Aspergillus sp. FEYQSJ2 and application of fermentation liquor crude extract of aspergillus sp. FEYQSJ2

Abstract

The invention discloses an aspergillus fungus FEYQSJ2 and application of a fermentation liquor crude extract of the aspergillus fungus FEYQSJ2, and belongs to the field of microbial secondary metabolites. The aspergillus fungus FEYQSJ2 is preserved in China General Microbiological Culture Collection Center (CGMCC) on May 27, 2024, the preservation number is CGMCC No.41243, the taxonomic name is Aspergillus fumigasus, the address of the preservation unit is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and a fermentation liquor crude extract of the aspergillus fungus FEYQSJ2 can be used for preparing a medicine for preventing and treating paris polyphylla rhizome rot. A new thought is provided for preventing and treating root rot and stem rot of the paris polyphylla var. Yunnanensis.

Description

Technical Field

[0001] The invention relates to an Aspergillus fungus FEYQSJ2 and application of a crude extract of its fermentation broth, and belongs to the field of microbial secondary metabolites. Background Art

[0002] Paris polyphylla var. yunnanensis. is a perennial herbaceous plant of the genus Paris L of the family Trilliaceae. It is a local medicinal material in Yunnan and has a long history of medicinal use in Yunnan. According to the "Southern Yunnan Materia Medica", "this is the best medicine for surgery, mainly treating all kinds of unknown swellings and toxins, and attacking various sores, carbuncles and ulcers". Paris polyphylla var. yunnanensis is included in the "Chinese Pharmacopoeia" of successive editions. Its dried rhizomes are used as medicine, and it has the effects of clearing away heat and detoxifying, reducing swelling and relieving pain, and cooling the liver and calming the nerves. Paris polyphylla var. yunnanensis is not only a commonly used medicine in traditional Chinese medicine prescriptions and folk families, but also the raw material for more than 40 famous Chinese patent medicines with national medicine approval numbers, such as Yunnan Baiyao series, Gongxuening capsules, Ji Desheng snake medicine tablets, Loulian capsules, and Reduqing. Modern research has also found that Paris polyphylla has anti-tumor, immune regulation, antibacterial and anti-inflammatory, hemostatic, vascular regulation and antioxidant effects. It is the main raw material for Jinfukang oral liquid (non-small cell lung cancer), Loulian capsules (liver cancer), Ganfule tablets (liver cancer), etc.

[0003] Yunnan is the origin and main production area of ​​Paris polyphylla, with a planting area and output accounting for more than 90% of the country. Artificial planting has become the main source of Paris polyphylla. However, with the rapid development of Paris polyphylla planting industry, due to blind introduction, planting patterns, climate, soil and other ecological environmental factors, the quality of Paris polyphylla is affected, and the disease problem of Paris polyphylla is becoming increasingly prominent. Among them, root rot and stem rot (called Paris polyphylla cancer in the place of origin) are the most harmful to Paris polyphylla planting. Fusarium oxysporum is one of the main pathogens causing root rot and stem rot of Paris polyphylla, which mainly infects the rhizome of Paris polyphylla, the medicinal part, causing the rhizome to blacken, soften and rot. In the early stage of the disease, there are no obvious symptoms. As the degree of rhizome rot worsens, the leaves above the ground begin to turn yellow, wither and dry up until the plant withers and dies.

[0004] In recent years, microbiological studies have found that the endophytes of many plants can produce the same medicinal ingredients as the host medicinal plants. The production of active substances by fermentation and cultivation of endophytes on a certain scale is considered to be a method for finding new antibacterial lead compounds, and Paris polyphylla has rich endophytic bacteria resources. Therefore, the present invention studies the endophytes collected from Paris polyphylla in Dali, Yunnan, explores the species diversity of Paris polyphylla endophytes by means of pure microbial culture and sequencing-based technology, and uses HPLC and UPLC-MS to screen the chemical diversity of the secondary metabolites of the strains, combined with the "micro broth dilution method" and other methods for antibacterial activity screening, and obtains an Aspergillus fungus that can produce antibacterial active substances. Summary of the invention

[0005] One of the objects of the present invention is to provide an Aspergillus fungus FEYQSJ2, wherein the Aspergillus fungus FEYQSJ2 is Aspergillus fumigatus, which was deposited in the General Microbiological Center of China Microorganism Culture Collection Administration on May 27, 2024, with a deposit number of CGMCC No.41243, a taxonomic name of Aspergillus fumigatus, and an address of the depository unit of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] Another object of the present invention is to provide a crude fermentation broth extract prepared by Aspergillus fungus FEYQSJ2.

