CN114214205B - Antrodia camphorata symbiotic fungus AcEF007 and separation method thereof - Google Patents
Antrodia camphorata symbiotic fungus AcEF007 and separation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses antrodia camphorata symbiotic fungus AcEF007 and a separation method thereof, wherein an ITS gene sequence of the antrodia camphorata symbiotic fungus AcEF007 comprises a nucleotide sequence shown as SEQ ID No.1, and the specific preservation number is CCTCC NO: M20211093. The antibacterial activity experiment shows that the AcEF007 bacterial liquid extract has better antibacterial activity on bacteria such as klebsiella pneumoniae, pseudomonas cupuresis, staphylococcus aureus, escherichia coli, acinetobacter baumannii, staphylococcus hemolyticus and the like, has better inhibition effect on various pathogenic bacteria, and provides a new way for relieving the threat of pathogenic bacteria drug resistance to human health and researching and developing new antibacterial drugs.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to antrodia symbiotic fungus AcEF007 and a separation method thereof.
Background
The screening of bioactive components or drug lead compounds from natural products is currently one of the effective ways to study the creation of new drugs, microorganisms are an important source of new natural products, and secondary metabolites are formed through a variety of biosynthetic and metabolic pathways. The plant endophytes invade into plant tissues to grow, but do not cause obvious negative effect on the host, and in the long-term co-evolution process, symbiotic bacteria and host plants form special physiological metabolism ways to generate active compounds with various structures, so that the host can be helped to enhance the resistance to insects, diseases, drought, plant pathogenic bacteria and the like, and the research on secondary metabolites of the symbiotic bacteria is important to the development of new antibacterial drugs.
Through long-term natural evolution, complex antagonism or symbiotic relation is established between pathogenic microorganisms and various species, so that the pathogenic microorganisms can not only cause certain specific diseases, but also serve as conditional pathogenic bacteria, and dysbacteriosis or bacterial translocation occurs when the host immunity is low and the barrier function of the organism is destroyed, so that serious infection of the organism is caused. In order to kill germs or inhibit the activity of germs, antibiotics and synthetic antibacterial drugs are invented, and with the wide application of the antibacterial drugs, pathogenic bacteria generate drug resistance to the antibiotics, even multiple drug resistance occurs, and the occurrence and spread of the drug resistance of pathogenic bacteria form a serious threat to human health. Based on market and research requirements, finding new biological resources with strong antibacterial activity and high-efficiency utilization becomes an important point of research.
Studies have shown that active substances similar to or the same as the active ingredients of host plants are often found in plant endophytic fungi metabolites, wherein part of the active substances are antibacterial substances, and natural active substances with antibacterial, antioxidant, antifungal and the like are found in various plant symbiotic bacteria such as chinaberry, red sage root, dendrobium candidum, shoe-shaped gold ingot and the like. Antrodia camphorate (Antrodia cinnamomea) is special rare medicinal fungus in Taiwan, and is parasitic on inner wall of trunk core material or wet surface of lodged trunk of wild Cinnamomum camphora (Cinnamomum kanehirai), contains various physiologically active substances such as terpenes, polysaccharides, sterols and fatty acids, and has anticancer and antiinflammatory effects.
Specific active strains are obtained through research and separation, which is beneficial to the further development of the pharmaceutical industry.
Disclosure of Invention
The invention mainly aims to provide the antrodia camphorata symbiotic fungus AcEF007 and the separation method thereof, and the antrodia camphorata symbiotic fungus AcEF007 and the culture or processed product thereof obtained by the method have wide antibacterial and anti-inflammatory effects, and provide a new way for researching and developing new antibacterial drugs.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the ITS gene sequence of the antrodia camphorata symbiotic fungus AcEF007 comprises a nucleotide sequence shown in SEQ ID No. 1.
Further, the preservation number of the Antrodia camphorata symbiotic fungus AcEF007 (phoma. Sp) is CCTCC NO: M20211093, and the preservation name is phoma. Sp of Phoma; the Chinese culture medium is preserved in China center for type culture Collection, and the preservation address is the university of Wuhan in China; preservation date: 2021, 09, 11.
