CN113481105B - Novel phomopsis fungus strain, preparation method and application - Google Patents
Novel phomopsis fungus strain, preparation method and application Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention provides a new strain of phomopsis fungi, belonging to the technical field of microorganisms, wherein the new strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2020394. The new strain of phomopsis has broad-spectrum antibacterial activity and antioxidant activity, and can be used for preparing agricultural or medical microbial antibacterial agent and antioxidant. The invention also provides a preparation method and application of the new strain of phomopsis fungi.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a novel phomopsis fungus strain, a preparation method and application thereof.
Background
Endophytes (endophytes) are fungi or bacteria that live inside the tissues and organs of healthy plants at some or all stages. Endophytic bacteria are ubiquitous in higher plants, woody, herbaceous, monocotyledonous and dicotyledonous plants. At present, endophytes of plants have become potential microbial pesticides in biological control, yield-increasing bacteria or are utilized as potential biocontrol carrier bacteria.
The national medicinal plant Yunnan stone catalpa (Gmelina arborea) is a Verbenaceae stone catalpa semi-deciduous tree, is a medicinal and edible plant of the Dai nationality, is also a precious fast-growing wood tree species, belongs to a national secondary protective plant, and has higher comprehensive utilization value. The Yunnan shicatalpa wood has excellent preservative property and is an important medicinal plant in Dai nationality and other minority regions. Dai nationality uses bark or heartwood as a medicine and is mainly used for treating cough, pharyngalgia, skin pruritus and eczema. The Hani nationality uses bark as medicine and has the functions of stopping bleeding, diminishing inflammation, eliminating swelling, etc. The Yunnan catalpa ovata has higher medicinal and economic values, but the research on the plant mainly focuses on the separation and identification of the pharmacological activity and chemical components of the extract, and the research report on the Yunnan catalpa ovata endophytic fungi is not found so far.
Disclosure of Invention
The invention aims to provide a new strain of phomopsis fungi, which has broad-spectrum antibacterial activity and antioxidant activity and can be used for preparing agricultural or medical microbial antibacterial agents and antioxidants.
The invention also provides a preparation method and application of the new strain of phomopsis fungi.
The invention is realized by the following technical scheme:
a new strain of phomopsis fungi is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2020394.
Furthermore, the ITS nucleotide sequence of the new strain is shown as SEQ ID NO. 1. The specific nucleotide sequence is as follows:
GATGGCTGGACGCGCTTCGGCGCACCCAGAAACCCTTTGTGAACTTATACCTATACTGTTGCCTCGGCGCTGGCCGGCCTCCTCACCGAGGCCCCCTGGAGACAGGGAGCAGCCCGCCGGCGGCCAAACAAACTCTTGTTTCTTAGTGAATCTCTGAGTAAAAAACATAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGAGGGCATGCCTGTTCGAGCGTCATTTCAACCCTCAAGCCTGGCTTGGTGTTGGGGCACCGCCTTTGGAAAAGGGCGGGCCCTGAAATCTAGTGGCGAGCTCGCCAGGACCCCGAGCGTAGTAGTTATATCTCGTTCTGGAAGGCCCTGGCGGTGCCCTGCCGTTAAACCCCCAACTTCTGAAATTTTGACCTCGGATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAAAAGGCCGGAGGAA。
based on the same inventive concept, the invention also provides the application of the novel phomopsis fungus strain in preparing the pathogenic microorganism antibacterial agent and/or antioxidant.
Based on the same invention concept, the invention also provides a preparation method of the new strain of phomopsis fungi, and the new strain of phomopsis fungi is extracted from catalpa yunnanensis.
Based on the same invention concept, the invention also provides a preparation method of the new strain of phomopsis fungi, and the preparation method comprises the following steps:
inoculating the Yunnan shicata fresh leaf slice tissues to a culture medium for culture;
and (3) selecting and culturing new hypha growing in the culture medium at the edge of the fresh Yunnan shicatalpa leaf slice tissue to obtain a new phomopsis fungus strain.
Based on the same invention concept, the invention also provides a preparation method of the fermentation crude extract of the new strain of the phomopsis fungi, and the preparation method comprises the following steps:
inoculating the new strain of the phomopsis fungi into a PDA culture medium, and performing activated culture to obtain an activated strain;
inoculating the activated strain to a PDB culture medium for culture to obtain a seed solution;
inoculating the seed solution into a PDB culture medium for shake cultivation, wherein the inoculation amount is 8-12% (v/v), and obtaining fermentation liquor;
adding an organic solvent into the fermentation liquor for extraction, taking the upper layer extract for decompression and concentration until the extract is dried, and adding sterile water to obtain a fermentation crude extract of the new strain of the phomopsis fungi.
