powder is a good hemostatic medicine. However, the difficulty of collecting spore powder in the field is high, the practical application progress is slow, the domestic reports about the xanthomonas fulva are few, and only 2 data on a Wanfang database are searched and are all resource introductions. The bacteria are not reported much in foreign countries, and the research on the extraction of antibacterial peptide functional substances such as chrysodin (chrysodein) and sepedonin (sepedonin) from the bacteria is mainly focused, so that the bacteria are not effectively developed and utilized at home and abroad. Along with the development of biotechnology, the development and utilization of fungal medicaments are more and more emphasized, compared with puffball, spore powder of the fulvia fulva has more obvious advantages as a traditional Chinese medicine raw material, sexual generation is not needed, the preparation period of the spore powder is obviously shortened, and in addition, the spores of the fulva also contain novel antibiotic substances such as flavonodin (chrysodin), flavonodinin (sepedonin) and the like, so the development and utilization prospect is wide. Therefore, research and development of a preparation method of the xanthomonas campestris spore powder are problems which need to be solved urgently.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a preparation method of a fulvia fulva spore powder. The xanthomonas campestris spore powder is produced from the HLB-01 of the xanthomonas campestris, and the preservation number is CGMCC No. 20259. The preparation method of the xanthomonas campestris spore powder comprises yellow strain breeding, seed preparation, liquid strain preparation, strain spore production culture and spore powder collection and preparation. The method utilizes an ultraviolet mutagenesis method to separate and breed strains to obtain the xanthomonas campestris strain, has stronger stress resistance, fast hypha growth and strong spore production capability, and the spore production can reach 21.15mg/cm2And a good technical foundation is laid for the preparation of the fulvia fulva spore powder product. The preparation method of the xanthomonas campestris spore powder provides new resources for developing and utilizing medicinal fungi, solves the problem of large-scale preparation of the xanthomonas campestris spore powder, and provides a technical basis for subsequent development and utilization.
In order to achieve the purpose, the invention adopts the following technical scheme.
The xanthomonas flavedo strain is a chrysosporium parasitic bacterium (Hypermyces chrysospermus), is preserved in China general microbiological culture Collection center (CGMCC) at 9-11 days 2020 at the preservation address of: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, with the collection number of CGMCC number 20259.
The method for breeding the xanthomonas comprises the following steps.
Step 1, strain separation: collecting Boletus edulis fruiting body infected with Xanthomonas flavivis in the field, placing under a 30W ultraviolet lamp, irradiating for 5min at an irradiation distance of 30cm, picking a ring of spore powder on the surface layer of the fruiting body, placing into a test tube filled with 10mL sterile water, oscillating uniformly, sucking 100 mu L of spore liquid by a pipette to a comprehensive PDA culture medium, scraping and coating uniformly, culturing at 30 ℃ for 4-7d, picking the edge of a target colony generating golden yellow spores, purifying for 2-3 times, and preserving for later use.
Step 2, strain mutagenesis: under aseptic condition, slowly adding 10mL of sterile water into the slant tube strain, slightly vibrating, pouring the spore suspension into a sterile plate with the concentration of about 1 × 107cfu/mL, placing under a 30W ultraviolet lamp, irradiating for 120s with the irradiation distance of 30 cm; under the aseptic condition, the spore suspension irradiated by ultraviolet is properly diluted and respectively absorbed by 100 mu L into a comprehensive PDA flat plate, the uniform smearing is carried out, the culture is carried out for 7d at the temperature of 30 ℃, the edge of a bacterial colony with the maximum spore yield is picked, the bacterial colony is purified for 2 times, and then the bacterial colony is transferred to a PDA inclined test tube to be cultured until the tube is full and preserved for later use; and continuously passaging for 5 times to check the spore yield of the strain.
A method for preparing spore powder of Thermomyces fulva comprises strain breeding, seed preparation, liquid strain preparation, strain spore production culture, and spore powder collection and preparation.
The preparation method of the fulvia fulva spore powder specifically comprises the following steps.
Step one, strain breeding.