Further, the invention provides a method for separating antrodia camphorata symbiotic fungus AcEF007, which comprises the following steps:
(1) Washing Antrodia camphorate fruiting body with tap water for 36h, transferring into a sterile workbench, and washing the fruiting body with 3L sterile water;
(2) Taking fruiting bodies by using a sterilizing knife, placing the fruiting bodies into a centrifuge tube, adding ultrapure water, vibrating and crushing in a cell crushing homogenizer, diluting the crushed liquid by one tenth, one hundredth, one thousandth and one ten thousandth, smearing the diluted fruiting body suspension on a MYKAS antibacterial culture medium, and placing the inoculated culture medium into a constant temperature incubator at 26 ℃ for culture;
(3) And (3) transferring the mycelium growing in the culture medium to a MY culture medium for culturing under a microscope, continuously observing for 10 days, and carrying out subsequent molecular identification on the selected strain to obtain the antrodia camphorata symbiotic fungus AcEF007.
Further, the present invention provides a process for preparing a fermentation broth extract of AcEF007 comprising the steps of:
(1) Adding 1000ul of PG liquid into 2ml centrifuge tubes, placing 3 sterilized steel balls in each centrifuge tube, scraping appropriate amount of grown Antrodia camphorata symbiotic fungus AcEF007 mycelium, placing into the centrifuge tubes, crushing for 90S, inoculating 500ul of crushed liquid into each conical flask filled with PG liquid culture medium, placing at 28deg.C at 150 r.min -1 Culturing for 8d in a shaker;
(2) Extracting mycelium of the culture, obtaining mycelium of Antrodia camphorata symbiotic fungi AcEF007 and a culture solution after fermentation culture is completed, separating the mycelium from the bacterial solution by using a separating funnel, adding ethyl acetate into the bacterial solution, shaking uniformly for 6min, standing for 12h after ultrasonic treatment for 30min, recovering layered lower-layer waste liquid, condensing, refluxing and drying an upper-layer extraction solution by using a rotary evaporator, and obtaining a fermentation solution extract of the Antrodia camphorata symbiotic fungi AcEF007 culture.
Preferably, in the step (2), ethyl acetate is added to the bacterial liquid according to a volume ratio of 1:1.
Preferably, the PG liquid culture medium comprises the following components: 5g/L potato soaked powder, 5g/L yeast powder, 20g/L glucose, 0.5g/L magnesium sulfate heptahydrate, 1g/L potassium dihydrogen phosphate and vitamin B 1 0.1g/L。
According to the invention, by utilizing an Antrodia camphorata screening technical scheme, symbiotic bacteria of Antrodia camphorata are separated and screened, and antibacterial activity detection is carried out on the symbiotic bacteria to obtain antibacterial active compounds generated by mycelium, so that a fungus AcEF007 with antibacterial activity is obtained. The antibacterial activity experiment shows that the AcEF007 bacterial liquid extract has better antibacterial activity on bacteria such as klebsiella pneumoniae, pseudomonas cupuresis, staphylococcus aureus, escherichia coli, acinetobacter baumannii, staphylococcus hemolyticus and the like, has better inhibition effect on various pathogenic bacteria, and provides a new way for relieving the threat of pathogenic bacteria drug resistance to human health and researching and developing new antibacterial drugs.
The fungus AcEF007 obtained by the invention grows well on the MY culture medium, the surface of the colony is villiated, hyphae grow radially and circumferentially in white in the early growth stage, hyphae are denser in the late growth stage, the fungus layer is thicker, the color is slightly reddish, the colony is in a regular circular shape, the fungus has special biological properties, the same properties as those of the fungus of the invention are not found in the existing same species, and the fungus has excellent antibacterial performance.
