CN116083243B - Preparation method of Antrodia camphorata and Phaliota joint-diamond-like-fungus composite biocontrol microbial inoculum - Google Patents
Preparation method of Antrodia camphorata and Phaliota joint-diamond-like-fungus composite biocontrol microbial inoculum Download PDFInfo
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- CAHGCLMLTWQZNJ-RGEKOYMOSA-N lanosterol Chemical compound C([C@]12C)C[C@@H](O)C(C)(C)[C@H]1CCC1=C2CC[C@]2(C)[C@H]([C@H](CCC=C(C)C)C)CC[C@@]21C CAHGCLMLTWQZNJ-RGEKOYMOSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- 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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Abstract
The invention belongs to the technical field of preparation of biocontrol microbial agents, and particularly discloses a preparation method of a composite biocontrol microbial agent of Antrodia camphorata and Arthropoda, which comprises the following specific steps: (a) Inoculating Antrodia camphorata fruiting body and Saprolegnia solani into an activation culture medium respectively for strain activation; (b) Inoculating the activated Antrodia camphorata and the activated Phaliota joint in a liquid culture medium of the same culture container for shake culture; (c) When a large amount of Antrodia mycelium appears in the liquid culture medium, collecting Antrodia and obtaining a product by the liquid culture medium; the preservation number of the section diamond spore mold is as follows: cctccc NO: m20211254, the deposit number of Antrodia camphorate is: the invention provides a new potato endophytic fungus, namely the sarcomyces (Arthrinium) strain, which has broad-spectrum antibacterial activity with an extract of a Antrodia camphorata co-culture, and active substances and an antibacterial mechanism of the strain can provide a new way for developing new medicines.
Description
Technical Field
The invention belongs to the technical field of preparation of biocontrol bactericides, and in particular relates to a preparation method of a composite biocontrol bactericides of Antrodia camphorata and Arthropoda, which is obtained by co-culturing Antrodia camphorata and Arthropoda.
Background
Large edible and medicinal fungi of the genus Phellinus (Antrodia) of the family Polyporaceae, class Anrodia camphorata (Antrodia cinnamomea) belonging to the genus Basidiomycota, order Anemone. Antrodia camphorate fruiting bodies are decomposed on the inner wall of a decayed heart wood with an altitude of 200-2000 m. In recent years, the research on chemical components and pharmacological actions of Antrodia camphorate has been greatly progressed, and the fruiting body of Antrodia camphorate contains a plurality of active substances such as triterpene, fungal glucan, maleic acid derivative, ubiquinone derivative and the like, and has the physiological functions of resisting tumor, resisting inflammation, protecting liver, regulating biological immunity and the like.
The important physiological active components of Antrodia camphorata are triterpene and polysaccharide compounds, more than 30 triterpene and steroid saponin compounds are found from Antrodia camphorata, and the content of triterpene substances in wild fruiting bodies is more than 10 percent and is far higher than 1-3 percent of ganoderma lucidum. As the Antrodia camphorata host is single and grows slowly, the Antrodia camphorata is endangered to be in a dead state because of being subjected to a large amount of theft and harvest, so the Antrodia camphorata is often praised as rubble in forests.
Submerged culture of Antrodia camphorate mycelia has been effectively used to produce extracellular bioactive metabolites, and co-culture is considered as a viable method to simulate the ecological environment and is an important emerging tool for triggering silencing gene expression under laboratory conditions. It has now been found that co-cultivation of fungi with bacteria can produce new compounds and induce biological activity that accumulates secondary metabolites. For example, the content of the compound of the constitutive antibiotic is obviously improved by co-culturing fusarium trilineum (Fusarium tricinctum) and bacillus subtilis (bacillus subtilis); co-cultivation of Fusarium trichlii with Streptomyces lividans (Streptomyces lividans) induces the production of related compounds such as naphthoquinone dimers (naphthoquinone dimers). Recent studies have shown that fungal co-cultivation is an effective strategy for inducing new secondary metabolite biosynthesis, improving productivity and improving bioactivity. For example, the co-culture of white rot fungi (Trametes versicolor) and ganoderma lucidum (Ganoderma applanatum) is a strong basidiomycete interaction mode, and can induce the production of compounds such as phenyllactic acid (3-PHENYLLACTIC ACID), and aglycone acid (orsellinic acid); inonotus obliquus (Inonotus obliquus) and Phellinus linteus (Phellinus punctatus) are co-cultured, and can accumulate compounds for scavenging free radicals and inhibiting tumor cell proliferation such as lanosterol, polyphenol, melanin, etc.; co-cultivation with Trichoderma reesei (Trichoderma Reesei) and Coprinus comatus (Coprinus comatus) is an important way to produce lignocellulose degrading enzymes. No report that Antrodia camphorate generates a new structural compound and induces the biological activity of the compound by co-culturing with other fungi is found at present.