(1) Strain separation: collecting Boletus edulis fruiting body infected with Xanthomonas flavivis in the field, placing under a 30W ultraviolet lamp, irradiating for 5min at an irradiation distance of 30cm, picking a ring of spore powder on the surface layer of the fruiting body, placing into a test tube filled with 10mL sterile water, oscillating uniformly, sucking 100 mu L of spore liquid by a pipette to a comprehensive PDA culture medium, scraping and coating uniformly, culturing at 30 ℃ for 4-7d, picking the edge of a target colony generating golden yellow spores, purifying for 2-3 times, and preserving for later use.
(2) Strain mutagenesis: under aseptic condition, slowly adding 10mL of sterile water into the slant tube strain, slightly vibrating, pouring the spore suspension into a sterile plate with the concentration of about 1 × 107cfu/mL, placing under a 30W ultraviolet lamp, irradiating for 120s with the irradiation distance of 30 cm; under the aseptic condition, the spore suspension irradiated by ultraviolet is properly diluted and respectively absorbed by 100 mu L into a comprehensive PDA flat plate, the uniform smearing is carried out, the culture is carried out for 7d at the temperature of 30 ℃, the edge of a bacterial colony with the maximum spore yield is picked, the bacterial colony is purified for 2 times, and then the bacterial colony is transferred to a PDA inclined test tube to be cultured until the tube is full and preserved for later use; and continuously passaging for 5 times to check the spore yield of the strain.
And step two, seed preparation.
Inoculating the strain of Thermomyces fulva to a mother strain eggplant flask culture medium under aseptic condition, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, culturing in a 30 deg.C constant temperature incubator for 7 days, and collecting seeds after the eggplant flask is full of slant.
And step three, preparing liquid strains.
And (3) inoculating when the temperature of the sterilized liquid culture medium is reduced to below 30 ℃, dividing the eggplant bottle inclined plane seeds into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by using an inoculation shovel, transferring the eggplant bottle inclined plane seeds into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, culturing for 5 days at the temperature of 30 ℃ and at the rpm/min of 170, and obtaining the liquid stock culture when the ratio of the bacterial balls reaches more than 80 percent in volume.
Step four, spore production and culture of the strains.
(1) Material preparation and sterilization: preparing a culture medium according to the culture medium formula A or the culture medium formula B, subpackaging and sterilizing, wherein the culture medium formula A needs 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 polypropylene plastic bags for producing edible fungi with the size of 35cm multiplied by 17cm, the filling amount is about 200g, and sterilization is carried out for 45 minutes at 121 ℃; the inoculation gun is simultaneously sterilized for use.
(2) Inoculation and culture: taking out the sterilized culture medium, inoculating the culture medium formula A to a sterilized inoculating gun device, inoculating the culture medium into a sterilized glass or plastic container, sealing with a sterile plastic film, adding 20mL of liquid strain into the container by using another inoculating gun after agar is solidified, and uniformly shaking; cooling the culture medium formula B to about 30 ℃, directly adding 10mL of liquid strain into the culture material under the aseptic condition, uniformly mixing, and flatly paving to the thickness of about 1.5 cm; after inoculation, the cells are placed in an incubator or a culture room at 30 ℃ for culture management.
And step five, collecting and preparing spore powder.
The culture medium formula A is agar plate culture medium, yellow spore is distributed on the surface layer of the culture medium, the yellow spore is collected by scraping and scraping, and then dried at 45 ℃ to obtain the spore powder of the xanthoma spore, and the spore yield is 20.15mg/cm2(ii) a The formula B of the culture medium 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 ℃, screened by a 60-mesh standard sieve, and undersize products are collected to be the spore powder of the xanthomonas mobilis, and the spore yield is 23.20mg/g dry material. And subpackaging the obtained xanthoma spore powder into plastic packaging bags, and carrying out vacuum packaging and storage.
Further, the raw material composition of the comprehensive PDA culture medium in the first step is that each liter of 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, and the sterilization condition of the culture medium is 121 ℃ and 30 minutes.