Drawings
FIG. 1 is a graph showing the growth of the hyphae of AcEF007 according to the present invention;
FIG. 2 is a diagram of mycelia under an AcEF007 fluorescence microscope;
FIG. 3 is a graph showing the inhibition of AcEF007 against Klebsiella pneumoniae in the present invention, wherein a, b, c represent control groups and d represents experimental groups;
FIG. 4 is a graph showing the inhibition of Pseudomonas cupurensis by AcEF007 according to the present invention, wherein a, b, c represent control groups and d represents experimental groups;
FIG. 5 is a graph showing the inhibition of Staphylococcus aureus by AcEF007 of the present invention, wherein a, b, c represent control groups and d represent experimental groups;
FIG. 6 is a graph showing the inhibitory effect of AcEF007 on Escherichia coli, wherein a, b and c represent control groups and d represents experimental groups;
FIG. 7 is a graph showing the inhibition of AcEF007 on Acinetobacter baumannii, wherein a, b and c represent control groups, and d represents experimental groups;
FIG. 8 is a graph showing the inhibition of Staphylococcus hemolyticus by AcEF007 of the present invention, wherein a, b, c represent control groups and d represent experimental groups;
FIG. 9 is a display diagram of a phylogenetic tree of Antrodia camphorata symbiotic fungi AcEF007 constructed based on ITS.
Detailed Description
The invention is further described below with reference to the drawings and examples, wherein the chemicals used in the invention are all of analytical grade and commercially available.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1
The invention screens out and obtains an Antrodia camphorata symbiotic fungus AcEF007 (Phoma. Sp.) which is preserved in China center for type culture collection, and the strain is obtained by separating and purifying Antrodia camphorata fruiting bodies. As shown in figures 1 and 2, fungi grow well on MY culture medium, the surface of the bacterial colony is villiated, hypha grows radially in white, the hypha grows densely in the late growth stage, the bacterial layer is thicker, the color is slightly reddish, the bacterial colony is in a regular round shape, as shown in figures 3-8, the fermentation broth extract of the bacterial strain culture has good antibacterial activity on pathogenic bacteria such as Klebsiella pneumoniae, pseudomonas cupurensis, staphylococcus aureus, escherichia coli, acinetobacter baumannii, staphylococcus haemolyticus and the like, and the detected pathogenic bacteria are purchased from the center for monitoring and controlling the drug resistance of the bacteria in Guangdong province. Provides a new way for researching and developing new antibacterial drugs in order to relieve the threat of pathogenic bacteria drug resistance to human health.
Example 2
Isolation and identification of Antrodia camphorate symbiotic fungus AcEF007 strain
1. Isolation of Antrodia camphorata symbiotic bacteria
Washing Antrodia camphorate fruiting body with tap water for 36h, transferring into a sterile workbench, and washing the fruiting body with 3L sterile water.
Placing 2g Antrodia camphorate fruiting body into a 2ml centrifuge tube with a sterilizing knife, adding 500 μl of sterile water, crushing in a cell crushing homogenizer, diluting the crushed solution by one tenth, one hundredth, one thousandth and one thousandth, plating 200ul suspension onto MYKAS (antibacterial) medium, and placing the grafted medium in a constant temperature incubator at 26 ℃ for culturing; and (3) transferring the mycelium growing in the culture medium to a MY culture medium for culturing under a microscope, continuously observing for 10 days, and carrying out subsequent molecular identification on the selected strain to obtain the antrodia camphorata symbiotic fungus AcEF007.
MYKAS antibacterial medium: 21g/L of yeast malt extract broth, 15g/L of agar, 50ug/mL of ampicillin, and 50ug/mL of kanamycin; MY medium: yeast malt extract broth 21 g/L+agar 15g/L (BD Co., USA)
2. Identification of Antrodia camphorata symbiotic bacteria
(1) Identification of morphology
Fungi grow well on a MY culture medium, the surface of a colony is villiated, hyphae grow radially to the periphery in white in the early growth stage, hyphae are denser in the later growth stage, a fungus layer is thicker, the color is slightly reddish, and the colony is in a regular round shape.