Optionally, the organic solvent is ethyl acetate.
Based on the same invention concept, the invention also provides the application of the fermentation crude extract of the new strain of the phomopsis fungus in inhibiting pathogenic microorganisms.
Optionally, the pathogenic microorganisms include at least one of staphylococcus aureus, salmonella typhi, methicillin-resistant staphylococcus aureus, candida albicans, fusarium solani, colletotrichum malorum, gibberella tritici, and botrytis cinerea.
Based on the same invention concept, the invention also provides the application of the fermentation crude extract of the new strain of the phomopsis fungus in removing DPPH free radicals and/or ABTS + free radicals.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
1. the invention relates to a new Phomopsis fungus strain, which is named as Phomopsis sp.YE3350, and a crude fermentation extract of the strain Phomopsis sp.YE3350 has obvious antibacterial activity on 8 pathogen indicator bacteria, has stronger scavenging capacity on DPPH free radicals and ABTS + free radicals, has stronger antioxidant activity, and has potential application in preparing new agricultural or medical microbial antibacterial agents and antioxidants.
2. The invention relates to a preparation method of a novel Phomopsis fungus strain, which is characterized in that a strain Phomopsis sp.YE3350 is extracted from catalpa yunnanensis and used as an endophyte of the catalpa yunnanensis, has broad-spectrum antibacterial activity and antioxidant activity, can be used for preparing an agricultural or medical microbial antibacterial agent and an antioxidant, and realizes the effective utilization of catalpa yunnanensis resources.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on the drawings without creative efforts.
FIG. 1 is a colony morphology of Phomopsis sp.YE3350 of the present invention on PDA medium;
FIG. 2 is a colony morphology of Phomopsis sp.YE3350 of the present invention on a check medium;
FIG. 3 is a colony morphology of Phomopsis sp.YE3350 of the present invention on MEA medium;
FIG. 4 is a colony morphology of Phomopsis sp.YE3350 of Phomopsis of the present invention on COB medium;
FIG. 5 is a colony morphology of Phomopsis sp.YE3350 of the present invention on CD medium;
FIG. 6 is a colony morphology of Phomopsis sp.YE3350 of the present invention on corn medium;
FIG. 7 is a phylogenetic tree of Phomopsis sp.YE3350 of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
in order to effectively develop plant endophyte resources, the applicant takes catalpa yunnanensis as a raw material, and separates and extracts a new endophyte Phomopsis fungus strain with the name of Phomopsis sp.YE3350, the preservation unit is China center for type culture Collection (CCTCC for short), the preservation address is China, Wuhan university, the preservation date is 2020, 8 and 3 days, the preservation number is CCTCC NO.M 2020394, and the taxonomic name is Phomopsis sp.
Phomopsis (Sacc.) Bub-k) is a large group of fungi, and plays an important role in the classification of fungi. The fungi of this genus have a wide distribution range, but are predominantly in tropical and subtropical regions. Phomopsis is diverse in species, is one of the ubiquitous main groups of endophytic fungi of plants, and can generate secondary metabolites with rich structural types and various biological activities, such as antibacterial, antiviral, anti-inflammatory, anti-tumor cells, phytotoxicity, enzyme inhibition activity and the like. Therefore, Phomopsis fungi are considered to be one of the important sources for obtaining pharmaceutically active natural products.
The Phomopsis sp.YE3350 and the crude fermentation extract thereof have antibacterial activity and antioxidant activity, and can be used for preparing agricultural or medical antibacterial agents and antioxidants through fermentation.
A novel strain of Phomopsis fungus of the present application will be described in detail below with reference to examples and experimental data.
Example 1
The method for separating and culturing the new strain of the phomopsis fungi specifically comprises the following steps:
(1) washing the leaves of freshly collected and disease-free Yunnan shicatalpa ovata with tap water to remove mud and dust attached to the surfaces. Placing the dried plant material into an ultra-clean workbench sterilized by ultraviolet rays, and selecting 75% of alcohol and 2% of sodium hypochlorite as surface disinfectants. Soaking plant material in 75% alcohol for 30-50s, and rinsing with sterile water for 2-3 times; treating with 2% sodium hypochlorite for 1-2min, and rinsing with sterile water for 3 times. Sterile filter paper blotted dry.
(2) Cutting the Yunnan stone catalpa leaves sterilized in the step (1) into small pieces of about 1 × 1cm, inoculating plant leaf tissues on a PDA culture medium, and culturing at constant temperature of 28 + -2 ℃. The PDA culture medium comprises 200g of peeled potatoes, 20g of glucose, 15g of agar and 1000mL of distilled water, and has natural pH.