Further, the raw materials of the improved comprehensive PDA culture medium in the step two comprise: each liter of culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of potassium dihydrogen phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water, and the sterilization condition of the culture medium is 121 ℃ and 30 minutes.
Further, 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 phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; the medium was sterilized at 21 ℃ for 30 minutes.
Further, the medium formula A in the fourth step comprises the following components in percentage by weight: potato juice 20%, brown sugar 0.3%, glucose 0.5%, dipotassium hydrogen phosphate 1.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.1%, ammonium chloride 0.5%, agar 2%, and water in balance.
Further, the medium formula B in the fourth step comprises the following components in percentage by weight: 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 bolete dry powder, 0.5% of dipotassium phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and a material-water ratio of 1: 1.
Further, the culture management in the fourth step comprises controlling the culture temperature to be 25-30 ℃, having no special requirement on air relative humidity, growing white hyphae in the initial stage, culturing until 3-5 days when yellow powdery spores are produced, culturing until 7-10 days when yellow powdery spores are produced, and collecting the spores.
The preparation method of the fulvia fulvidraco spore powder has the purity higher than 99%.
The preparation method of the fulvia fulvidraco spore powder is used for preparing a medicament for stopping bleeding of trauma.
Compared with the prior art, the invention has the following beneficial effects.
1) The method utilizes an ultraviolet mutagenesis method to separate and breed strains to obtain the xanthomonas campestris strain, has stronger stress resistance, fast hypha growth and strong spore production capability, and the spore production can reach 21.15mg/cm2And a good technical foundation is laid for the preparation of the fulvia fulva spore powder product.
2) The preparation method of the xanthomonas campestris spore powder provides new resources for developing and utilizing medicinal fungi, solves the problem of large-scale preparation of the xanthomonas campestris spore powder, and provides a technical basis for subsequent development and utilization.
3) The preparation method of the xanthomonas campestris spore powder provides a xanthomonas campestris strain formula and a spore production culture technology, and can flexibly shorten the production period according to the production conditions.
Drawings
FIG. 1 shows the sequence of Thermomyces flavus rDNA-ITS of example 1.
Detailed Description
The invention is further described below with reference to the accompanying drawings and specific embodiments.
Example 1 isolation and characterization of the Chrysosporium parasiticum Hypomyces chrysospermus and study of its biological properties.
In the investigation research on western Liaoning macrofungi resources, a strain of boletus pathogenic bacteria is separated in 2012, the hypha grows flatly on the fruiting body of boletus, the initial infection stage is white, and the hypha stage is mainly adopted at the moment; in the late stage of infection, the fruiting body gradually decays to generate a large amount of yellow powder, and the spore production stage is carried out. 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.
Firstly, a culture medium.
Comprehensive PDA culture medium: the culture medium contains 200g of potato juice, 1.5 g of magnesium sulfate, 3 g of potassium dihydrogen phosphate, 20 g of glucose and 20 g of agar in percentage by mass, the balance is water, after the culture medium is subpackaged into triangular bottles, the pH of the culture medium is adjusted to 4.5, 5.5, 6.5, 7.5, 8.5, 9.5 and 10.5 by using 40% NaOH and 0.1mol/LHCl respectively in 7 bottles, and the culture medium is sterilized for 30 minutes at 121 ℃.
10 tubes of 10mL sterile water were filled for use.
Secondly, separating and identifying the strain.
(1) Strain separation: collecting Boletus edulis fruiting body infected with Xanthomonas flavivis in the field, placing under a 30W ultraviolet lamp, irradiating for 5min at an irradiation distance of 30cm, picking a ring of spore powder on the surface layer of the fruiting body, placing into a test tube filled with 10mL sterile water, oscillating uniformly, sucking 100 mu L of spore liquid by a pipette to a comprehensive PDA culture medium, scraping and coating uniformly, culturing at 30 ℃ for 4-7d, picking the edge of a target colony generating golden yellow spores, purifying for 2-3 times, and preserving for later use.