(2) DNA extraction
Before the test, a CTAB water bath is used for more than 30min at 65 ℃, and a quick-freezing centrifuge is started 20min in advance; taking mycelium of the isolation and purification culture of Antrodia camphorata symbiotic bacteria, adding 3 sterilized steel balls into a 2ml centrifuge tube, soaking the centrifuge tube in a stainless steel tank filled with liquid nitrogen, cooling for 6min, crushing for 1.5min by a crusher, and crushing the mycelium; adding 1ml of CTAB and 20ul of beta-mercaptoethanol into a centrifuge tube, shaking uniformly (10 min), placing into a water bath for 5h, shaking uniformly for 1 time every 10min; after the water bath is finished, putting the mixture into a quick-freezing centrifuge at the temperature of 4 ℃ and 12000r/min for centrifugation for 10min; transferring 1ml of the supernatant into a new 2ml centrifuge tube, adding 1ml (phenol: chloroform: isoamyl alcohol=25:24:1), shaking for 10min, and centrifuging for 10min (repeated twice); transferring 1ml of supernatant into a new 2ml centrifuge tube, adding 1ml (chloroform: isoamyl alcohol=24:1), shaking uniformly for 10min, and centrifuging in a quick-freezing centrifuge at 4deg.C 12000r/min for 10min; transferring 500ul of supernatant into a new 1.5ml centrifuge tube, adding 50ul NaAc and 1ml absolute ethanol (-20 ℃), shaking, marking, and precipitating in a refrigerator at-20deg.C for 12 hr; centrifuging the precipitate in a quick-frozen centrifuge at 4deg.C/12000 r/min for 10min, adding 500ul 75% ethanol, slightly shaking for 5-10 times, standing for 3min, and pouring ethanol to complete elution (total elution for 2 times); adding 500ul of absolute ethyl alcohol, eluting for 1 time (pouring absolute ethyl alcohol after standing for 3 min), placing into a quick-freezing centrifuge at 4 ℃ and 12000r/min, centrifuging for 3min, pouring absolute ethyl alcohol, and standing to dry; 80ul of elution buffer (TE buffer) was added and the mixture was subjected to transient centrifugation for 1min to obtain fungal AcEF007 genomic DNA.
(3) ITS analysis and identification
The sequence of the interval sequence (containing ITS1 region, 5.8S region and ITS2 region) of fungus rDNA is amplified by using fungus universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), and the obtained ITS sequencing sequence is shown as SEQ ID No. 1.
(4) Construction of developmental trees
As shown in FIG. 9, a phylogenetic tree of Antrodia camphorata symbiotic fungus AcEF007 was constructed based on ITS, and the results of morphological identification and molecular biological identification of the strain were combined, and the fungus was closest to the genus Phoma (phoma. Sp), so that Antrodia camphorata symbiotic fungus AcEF007 was identified as the genus Phoma (phoma. Sp).
Example 3
Preparation of active substance of Antrodia camphorata symbiotic fungus AcEF007
1. Liquid fermentation culture
Liquid fermentation medium PG: potato soaked powder 5g/L, yeast powder 5g/L, glucose 20g/L, magnesium sulfate heptahydrate 0.5g/L, potassium dihydrogen phosphate 1g/L, vitamin B1.1 g/L, purified water 1L, and packaging the culture medium into 100mL triangular flask.
Adding 1000ul of PG liquid into 2ml centrifuge tubes, placing 3 sterilized steel balls in each centrifuge tube, scraping appropriate amount of grown Antrodia camphorata symbiotic fungus AcEF007 mycelium, placing into the centrifuge tubes, crushing for 90S, inoculating 500ul of crushed liquid into each conical flask filled with PG liquid culture medium, placing at 28deg.C at 150 r.min -1 Is cultured for 8 days in a shaking table.