(3) Observing the growth condition of the bacterial colony on the surface of the culture medium of the plant tissue block, picking up the hypha newly grown at the edge of the tissue block onto a fresh PDA culture medium plate by using a sterile bamboo stick, culturing at the constant temperature of 28 +/-2 ℃, taking a picture of the plate when a single colony of the picked endophytic fungus Phomopsis sp.YE3350 grows to the diameter of about 4-5cm in the plate, and transferring to an inclined plane for preservation.
(4) The endophytic fungus Phomopsis sp.YE3350 grows faster on a PDA culture medium, and after the endophytic fungus is cultured for 3-4 days at 28 ℃, bacterial colonies begin to thicken and become rough; culturing for 7-8 days, wherein the diameter of the colony reaches 7.6cm, the colony is grey white, the aerial hyphae are dense, the colony is round and has irregular edges, and is wavy, and a black carrier begins to form on the colony about 10 days. The colony morphology of the endophytic fungus Phomopsis sp.YE3350 in PDA medium is shown in FIG. 1.
(5) The endophytic fungus Phomopsis sp.YE3350 grows slowly on a check culture medium, is cultured for 10 days at 28 ℃, has the diameter of a bacterial colony of 6.3cm, has uneven thickness growth of the bacterial colony, is in a state of gradually thickening from the middle to the outer edge, and has irregular edges; the hyphae are grey white velvet, and no black carrier is seen after 15 days of culture. The colony morphology of the endophytic fungus Phomopsis sp.YE3350 in the look-up medium is shown in FIG. 2.
(6) The endophytic fungus Phomopsis sp.YE3350 grows faster on an MEA culture medium, the culture is carried out for 7-8 days at the temperature of 28 ℃, the diameter of a bacterial colony reaches 7.8cm, the bacterial colony forms a concentric circle, and the edge is in a regular wave shape; white hyphae appear; after 15 days of culture, sporangia appeared. The colony morphology of the endophytic fungus Phomopsis sp.YE3350 in MEA medium is shown in FIG. 3.
(7) The endophytic fungus Phomopsis sp.YE3350 grows faster on a COB culture medium, is cultured and cultured for 7-8 days at the temperature of 28 ℃, the diameter of a colony reaches 8.1cm, the hyphae are compact, and the aerial hyphae are flocculent and are easy to pick up; the bacterial colony is white and the surface is not smooth; no black colonies appeared after 15 days of culture. The colony morphology of Phomopsis sp.YE3350 on COB medium is shown in FIG. 4.
(8) The endophytic fungus Phomopsis sp.YE3350 grows slowest on a CD culture medium, is cultured and cultured for 10 days at the temperature of 28 ℃, the diameter of a colony reaches 5.7cm, the surface of hypha is smooth, and an obvious white flocculent colony with the diameter of 1cm is formed in the middle; the surrounding bacterial colonies are loose and have irregular edges, and hyphae are thin; no black colonies appeared after 15 days of culture. The colony morphology of Phomopsis sp.YE3350 in CD medium is shown in FIG. 5.
(9) The endophytic fungus Phomopsis sp.YE3350 grows faster on a corn culture medium, is cultured and cultured for 7-8 days at 28 ℃, the diameter of a colony reaches 7.4cm, the surface is not smooth, and hyphae are villous; the bacterial colony grows unevenly, the periphery is faster, the hyphae are thinner, and the growth is looser; no black colonies appeared after 15 days of culture. The colony morphology of Phomopsis sp.YE3350 in corn medium is shown in FIG. 6.
(10) Molecular biological characteristics of the endophytic fungus Phomopsis sp.YE3350: by adopting molecular biology PCR technology and DNA sequence determination analysis, the I TS rDNA genome of the strain YE3350 consists of 554 bases (bp). A phylogenetic tree was constructed using Mega 6.0 software, and strain YE3350 was assigned to the genus Phomops i s. The phylogenetic tree of strain YE3350 is shown in FIG. 7.
Example 2
A preparation method of a Phomopsis sp.YE3350 fermentation crude extract comprises the following steps:
(1) activation of the strain: taking out the test tube in which the strain Phomopsis sp.YE3350 is stored, picking out a small amount of hypha with a bamboo stick under aseptic condition, inoculating into the test tube filled with sterilized PDA culture medium, and culturing in an incubator at 28 + -2 deg.C for 7 days to obtain activated strain.