(2) Strain mutagenesis: under aseptic conditions, 10mL of sterile water was slowly added to the slant tube strain, after gentle shaking, the spore suspension was poured into a sterile plate (concentration about 1X 10)7cfu/g), placing under a 30W ultraviolet lamp, irradiating for 120s at a distance of 30 cm. Under aseptic condition, respectively sucking 100 μ L of spore suspension irradiated by ultraviolet into a comprehensive PDA plate, uniformly coating, culturing at 30 deg.C for 7d, picking colony edge with maximum spore yield, purifying for 2 times, and transferring to PDA slantCulturing until the tube is full, and preserving for later use. And sending the slant strains to Shanghai workers for ITS sequence analysis.
The bacterial colony is white in the initial stage and regular in edge, yellow spores begin to be generated after being cultured on PDA for 4-5 days, and the surface layer of the bacterial colony in the later stage is full of the yellow spores; its microscopic morphology is characterized by hypha with diaphragm, developed branch, creeping growth and nearly colorless. Conidiophores are short and have multiple branches. Conidium is born on the top of the sporophores, and the conidium is cylindrical or elliptical and has a diameter of 3.1-5.4 multiplied by 4.5-7.3 mu m; chlamydospores are unipartite, end on hyphal lateral branches, are spherical, have nodular protrusions on the surface layer, are colorless at first time, and then are yellow to golden yellow, and have extremely large number and diameter of 12.2-18.5 mu m. According to the characteristic of fungus morphology identification and the analysis result of rDNA-ITS sequence (shown in figure 1), the fungus is identified as a Chrysosporium parasitic fungus (Hypermyces chrysospermus) and the sexual generation thereof is a Chrysosporium parasitic fungus (Hypermyces chrysospermus Tul. & C.
And thirdly, researching the biological characteristics of the strains.
The biological characteristics of the strain are researched, and the test result shows that the optimum growth temperature of the xanthomonas is 25-30 ℃, the optimum pH value is 6.5, the strain is cultured on a PDA culture medium for 96 hours, the diameter of a bacterial colony can reach 69.18mm, and a large amount of yellow spores are generated. Above 35 deg.C, the strain is limited in growth, does not grow at 40 deg.C, but can recover growth when the temperature is reduced to below 30 deg.C, as shown in Table 1-2.
TABLE 1. influence of different culture temperatures on the growth of Onema flava.
Note: "-" indicates no growth; "+" indicates a small amount of sporulation, and "+ + +" indicates a large amount of sporulation.
TABLE 2. effect of growth of Onema fulva at different pH.
Note: "-" indicates no growth; "+" indicates low spore production, "+ + + +" indicates medium spore production, and "+ + + +" indicates high spore production.
Example 2 influence of different species types on sporulation.
Firstly, 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 potassium dihydrogen phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water, and after the culture medium is prepared, the culture medium is respectively filled into eggplant bottles, and each bottle contains 70 mL.
The formula of the liquid strain culture medium comprises: the potato juice beverage 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 monopotassium phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water, wherein each bottle is 50 mL.
Spore production medium formula a: potato juice 20%, brown sugar 0.3%, glucose 0.5%, dipotassium hydrogen phosphate 1.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.1%, ammonium chloride 0.5%, agar 2%, and water in balance.
And (3) sterilizing conditions of the culture medium: 121 ℃ for 30 minutes.
And II, seed preparation.
(1) The preparation method of eggplant bottle seeds comprises the following steps: inoculating the strain of Thermomyces fulva to the mother strain eggplant flask culture medium under aseptic condition, culturing in 30 deg.C constant temperature incubator for 7 days until the eggplant flask slant is full of strain to obtain seed; the liquid strain preparation method comprises the following steps: and (3) inoculating when the temperature of the sterilized liquid culture medium is reduced to below 30 ℃, dividing the eggplant bottle inclined plane seeds into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by using an inoculation shovel, transferring the eggplant bottle inclined plane seeds into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, culturing for 5 days at the temperature of 30 ℃ and at the rpm/min of 170, and obtaining the liquid stock culture when the ratio of the bacterial balls reaches more than 80 percent in volume.