2. Culture mycelium extraction
After fermentation culture is completed, the obtained fungus mycelium and culture solution are obtained. Separating mycelium from bacterial liquid by using a separating funnel, adding ethyl acetate (1:1) into the bacterial liquid, shaking uniformly for 6min, standing for 12h after shaking uniformly, then recovering layered lower-layer waste liquid, condensing, refluxing and drying an upper-layer extraction solution by using a rotary evaporator, and obtaining a fermentation liquor extract cultured by Antrodia camphorata symbiotic fungi AcEF007.
Test example 1
Antibacterial activity detection of fermentation broth extract of Antrodia camphorata symbiotic fungus AcEF007 culture
1. Pathogenic bacteria activation and dilution
Pathogenic bacteria such as slow bacillus, vibrio parahaemolyticus, staphylococcus haemolyticus, pseudomonas cuprina, shigella flexneri, bacillus subtilis, escherichia coli and the like stored in a laboratory are respectively taken to be small in quantity (5-6 ul) and added into a 2ml centrifuge tube, 700ul of LB liquid medium is added into the centrifuge tube, and then the centrifuge tube is put into 37 ℃ and the rotating speed is 180 r.min -1 Culturing in a constant temperature shaking table for 12h to obtain pathogenic bacteria liquid, placing in a refrigerator at 4deg.C for use, and taking out the activated bacteria for dilution by 1/10 times (if the activated bacteria is stored at 4deg.C for more than 12h, no dilution is needed).
2. Antibiotics for positive control and preparation thereof
Ampicillin (Ampicillin) (lot number: 0339, purity: 95%) was purchased from Kunming Shuoyang technologies Co. 50mg of ampicillin is precisely weighed, placed in a centrifuge tube respectively, and 1mL of DMSO solution is added to prepare 50mg/mL of antibiotic DMSO solution for later use.
3. Filter paper sheet method for detecting antibacterial activity
Preparing a Luria-Bertani solid culture medium, pouring 20mL of the culture medium which is not solidified after sterilization into a culture dish, cooling and solidifying, absorbing 300uL of corresponding test bacterial suspension, dripping the corresponding test bacterial suspension onto the solid culture medium, uniformly coating the surfaces of the culture medium by using a sterile cotton swab to prepare a bacteria-containing flat plate, weighing a proper amount of Antrodia camphorata symbiotic fungus AcEF007 fermentation liquor extract, adding a proper amount of DMSO (dimethyl sulfoxide) solution after weighing, diluting to 50mg/mL, airing the bacteria-containing LB flat plate, drying filter paper wafers with the diameter of 5mm after autoclaving, dripping 10uL of test liquid as test sample pieces, dripping PG pure culture medium and ampicillin sample pieces as positive control, dripping the DMSO liquid as negative control sample pieces, pasting the test sample pieces and 3 control sample pieces onto the surfaces of each culture dish by using sterile forceps, covering the culture dish after marking, placing the culture medium in a constant temperature incubator at 37 ℃ for culturing for 12h, observing whether a bacteria inhibition zone appears or not and measuring the diameter of the bacteria inhibition zone by a cross method. Results antibacterial activity of broth extract (50 mg/ml) of Antrodia camphorata symbiotic fungus AcEF007 culture is shown in Table 1:
bacillus lentus, vibrio parahaemolyticus, staphylococcus haemolyticus, pseudomonas cuprina, shigella flexneri, bacillus subtilis and Escherichia coli
TABLE 1 antibacterial Activity of Antrodia camphorate symbiotic fungus AcEF007 extract compared to positive control
As can be seen from Table 1 and the antibacterial effects shown in figures 3-8, the fermentation broth extract cultured by the Antrodia camphorata symbiotic fungus AcEF007 (Phoma. Sp. Phoma) obtained by screening has better antibacterial activity on pathogenic bacteria such as Klebsiella pneumoniae, pseudomonas cuprina, staphylococcus aureus, escherichia coli, acinetobacter baumannii, and Staphylococcus haemolyticus, and provides a new way for researching and developing new antibacterial drugs for relieving the threat of pathogenic bacteria resistance to human health.