[0007] Preferably, the method for preparing the crude extract of fermentation broth is as follows:

[0008] (1) Cultivate and obtain the fermentation broth of Aspergillus fungus FEYQSJ2.

[0009] (2) Separating the fermentation broth and the bacterial cells, extracting with ethyl acetate, combining the extracts, concentrating under reduced pressure, and drying to obtain the crude extract.

[0010] Preferably, the method for preparing the fermentation liquid in step (1) is: inoculating the Aspergillus fungus FEYQSJ2 into PDB culture medium, and culturing at 28°C in a shaking incubator for 3 days to obtain seed liquid; transferring the seed liquid to rice culture medium, and culturing at 28°C for 28-30 days to obtain a fermentation product.

[0011] Preferably, the rotation speed of the shaking culture in step (1) is 150-180 r / min.

[0012] Preferably, the transfer amount of the seed liquid in step (1) is preferably 10%-12%.

[0013] Preferably, the amount of ethyl acetate used in step (2) is 1-2 times the volume of the fermentation broth.

[0014] The present invention also provides an application of a crude extract of fermentation liquid prepared by the Aspergillus fungus FEYQSJ2 in preparing an agent for preventing and treating Paris polyphylla root rot or stem rot.

[0015] Preferably, the Paris polyphylla root rot is caused by Fusarium oxysporum.

[0016] Beneficial Effects of the Invention

[0017] (1) The present invention is the first to discover an Aspergillus fungus FEYQSJ2 that can prevent and control root rot and stem rot of Paris polyphylla, thus broadening the biological control methods for root rot and stem rot of Paris polyphylla.

[0018] (2) The present invention utilizes microorganisms to prevent and treat root rot and stem rot of Paris polyphylla, without causing any impact or damage to the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a morphological picture of the Aspergillus fungus FEYQSJ2 isolated in Example 1 on the culture medium.

[0020] Figure 2 This is a morphological characteristic diagram of the Aspergillus fungus FEYQSJ2 isolated in Example 1 under a microscope.

[0021] Figure 3 is the HPLC chart of the crude extract prepared in Example 1.

[0022] Figure 4 is a total ion current (TIC) graph of the crude extract prepared in Example 1.

[0023] Figure 5 The crude extract prepared in Example 3 shows resistance to Fusarium oxysporum, the pathogenic fungus of root and stem rot, wherein Figure A is the experimental group and Figure B is the blank control. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are only simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0025] Example 1

[0026] Isolation, Purification and Analysis of Aspergillus FEYQSJ2

[0027] The Aspergillus fungus FEYQSJ2 was isolated and purified from the rhizomes of the Paris polyphylla plant of Qishengjiankang in Yousuo Town, Eryuan County, Yunnan Province. The isolated FEYQSJ2 was sequenced by ITS rDNA and was identified as an Aspergillus fumigatus strain through BLAST comparison analysis on the NCBI website.

[0028] The Aspergillus fungus FEYQSJ2 has the following characteristics:

[0029] (1) The Aspergillus fungus FEYQSJ2 obtained by screening was streaked on the culture medium and cultured at 28°C for 3-5 days. It was found that the fungus was gray-green, large in shape, dry in appearance, opaque, and the connection between the colony and the culture medium was tight and difficult to pick up. Figure 1 shown.

[0030] (2) Observe the vegetative hyphae, aerial hyphae, and conidia using a 10×40 optical microscope. Figure 2 shown.

[0031] Example 2

[0032] The preparation of the crude extract of the fermentation broth of Aspergillus fungus FEYQSJ2 is as follows:

[0033] (1) Aspergillus fungus FEYQSJ2 was inoculated into PDB medium (purchased from Aladdin Reagent (Shanghai) Co., Ltd.) and cultured in a shaking incubator at 28°C and 150 rpm for 3 days to obtain seed solution.

[0034] (2) The seed solution obtained in step (1) was transferred to a rice culture medium (inoculation amount 10%), and the culture was placed at 28° C. for 28 days to obtain a fermentation product.

[0035] (3) separating the fermentation broth and the bacterial cells in the fermentation product obtained in step (2), wherein the fermentation broth is extracted twice with 1.5 times the volume of ethyl acetate, and the extracts are combined, concentrated under reduced pressure, and dried to obtain the crude extract.