Disclosure of Invention
The invention mainly aims to provide a preparation method of a composite biocontrol microbial inoculum of Antrodia camphorata and Phaliota joint-tsubishi, and provides a new technical scheme for germ control.
In order to achieve the above object, the present invention provides the following technical solutions:
a preparation method of a compound biological antibacterial agent of Antrodia camphorata and Phaliota glabrous, which comprises the following steps,
(A) Inoculating Antrodia camphorata fruiting body and Saprolegnia solani into an activation culture medium respectively for strain activation;
(b) Inoculating the activated Antrodia camphorata and the activated Phaliota joint in a liquid culture medium of the same culture container for shake culture;
(c) When a large amount of Antrodia mycelium appears in the liquid culture medium, collecting Antrodia and obtaining a product by the liquid culture medium;
The preservation number of the section diamond spore mold is as follows: cctccc NO: m20211254, the deposit number of Antrodia camphorate is: CGMCC No.20234.
Preferably, in the step (a), the activated medium of Antrodia camphorata is BD liquid medium, and the BD liquid medium comprises: BD culture medium powder finished product 15g/L.
Preferably, in the step (a), the activation medium of the tsubishi is a MY medium, and the MY medium includes: 21g/L of yeast malt extract broth and 15-20g/L of agar.
Preferably, in the step (b), the liquid medium is potato medium.
Further, the potato culture medium comprises: potato powder 5g/L, glucose 1g/L, yeast powder 5g/L, mgSO4.7H2O 0.5g/L, KH2PO 41 g/L and vitamin B 1 0.1.1 g/L.
Preferably, in the step (b), the shaking culture temperature is 25 to 28℃and the speed is 100 to 150rpm/min.
The invention has the beneficial effects that:
(1) The method for separating symbiotic bacteria by using the method for crushing the liquid coated plates after crushing the fruiting bodies is simple and convenient to operate and has obvious effect compared with the common separation method.
(2) The invention provides a new potato endophytic fungus, namely a strain of the arthrosporium (Arthrinium), and the applicant discovers that an extract of a co-culture of the strain and Antrodia camphorata has broad-spectrum antibacterial activity, and active substances and an antibacterial mechanism of the extract can provide a new way for developing new medicines.
(3) The composite biocontrol microbial inoculum obtained by the invention has remarkable inhibition effect on bacillus cereus.
Drawings
FIG. 1 is a graph showing the inhibition effect of the biocontrol agent of the present invention on Bacillus cereus.
Detailed Description
The invention, together with further advantages, may be best understood from the following detailed description of the invention taken in conjunction with the accompanying drawings and detailed description. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention, and that the experimental methods used in the examples described below are conventional, unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
The strain of the siderochanterium used in the invention is preserved in China center for type culture Collection, with the preservation name Arthrinium SP.YAFEF012, and the preservation number is as follows: cctccc NO: m20211254, the strain was received from the China center for type culture collection on day 10 and 11 of 2021, and was registered, and stored for thirty years from that day, and the viability of the culture was obtained by the center for viability at day 10 and 26 of 2021.