In addition to fermentation broth extraction by culturing strains, other culturing or processing methods, such as ultrasonic lysis supernatant of Antrodia camphorate symbiotic fungus AcEF007 cells; the ultrasonic pyrolysis precipitation of the Antrodia camphorata symbiotic fungus AcEF007 cells, and the processed products or cultures of the Antrodia camphorata symbiotic fungus AcEF007 cells have corresponding antibacterial activity under the antibacterial effect of the fungus AcEF007, and the preparation of related antibacterial active medicaments, bacterial agents and the like by using the bacteria are all within the protection scope of the invention.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the foregoing embodiments, which have been described in the foregoing embodiments and description merely illustrates the principles of the invention, and various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Sequence listing
<110> Wang Yi
<120> Antrodia camphorata symbiotic fungus AcEF007 and separation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 541
<212> DNA
<213> Phoma (sp)
<400> 1
tcctactgat ccgaggtcaa gagtgtaaaa atgtactttt ggatgtcgtc gttatgagtg 60
caaagcgcga gatgtactgc gctccgaaat caatacgccg gctgccaatt gttttaaggc 120
gagtctacag gagacaaaca cccaacacca agcagagctt gaaggtacaa atgacgctcg 180
aacaggcatg ccccatggaa taccaagggg cgcaatgtgc gttcaaagat tcgatgattc 240
actgaattct gcaattcaca ctacttatcg catttcgctg cgttcttcat cgatgccaga 300
accaagagat ccgttgttga aagttgtaac tattaagttt tttcagacgc tgatttcaac 360
tgcaaagggt ttaaatttgt ccaatcggtg ggcgaaccca ccgaggaaac gtaaggtact 420
caaaagacat gggtaagaga tagcaggcaa agcctacaac tctaggtaat gatccttccg 480
caggttcacc tacggaaacc ttgttacgac ttttacttcc tctaattgga accaagagat 540
a 541
Claims (4)
1. The Antrodia camphorata symbiotic fungus is characterized in that the Antrodia camphorata symbiotic fungus is Phoma sp. ACEF007, and the preservation number is: cctccc NO: m20211093; preservation address: china center for type culture Collection.
2. Use of the antrodia camphorate symbiotic fungus according to claim 1 in the preparation of antibacterial drugs, wherein the bacterium is pseudomonas cupurensis or escherichia coli.
3. The microbial inoculum is characterized in that the active ingredient of the microbial inoculum is the fermentation liquor extract of the antrodia camphorate symbiotic fungus as claimed in claim 1, and the preparation method of the fermentation liquor extract comprises the following steps:
(1) Adding 1000uL of PG liquid culture medium into 2mL centrifuge tubes, placing 3 sterilized steel balls in each centrifuge tube, scraping appropriate amount of grown Antrodia camphorata symbiotic fungus AcEF007 mycelium, placing into the centrifuge tubes, crushing for 90S, inoculating 500uL of crushed liquid into each conical flask filled with PG liquid culture medium, placing at 28deg.C at 150 r.min -1 Culturing for 8d in a shaker;
(2) Extracting mycelia of a culture, obtaining mycelia and a culture solution of Antrodia camphorata symbiotic fungi AcEF007 after fermentation culture is completed, separating the mycelia and the culture solution by using a separating funnel, adding ethyl acetate into the culture solution, shaking uniformly for 6min, standing for 12h after ultrasonic treatment for 30min, recovering layered lower-layer waste liquid, condensing, refluxing and drying an upper-layer extraction solution by using a rotary evaporator, and obtaining a fermentation solution extract of the Antrodia camphorata symbiotic fungi AcEF007 culture;
the PG liquid culture medium comprises the following components: potato soaking powder 5g/L and yeast powder5g/L, 20g/L glucose, 0.5g/L magnesium sulfate heptahydrate, 1g/L potassium dihydrogen phosphate and vitamin B 1 0.1g/L, and the balance of water.
4. A microbial inoculum according to claim 3, wherein in step (2), ethyl acetate is added to the microbial inoculum according to a volume ratio of 1:1.
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