Thirdly, spore production and culture.
And pouring the spore production culture medium into a sterilized plate with the diameter of 90mm, and cooling the plate for later use. Adding 100mL sterile water into eggplant bottle strain, scraping off superficial layer spore with inoculating needle, and making into spore suspension with concentration of 1 × 106cfu/mL, based onDiluting to 1 × 102cfu/mL. Adding 100mL of sterile water into the liquid strain, diluting to 1 × 102Individual pellet/mL. 0.1mL of the bacterial suspension or 0.1mL of the liquid strain diluent is added into each dish and is evenly smeared by a spatula. After inoculation, the mixture is placed in an incubator at the temperature of 30 ℃ for constant-temperature culture. The test results show that the liquid strains are obviously superior to spore suspension, the germination is fast, the growth is rapid, the spore yield is large, and the yield can be obviously reduced, which is shown in Table 3.
TABLE 3 influence of different strains on the growth and sporulation of P.xanthomatosa.
Note: "-" indicates no growth; "+" indicates low spore production, "+ + + +" indicates medium spore production, and "+ + + +" indicates high spore production.
Example 3 Effect of different culture methods and media on sporulation.
Firstly, preparing a culture medium.
(1) Mother culture medium composition: each liter of culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of potassium dihydrogen phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water. And (3) sterilizing conditions of the culture medium: 121 ℃ for 30 minutes.
(2) The formula of the liquid strain culture medium comprises: the potato juice beverage 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 monopotassium phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; and (3) sterilizing conditions of the culture medium: 121 ℃ for 30 minutes.
Spore production medium formula a: potato juice 20%, brown sugar 0.3%, glucose 0.5%, dipotassium hydrogen phosphate 1.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.1%, ammonium chloride 0.5%, agar 2%, and water in balance. Placing into a high temperature and high pressure resistant glass container with a filling amount of 50% of the container volume, and sterilizing at 121 deg.C for 30 minutes.
Spore production medium formula 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 bolete dry powder, 0.5% of dipotassium phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride, and a material-water ratio of 1: 1. placing into polypropylene plastic bags with size of 35cm × 17cm for edible fungus production, loading amount of about 200g, and sterilizing at 121 deg.C for 45 min.
The inoculation gun is simultaneously sterilized for use.
Secondly, preparing liquid strains.
Inoculating the strain of Thermomyces fulva to a mother strain eggplant flask culture medium under aseptic conditions, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, and culturing in a 30 deg.C constant temperature incubator for 7 days until the eggplant flask slope is full to obtain seeds; and (3) inoculating when the temperature of the sterilized liquid culture medium is reduced to below 30 ℃, dividing the eggplant bottle inclined plane seeds into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by using an inoculation shovel, transferring the eggplant bottle inclined plane seeds into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, culturing for 5 days at the temperature of 30 ℃ and at the rpm/min of 170, and obtaining the liquid stock culture when the ratio of the bacterial balls reaches more than 80 percent in volume.
Thirdly, spore production and culture of the strain.
Spore production medium formula a: taking out the sterilized culture medium, inoculating with sterilized inoculating gun, 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.
Spore production medium formula B: cooling to about 30 ℃, directly adding 10mL of liquid strain into the culture material under aseptic condition, uniformly mixing, and flatly paving to the thickness of about 1.5 cm.
After inoculation, the cells were placed in an incubator at 30 ℃. Sampling is carried out periodically in the peak period of sporulation and is reserved 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 collected by scraping and scraping with a scraper and then dried at 45 ℃ to obtain a sample of the spore powder of the xanthomonas campestris.
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 ℃, screened by a 60-mesh standard sieve, and the undersize product is collected to be the xanthomonas powder sample obtained from the xanthomonas spore powder.
And weighing the obtained xanthomonas campestris spore powder samples sampled in different culture periods, and calculating according to the following formula to obtain the yield of the xanthomonas campestris.
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 quantity is measured according to the change of the spore production quantity of a unit area.