The strain is obtained by separating and purifying potato tubers collected in the Hongmang city of Yunnan province in 5 months of 2020. Repeatedly washing potato tuber with tap water for 4 hr to remove fungi or other microorganisms on the surface of the material as much as possible. Placing the material in an ultra-clean workbench for disinfection treatment, washing the material with 1L of sterile water, placing the material in a culture dish, and shearing the material into small sections of 3-5mm by scissors; placing the small segments into a 50mL sterile centrifuge tube, soaking in 75% ethanol for 1min, continuously shaking for 1min, pouring out ethanol, washing with sterile water for 2 times, pouring 10mL of prepared 5%H 2O2, soaking for 3min, and continuously shaking; pouring hydrogen peroxide, washing with sterile water for 3 times to sufficiently wash the hydrogen peroxide, placing the sterilized tissue on sterile filter paper to suck the superfluous water on the surface, and airing for later use. The sterilized potato tubers were placed in MY medium, two replicates were set, labeled and cultured in a 26℃incubator, and the colony growth state in the medium was observed. Observing whether colonies appear below or at the edge of the tissue, picking fresh mycelia of single colonies in time by adopting a tip mycelia picking method according to the growth form, color, time and the like of the colonies, transferring the fresh mycelia into a PDA culture dish, culturing the fresh mycelia in a biochemical incubator at 26 ℃ in an inverted manner, and repeatedly transferring and culturing the fresh mycelia for multiple times to obtain single colonies with consistent forms, namely the purified endophytic fungus strain. The separated endophytic fungi are subjected to morphological description, DNA is extracted, PCR amplification reaction is carried out on the extracted DNA through universal primers ITS1 and ITS4, a PCR product is entrusted to Shanghai biological engineering Co., ltd to determine an 18S rDNA sequence, the ITS sequence is shown as SEQ ID No.1, and the product is identified as the Phascosporium nodosum according to morphological identification and molecular biological technology, and is named YAFEF012.
Example 2
The Antrodia camphorate used in the invention is preserved in China general microbiological culture Collection center with the preservation number CGMCC NO.20234, the classification name is Antrodia camphorate (Taiwanofungus camphoratus), the biological material is received by the China general microbiological culture collection center on the 08 th month 07 of 2020, and is registered in a book, and is preserved for thirty years from the date, and the viability of the biological material is detected by the preservation center on the 08 th month 07 of 2020, so that the biological material is viable.
The Antrodia camphorate strain activation solid culture medium is Potato Dextrose Agar (PDA), and is sterilized by high-pressure moist heat at 121 ℃ for 20-30 minutes. Pouring into a 90mm dish, inoculating Antrodia camphorate mycelium, standing at 26deg.C for 10-15 days, and preserving at 4deg.C for inoculating.
Antrodia camphorate strain is cultivated into BD liquid culture medium, and each 1L of liquid culture medium is prepared according to the following method: 15g of BD culture medium powder finished product, adding distilled water to 1L, subpackaging into 250ml volumetric flask, naturally sterilizing at 121 ℃ under high pressure and moist heat for 20-30 minutes, culturing at 25-28 ℃, then crushing the cultured antrodia mycelium, inoculating into BD liquid culture medium, oscillating by a shaking table at 100-150 rpm/min, and culturing for 4-7 days. BD liquid medium includes: BD culture medium powder finished product 15g/L.
Example 3
The activation culture medium of the siderochanterium strain used in the invention is MY culture medium, and each 1L of solid culture medium is prepared according to the following method: 21g of yeast malt extract broth and 15-20g of agar. The components are added in sequence, distilled water is added to 1L, the pH value is natural, and the high-pressure moist heat sterilization is carried out for 20-30 minutes at 121 ℃. Pouring into a 90mm dish, standing at 26 ℃ for 4-7 days, and preserving at 4 ℃ for culture inoculation. The MY medium comprises: 21g/L of yeast malt extract broth and 15-20g/L of agar.
Example 4
The co-culture method of Antrodia camphorata and the Phaliota glabra used in the invention comprises the following steps: 0.5-1g of Antrodia camphorate mycelium under BD liquid culture condition is added with 500 mu l of sterile water for homogenate, inoculated into potato culture medium, and cultured at 25-28 ℃ for 3 days by shaking 100-150 rpm/min; taking 1cm 2 of Saprolegnia solani under solid culture condition, adding 500 μl of sterile water for homogenizing, and inoculating into liquid cultured Antrodia camphorata for 3-6 days.
The potato culture medium comprises: potato powder 5g/L, glucose 1g/L, yeast powder 5g/L, mgSO 4·7H2O 0.5g/L,KH2PO4 g/L, vitamin B 1 0.1g/L.
Example 5
Compound extraction was performed on the obtained mycelium and medium from co-cultivation of antrodia camphorata and tsubishi for 4 to 7 days. Adding equal proportion of ethyl acetate into the liquid culture medium for ultrasonic extraction, collecting the extract, performing rotary evaporation (the temperature is 55 ℃, the rotating speed is 90rpm, the vacuum degree is less than 3 mmHg), and dissolving with 0.30-0.60mL of acetone after evaporating.