[0036] (4) The crude extract obtained in step (3) was analyzed by HPLC, with blank rice culture medium as control. The HPLC conditions were as follows: high performance liquid chromatograph: Agilent 1260; chromatographic column: Agilent ZORBAX Eclipse XDS-C18 (specification: 5 μm, 4.6 mm×250 mm); chromatographic conditions: flow rate: 1.0 mL / min, injection volume: 5 μL; detection wavelengths: 210, 254 and 330 nm; mobile phase: water (A)-methanol (B), gradient elution program: 0-45 min, 5%-100% B; 45-50 min, 100%-100%

[0037] B; 50-51min, 100%-5%B, 52-57min, 100%B. The results are as follows Figure 4 , the secondary metabolites of the crude extract of the fermentation broth of Aspergillus fungus JCG2 appeared at 30min to 35min and had medium polarity.

[0038] (5) The crude extract obtained in step (3) was analyzed by UPLC-ESI-Q-TOF-MS / MS, and the ultra-high performance liquid chromatography conditions were as follows: ultra-high performance liquid chromatograph: UltiMate 3000 ultra-high performance liquid chromatograph; mass spectrometer: Compact mass spectrometer system; chromatographic column: Thermo C18 chromatographic column (specification 5 μm, 4.6 mm×250 mm); chromatographic conditions: flow rate 0.2 mL / min, injection volume 2 μL; detection wavelengths: 210, 254 and 330 nm; mobile phase: water (A)-methanol (B), gradient elution program: 0-45 min, 5%-100% B; 45-50 min, 100%-100% B; 50-51 min, 100%-5% B, 52-57 min, 5%-5% B. Mass spectrometry conditions: ESI as electrospray ion source, scanning in negative ion mode, 253.8 kPa nebulizer pressure, He as drying gas, 8 L / min gas flow rate, 220 °C ion source temperature, 350 °C drying temperature, 4 kV capillary voltage, scanning in the range of m / z 50 to 2000, Ar as collision gas, 8 eV scanning collision energy and quadrupole ion energy, 70 Vpp hexapole radio frequency, Na[NaCOOH] as calibration solution, Figure 3 This is the total ion chromatogram of the crude extract of the fermentation broth of Aspergillus fungus FEYQSJ2 in negative ion mode by UPLC-ESI-Q-TOF-MS / MS.

[0039] Example 3

[0040] Inhibitory effects of crude extracts from fermentation broth of Aspergillus FEYQSJ2 on Fusarium oxysporum, the pathogenic fungus for root and stem rot of Paris polyphylla

[0041] The experiment in this example was conducted by the "filter paper method". The pathogenic fungus used was Fusarium oxysporum G1F2, which was a fungus causing rhizome disease of Paris polyphylla obtained by separation, purification and identification from the diseased Paris polyphylla rhizome species in Yunlong Guanping. The sequencing results were analyzed by BLAST on the NCBI website. The similarity between G1F2 and the reference strain (GenBank MT530243.1) was 100%. The specific steps are as follows:

[0042] The fungus cake of the pathogenic fungus was taken with a hole puncher with a pore size of 5 mm, and the cake was placed in the center of the PDA plate using sterile tweezers. Four filter papers with a diameter of 6 mm were attached at a distance of 2.5 cm from the cake, and 4 μL of dimethyl sulfoxide (DMSO) containing crude extract (25 mg of crude extract per ml of DMSO) was dripped on each paper. Only the pathogenic fungus to be tested was inoculated as a control, and three replicates were used for each treatment. The culture was inverted at 28° C. for 7 days. The diameter of the pathogenic fungus colony was measured by the cross method, and the inhibition rate of hyphae growth was calculated according to the following formula. The inhibition rate of the crude extract of the present invention on Fusarium oxysporum was calculated to be 60.34%.

[0043]

[0044] The antibacterial results showed that the crude extract prepared in Example 1 had an antibacterial effect on the root rot pathogenic fungus F.oxysporum ( Figure 5 ).

Claims

1. An Aspergillus fungus FEYQSJ2, characterized in that: The Aspergillus fungus FEYQSJ2 is Aspergillus fumigatus, with a preservation number of CGMCC No. 41243 and a taxonomic name of Aspergillus fumigatus.

2. A crude fermentation broth extract prepared using the Aspergillus fungus FEYQSJ2 according to claim 1.

3. Use of the crude extract of fermentation broth prepared by the Aspergillus fungus FEYQSJ2 according to claim 2 in the preparation of an agent for preventing and treating root rot or stem rot of Paris polyphylla.

4. The method for preparing the crude extract of fermentation broth prepared by the Aspergillus fungus FEYQSJ2 according to claim 2, characterized in that: The preparation method specifically comprises the following steps: (1) culturing to obtain a fermentation liquid of Aspergillus fungus FEYQSJ2; (2) Separating the fermentation liquid from the bacterial cells, extracting with ethyl acetate, combining the extracts, concentrating under reduced pressure, and drying to obtain the crude extract.

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