The spore production efficiency of the culture medium formula B is closely related to the weight of the material, and the spore production is mainly measured by the change of the spore production of unit weight.
Test results show that the two culture modes can obtain better yield of the spore powder of the fulvia fulva, wherein the culture medium A is adopted for growth and spore production on the surface layer of the culture medium, so that the spore powder of the fulva with higher purity can be obtained, the spore production period is suitable for 7 days considering the production cost, and the spore production amount is 20.15mg/cm2The production period is short; although the growth cycle of the culture medium formula B is slightly long, the culture medium formula B has the advantages of simplicity and convenience in operation, wide applicability and the like, the production cycle is preferably 10 days by combining the production cost, and the spore yield is 23.20mg/g of dry materials, which is shown in Table 4.
TABLE 4. Effect of different strains on the growth and sporulation of P.xanthomatosa.
Note: "-" indicates no growth; "+" indicates low spore production, "+ + + +" indicates medium spore production, and "+ + + +" indicates high spore production.
Example 4 application of spore powder of fulvestrant.
The xanthomonas campestris spore powder obtained according to example 3 can be used for trauma hemostasis.
Can provide raw materials with the purity higher than 99 percent for the medicinal development of the spore powder of the xanthoma spore.
Sequence listing
<110> Liaoning province institute of microbiology and science
<120> preparation method of xanthoma 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 (10)
1. The fulvia fulvidraco strain is fulvia fulvidraco HLB01, and is preserved in China general microbiological culture Collection center at 9-11 th of 2020 with the preservation addresses as follows: the microbial research institute of China academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, with the collection number of CGMCC 20259.
2. A preparation method of a fulvia fulvidraco spore powder is characterized by comprising the following steps:
step one, strain breeding
(1) Strain separation: collecting Boletus edulis fruiting body infected with Thermomyces flavus in the field, placing under a 30W ultraviolet lamp, irradiating for 30cm, irradiating for 5min, picking a ring of spore powder on the surface layer of the fruiting body, placing into a test tube filled with 10mL sterile water, oscillating uniformly, sucking 100 mu L of spore liquid to a comprehensive PDA culture medium by using a pipettor, scraping and coating uniformly, culturing at 30 ℃ for 4-7d, picking the edge of a target colony generating golden yellow spores, purifying for 2-3 times, and preserving for later use;
(2) strain mutagenesis: under aseptic condition, slowly adding 10mL of sterile water into the slant tube strain, slightly vibrating, pouring the spore suspension into a sterile plate with the concentration of about 1 × 107cfu/mL, placing under a 30W ultraviolet lamp, irradiating for 120s with the irradiation distance of 30 cm; under the aseptic condition, after being properly diluted, the spore suspension subjected to ultraviolet irradiation is respectively sucked into a comprehensive PDA flat plate by 100 mu L, uniformly smeared, cultured at 30 ℃ for 7d, the edge of a bacterial colony with the maximum spore yield is picked, purified for 2 times, transferred to a PDA inclined plane test tube to be cultured until the tube is full, and preserved for later use; continuously carrying out passage for 5 times, and inspecting the spore yield of the strain;
step two, seed preparation
Inoculating the strain of Thermomyces fulva to a mother strain eggplant flask culture medium under aseptic conditions, wherein the mother strain culture medium is a comprehensive PDA enriched culture medium, and culturing in a 30 deg.C constant temperature incubator for 7 days until the eggplant flask slope is full to obtain seeds;
step three, liquid strain preparation
Inoculating when the temperature of the sterilized liquid culture medium is reduced to below 30 ℃, dividing the eggplant bottle slope seeds into inoculation blocks with the size of about 0.5cm multiplied by 0.5cm by using an inoculation shovel, transferring the eggplant bottle slope seeds into 5-6 blocks to 150mL of liquid strain culture medium under the aseptic condition, culturing for 5 days at the temperature of 30 ℃ and the rpm/min, wherein the ratio of bacteria balls reaches more than 80 percent of the volume ratio, and obtaining liquid stock seeds;
step four, spore production culture of strains