(2) Preparing a PDB culture medium: weighing 200g of peeled potato, cutting into small pieces, adding 1000mL of distilled water, boiling, filtering with gauze to obtain a potato filtrate, adding 20g of glucose, naturally adjusting pH, and sterilizing at 121 ℃ for 30min to obtain a PDB culture medium for later use, wherein the concentration of the potato in the PDB culture medium is 0.2g/mL, and the concentration of the glucose is 0.02 g/mL.
(3) Preparing a seed solution: under aseptic condition, picking a small amount of hypha from the activated strain with bamboo stick, inoculating into sterilized PDB culture medium, and culturing at 28 + -2 deg.C for 3-4 days to obtain seed liquid of Phomopsis sp.YE3350 strain.
(4) Fermentation: 100ml of the prepared PDB culture medium is filled into a 500ml triangular flask and sterilized for 30 minutes at 121 ℃ for later use. A seed solution of Phomopsis sp.YE3350 strain is taken and inoculated into a 500ml triangular flask filled with 100ml PDB culture medium under the aseptic condition, the inoculum size is calculated according to 10 percent (v/v), and the mixture is placed into a shaking table (200r/min) at 28 +/-2 ℃ for culture for 7 days, and the existence of the contamination phenomenon is checked during the culture.
(5) After fermentation, adding equal volume of ethyl acetate into the fermentation liquor for extraction, taking the upper layer extract, concentrating under reduced pressure until the extract is dry, and adding sterile water to prepare a fermentation crude extract.
Example 3
The strain Phomopsis sp.YE3350 is used for measuring the anti-pathogenic activity and the in vitro antioxidant capacity of a fermentation crude extract:
(1) and (3) determination of antibacterial activity:
the 8 pathogenic indicator bacteria used include 3 pathogenic bacteria of human body: staphylococcus aureus (Staphylococcus aureus), Salmonella typhi (Salmonella typhimurium), methicillin-resistant Staphylococcus aureus (MRSA); 1 pathogenic fungi of human body: candida albicans (Candida albicans) and 4 phytopathogenic fungi: fusarium solani (Fusarium solani), apple anthracnose (Glomeella cingulata), Gibberella tritici (Gibberella sautiniii), and Botrytis cinerea (Botrytis cinerea). The fungus culture medium is a PDA culture medium; the bacterial culture medium is LB solid culture medium.
The growth inhibition activity of the crude fermentation extract of the strain Phomopsis sp.YE3350 on 8 pathogenic microorganisms is measured by adopting a filter paper diffusion method, and the diameter of a filter paper is 7 mm. Respectively sucking 0.2mL of test indicator bacterial suspension under aseptic condition, adding corresponding solid culture medium to prepare a bacterial-containing plate, then putting a sterile filter paper sheet dipped with a fermentation liquid sample into the plate, placing bacteria in a temperature of 37 +/-1 ℃, placing fungi in a temperature of 28 +/-1 ℃, culturing for 48-72h, and respectively measuring the diameter of a bacteriostatic zone.
(2) DPPH radical scavenging capacity:
1mg of a sample to be measured was weighed and dissolved in 2mL of DMSO (dimethyl sulfoxide) to obtain a sample mother solution of 0.5 mg/mL. Then, the prepared mother liquor is diluted to 5-50 mu g/mL. 7.886mg of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) were weighed out and dissolved in 100mL of DMSO solution to give a 0.2mM DPPH stock solution. 1mL of the prepared DPPH mother solution was added to compound solutions of different concentrations (3mL, 5-50. mu.g/mL) and reacted for 30 minutes in the dark. The reacted sample (200. mu.L) was put in a 96-well plate, and the absorbance at 517nm was measured with a microplate reader. DMSO as negative control, vitamin C (V)C) And vitamin E (V)E) Is a positive control.
The measured data are expressed by the formula [ (Ac-As)/Ac]100% DPPH radical clearance K (%) of the samples tested was calculated. Ac is the absorbance of the negative control DMSO at 517nm, As is the addition of the test sample or the positive control VCAnd VEAbsorbance measured at 517 nm. According toCalculating IC of the test sample from its free clearance data50(median inhibitory concentration) value.
(3) ABTS + radical scavenging ability:
weighing 1mg of sample to be detected, adding 2mL of DMSO, and dissolving to obtain 0.5mg/mL of sample mother solution. Then, the prepared mother liquor is diluted to 5-50 mu g/mL. Weighing 109.736mg ABTS+(2, 2-Aza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) and 66.2284mg of potassium persulfate were dissolved in 100mL of water and reacted at room temperature in the dark for 12 hours to obtain ABTS+The mother liquor was at a concentration of 0.2 mM. Absorb 1mL of configured ABTS+The mother liquor was added to diluted test sample solutions of various concentrations (3mL,5-50g/mL) and reacted for 10 minutes under dark conditions. 200. mu.L of the reacted sample was placed in a 96-well plate, and the absorbance at 734nm was measured using a microplate reader. DMSO was a negative control in this assay, VCAnd VEIs a positive control. The measured data are expressed by the formula [ (Ac-As)/Ac]X 100% DPPH radical clearance K (%) of the test samples was calculated, respectively. Ac is the absorbance of the negative control DMSO at 517nm, As is the addition of the test sample or the positive control VCAnd VEAbsorbance measured at 734 nm. Calculating the IC of the test sample from its free clearance data50The value is obtained.