Example 6
And adding a small amount of pathogenic bacillus cereus stored in a laboratory into a 2ml centrifuge tube, adding an LB liquid culture medium, and placing the centrifuge tube into a constant temperature shaking table at 37 ℃ for culturing for 12 hours to obtain pathogenic bacterial liquid. Taking a proper amount of Antrodia camphorata and Saprolegnia solani co-culture fermentation broth extract, weighing, adding a proper amount of DMSO (dimethyl sulfoxide) solution, and diluting to 50mg/ml. Uniformly smearing the activated pathogenic bacteria liquid on LB solid culture medium, airing, placing a 5mm filter paper wafer which absorbs the dissolved co-culture fermentation liquid extract at a corresponding mark of the culture medium, dipping the DMSO solution with the 5mm filter paper wafer as negative control, placing the culture medium in a constant temperature incubator at 37 ℃ for culturing for 12 hours, observing whether a bacteriostasis ring appears or not, and measuring the diameter of the bacteriostasis ring by a crisscross method. The results show in figure 1 that the antibacterial activity of a fermentation broth extract (50 mg/ml) of the co-culture of Antrodia camphorata and Arthrospora is as follows, wherein A is the Arthrospora cultivated by a potato culture medium, B is the Antrodia camphorata cultivated by a potato culture medium, C is a control DMSO solution, D is the co-culture of Antrodia camphorata and Arthrospora, the experiment is repeated 3 times, the average antibacterial circle of A is 6mm, B is 7mm, and D is 21mm, and the antibacterial circle is obviously increased after the co-culture, which indicates that the co-culture of Antrodia camphorata and Arthrospora has obvious antibacterial effect on bacillus cereus.
Sequence listing
<110> Zheng Yuan Wang Yi
<120> Preparation method of Antrodia camphorata and Phaliota joint-diamond-back composite biocontrol microbial inoculum
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 610
<212> DNA
<213> Saprolegnia festival (Arthrinium SP)
<400> 1
atttctacct gatccgaggt caaccactaa aaagatgggg gttttatggc gggaggacgg 60
agcctgacaa aagcgagaaa taaattacta cgctcagagg actaccgccg ctccgccact 120
gtctttaagg aactgcagta cagcagattc ccaacactaa gctaggctta agggttgaaa 180
tgacgctcga acaggcatgc ccaccagaat actgatgggc gcaatgtgcg ttcaaagatt 240
cgatgattca ctgaattctg caattcacat tacttatcgc atttcgctgc gttcttcatc 300
gatgccagaa ccaagagatc cgttgttgaa agttttaact tattaaaata agacgctcag 360
aagatacaat aaaacatagt tttgcgtacc gccggcgggc cgcggctgga tgtggcagcc 420
cgcaacttca gggtagcaag ctacagggta gctagctaca gggtagctgc gggtgcctcc 480
aaccgagctt acgccgaggc ataactgggt aggttcacag atggtatggg agttgtataa 540
ctctgtaatg atccctccgc tggttcacca acggagacct tgttacgact tttacttcct 600
caatgggaca 610
Claims (3)
1. The non-therapeutic purpose application of the Antrodia camphorata and Phaliota glabrous in inhibiting bacillus cereus is characterized in that the preparation method of the compound biocontrol microbial inoculum comprises the following steps,
(A) Inoculating Antrodia camphorata fruiting body and Saprolegnia solani into an activation culture medium respectively for strain activation;
(b) Inoculating the activated Antrodia camphorata and the activated Phaliota joint in a liquid culture medium of the same culture container for shake culture; the liquid culture medium is a potato culture medium;
(c) Co-culturing Antrodia camphorata and Phaliota joint for 4-7 days; harvesting the co-cultivated liquid culture medium;
the preservation number of the section diamond spore mold is as follows: cctccc NO: m20211254, the deposit number of Antrodia camphorate is: CGMCC No.20234;
the potato culture medium contains 5g/L of potato powder, 1g/L of glucose, 5g/L of yeast powder, 4·7H2O 0.5g/L,KH2PO4 g/L of MgSO and 0.1g/L of vitamin B.
2. The use according to claim 1, wherein in step (a), the activation medium of the sideromyces is a MY medium comprising: 21g/L of yeast malt extract broth and 15-20g/L of agar.
3. The use according to claim 1, wherein in the step (b), the shaking culture temperature is 25 to 28℃and the speed is 100 to 150 rpm.
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