(1) Material preparation and sterilization: preparing a culture medium according to the culture medium formula A or the culture medium formula B, subpackaging and sterilizing, wherein the culture medium formula A needs 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 polypropylene plastic bags for producing edible fungi with the size of 35cm multiplied by 17cm, the filling amount is about 200g, and sterilization is carried out for 45 minutes at 121 ℃; simultaneously sterilizing the inoculation gun for later use;
(2) inoculation and culture: taking out the sterilized culture medium, inoculating the culture medium formula A to a sterilized inoculating gun device, inoculating the culture medium into a sterilized glass or plastic container, sealing with a sterile plastic film, adding 20mL of liquid strain into the container by using another inoculating gun after agar is solidified, and uniformly shaking; cooling the culture medium formula B to about 30 ℃, directly adding 10mL of liquid strain into the culture material under the aseptic condition, uniformly mixing, and flatly paving to the thickness of about 1.5 cm; after inoculation, placing the mixture in an incubator or a culture room at 30 ℃ for culture management;
step five, collecting and preparing spore powder
The culture medium formula A is an agar plate culture medium, yellow spores are distributed on the surface layer of the culture medium, are collected by scraping and scraping with a scraper and then are dried at 45 ℃ to obtain the xanthomonas campestris 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 ℃, screened by a 60-mesh standard sieve, and undersize products are collected to obtain the xanthomonas powder;
and subpackaging the obtained xanthoma spore powder into plastic packaging bags, and carrying out vacuum packaging and storage.
3. The method for preparing a powder of spore of Thermomyces fulva according to claim 2, wherein the raw materials of the integrated PDA culture medium in the first step comprise, per liter of the culture medium, 200g of potato juice, 1.5 g of magnesium sulfate, 3 g of potassium dihydrogen phosphate, 20 g of glucose, 20 g of agar, and the balance of water, and the sterilization condition of the culture medium is 121 ℃ for 30 minutes.
4. The method for preparing the xanthomonas powder of claim 2, wherein the raw materials of the improved comprehensive PDA culture medium in the second step comprise: each liter of culture medium contains 200g of potato juice, 2.5 g of peptone, 15 g of glucose, 3.6 g of potassium dihydrogen phosphate, 1.4 g of magnesium sulfate, 2.0 g of bolete dry powder, 20 g of agar and the balance of water, and the sterilization condition of the culture medium is 121 ℃ and 30 minutes.
5. The method for preparing the xanthomonas powder of claim 2, wherein the liquid strain culture medium 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 phosphate, 0.5% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and the balance of water; the medium was sterilized at 21 ℃ for 30 minutes.
6. The method for preparing the xanthomonas powder of claim 2, wherein the culture medium formula a in the fourth step comprises the following components in percentage by weight: potato juice 20%, brown sugar 0.3%, glucose 0.5%, dipotassium hydrogen phosphate 1.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.1%, ammonium chloride 0.5%, agar 2%, and water in balance.
7. The method for preparing the xanthomonas powder of claim 2, wherein the culture medium formula B in the fourth step comprises the following components in percentage by weight: 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 bolete dry powder, 0.5% of dipotassium phosphate, 0.3% of potassium dihydrogen phosphate, 0.1% of magnesium sulfate, 0.5% of ammonium chloride and a material-water ratio of 1: 1.
8. The method for preparing the xanthomonas campestris spore powder according to claim 2, wherein the culturing management in the fourth step comprises controlling the culturing temperature to be 25-30 ℃, no special requirement for the relative humidity of air, growing white hyphae at the initial stage, culturing until 3-5 days to show the generation of yellow powdery spores, culturing until 7-10 days to generate a large amount of yellow powdery spores, and collecting the spores.
9. The method for preparing the spore powder of the fulvia fulva according to the claims 2-8, wherein the purity of the spore powder of the fulva is higher than 99%.
10. The use of the spore powder of Thermomyces fulva prepared by the method of claims 2-8 in the preparation of a medicament for hemostasis of trauma.
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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 |
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