The results of the diameter of the inhibition zone of the crude fermentation extract of the endophytic fungi Phomopsis sp.YE3350 of the invention on 8 pathogenic indicator bacteria are shown in Table 1.
TABLE 1 inhibitory Activity of crude fermentation extract of Phomopsis sp.YE3350 on 8 pathogenic indicator bacteria
The crude fermentation extract of the endophytic fungi Phomopsis sp.YE3350 has the effects of removing DPPH free radicals and ABTS+The results of radical scavenging are shown in table 2.
TABLE 2 free radical scavenging ability of crude fermentation extract of the strain Phomopsis sp.YE3350
The experimental results show that the crude fermentation extract of the strain Phomopsis sp.YE3350 has obvious antibacterial activity on 8 pathogenic indicator bacteria and can resist DPPH free radicals and ABTS+Has stronger scavenging ability of free radicals and stronger antioxidant activity, and has potential application in preparing novel agricultural or medical microbial antibacterial agents and antioxidants.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<110> university of Yunnan
<120> a new strain of phomopsis fungus, preparation method and application
<160> 1
<170> SIPOSequenceListing 1.0
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<213> ITS sequence (1)
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gatggctgga cgcgcttcgg cgcacccaga aaccctttgt gaacttatac ctatactgtt 60
gcctcggcgc tggccggcct cctcaccgag gccccctgga gacagggagc agcccgccgg 120
cggccaaaca aactcttgtt tcttagtgaa tctctgagta aaaaacataa tgaatcaaaa 180
ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcgaa atgcgataag 240
taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgccctctg 300
gtattccgga gggcatgcct gttcgagcgt catttcaacc ctcaagcctg gcttggtgtt 360
ggggcaccgc ctttggaaaa gggcgggccc tgaaatctag tggcgagctc gccaggaccc 420
cgagcgtagt agttatatct cgttctggaa ggccctggcg gtgccctgcc gttaaacccc 480
caacttctga aattttgacc tcggatcagg taggaatacc cgctgaactt aagcatatca 540
aaaggccgga ggaa 554
Claims (6)
1. Phomopsis (A. phomopsis)Phomopsissp.) YE3350, which is characterized in that the phomopsis YE3350 is preserved in China center for type culture Collection with the preservation number of CCTCC NO. M2020394.
2. Use of a phomopsis YE3350 as defined in claim 1 for the preparation of an antimicrobial agent and/or an antioxidant agent against pathogenic microorganisms.
3. A method for preparing a crude fermentation extract of phomopsis YE3350 according to claim 1, which comprises:
inoculating phomopsis fungi YE3350 into a PDA culture medium, and performing activated culture to obtain activated strains;
inoculating the activated strain to a PDB culture medium for culture to obtain a seed solution;
inoculating the seed solution into a PDB culture medium for shake culture, wherein the inoculation amount is 8-12% of the volume percentage of the seed solution to the PDB culture medium, and obtaining fermentation liquor;
and adding an organic solvent into the fermentation liquor for extraction, wherein the organic solvent is ethyl acetate, taking the upper layer of extraction liquid, concentrating under reduced pressure until the extraction liquid is dried, and adding sterile water to obtain a fermentation crude extract of phomopsis YE 3350.
4. Use of a crude fermentation extract based on phomopsis YE3350 prepared according to claim 3 for the inhibition of pathogenic microorganisms.
5. Use according to claim 4, characterised in that said pathogenic microorganisms comprise Staphylococcus aureus (S.aureus)Staphylococcus aureus) Salmonella typhi (A), (B)Salmonella typhimurium) Candida albicans (C.albicans) (C.albicans)Candida albicans) Fusarium solani (F.sp.), (Fusarium solani) Pleurotus ostreatus (A), (B)Glomerella cingulata) And Gibberella (C.elegans)Gibberella saubinetii) And Botrytis cinerea (Botrytis cinerea) At least one of (1).
6. Use of a crude fermentation extract based on phomopsis YE3350 prepared according to claim 3 for scavenging DPPH free radicals and/or ABTS + free radicals.
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