Bacillus sp. BLLGPG27 and application of fermentation liquor crude extract of Bacillus sp. BLLGPG27
By screening and preparing crude extracts of Bacillus BLLGPG27 fermentation broth, the problems of root and stem rot and drug-resistant bacterial infection in Paris polyphylla have been solved, providing an effective means of prevention and treatment.
Patent Information
- Application Number
- CN202511111662.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-08
- Publication Date
- 2025-11-14
AI Technical Summary
Root and stem rot is a serious problem in the cultivation of Paris polyphylla, and traditional antibiotics are ineffective against drug-resistant bacteria, so new prevention and treatment strategies need to be found.
A strain of Bacillus BLLGPG27 and its crude fermentation broth were screened out and prepared by ethyl acetate extraction. This preparation was used to develop a drug for preventing and treating root and stem rot of Paris polyphylla and for treating Gram-positive bacterial infections.
This study provides a new source of antibacterial active substances for the effective prevention and treatment of root and stem rot in Paris polyphylla and for the treatment of infections caused by Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus.
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Abstract
Description
Technical Field
[0001] This invention relates to the application of Bacillus BLLGPG27, a bacterium of the genus Bacillus, and its crude fermentation broth extract, belonging to the field of microbial secondary metabolites. Background Technology
[0002] *Paris polyphylla* var. *yunnanensis*, a perennial herb belonging to the genus *Paris* L of the family Trilliaceae, is a traditional Chinese medicine from Yunnan Province with a long history of medicinal use in the region. According to the *Diannan Materia Medica*, *Paris polyphylla* is "an essential medicine for surgery, primarily treating all kinds of unnamed swellings and boils, and attacking various sores and carbuncles." This medicinal herb is included in all editions of the *Chinese Pharmacopoeia*. The dried rhizome is used medicinally, possessing the effects of clearing heat and detoxifying, reducing swelling and relieving pain, and cooling the liver and calming the nerves. *Paris polyphylla* is not only a commonly used medicine in traditional Chinese medicine prescriptions and in folk households, but also a raw material for more than 40 well-known nationally approved traditional Chinese medicines, including Yunnan Baiyao series, Gongxue Ning capsules, Jidesheng snake medicine tablets, Loulian capsules, and Reduqing. Modern research has also found that Paris polyphylla has anti-tumor, immunomodulatory, antibacterial and anti-inflammatory, hemostatic, vascular-regulating and antioxidant effects. It is the main ingredient in Jinfukang oral liquid (for non-small cell lung cancer), Loulian capsules (for liver cancer), and Ganfule tablets (for liver cancer).
[0003] Yunnan is the origin and main production area of Paris polyphylla, accounting for over 90% of the national planting area and output. Artificial cultivation has become the primary source of Paris polyphylla. However, with the rapid development of the Paris polyphylla cultivation industry, changes in ecological factors such as indiscriminate introduction of new varieties, planting methods, climate, and soil have not only affected the quality of Paris polyphylla but also led to increasingly prominent diseases. Among these, root and stem rot (known locally as "Paris polyphylla cancer") poses the greatest threat to Paris polyphylla cultivation. Root and stem rot: Fusarium oxysporum and Fusarium solani are the main pathogens causing root and stem rot in Paris polyphylla. They primarily infect the medicinal parts of the plant, the rhizomes, causing the rhizome epidermis to blacken, soften, and rot. In the early stages, there are no obvious symptoms. As the root and stem rot worsens, the above-ground leaves begin to yellow, wither, and dry out, eventually leading to the death of the plant.
[0004] The spread of antibiotic resistance has become a major threat to human health. The US Centers for Disease Control and Prevention (CDC) report on the threat of antibiotic resistance indicates that more than 2.8 million people are infected with drug-resistant pathogens each year, and more than 35,000 die from the lack of effective control of these resistant bacteria. In particular, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF) were listed by the WHO as drug-resistant pathogens urgently requiring new antibiotics as early as 2017. Treatment with traditional antibiotics often requires prolonged high-dose use and is ineffective against persistent biofilm-related infections. It is also noteworthy that Enterococcus faecalis, Enterococcus faecium, and Staphylococcus epidermidis are important pathogens causing infections in newborns and adults, and their prevalence is increasing, necessitating the search for new prevention and treatment strategies.
[0005] Recent microbiological studies have discovered that endophytic bacteria in many plants can produce the same medicinal components as their host medicinal plants. Producing active substances through large-scale fermentation of endophytic bacteria is considered a method for finding novel antibacterial lead compounds, and *Paris polyphylla* possesses abundant endophytic bacterial resources. Therefore, this invention studies endophytic bacteria collected from *Paris polyphylla* in Dali, Yunnan Province. Species diversity of the endophytic bacteria was explored using pure microbial culture methods and sequencing-based technology. Chemical diversity screening of the secondary metabolites of the strains was performed using HPLC, and antibacterial activity screening was conducted using methods such as the "micro-broth dilution method" and the "filter paper disc method." A *Bacillus* strain capable of producing antibacterial active substances was identified. Summary of the Invention
[0006] One of the objectives of this invention is to provide a strain of Bacillus BLLGPG27, which was deposited on December 31, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33259 and taxonomic name Bacillus subtilis. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
[0007] Another objective of this invention is to provide a crude extract of fermentation broth prepared from Bacillus BLLGPG27.
[0008] Preferably, the method for preparing the crude extract of the fermentation broth is as follows:
[0009] (1) The fermentation broth of Bacillus BLLGPG27 was obtained by culturing.
[0010] (2) Separate the fermentation broth and bacterial cells, extract with ethyl acetate, combine the extracts, concentrate under reduced pressure, and dry to obtain the crude extract. Preferably, the fermentation broth in step (1) is prepared as follows: Bacillus spp. BLLGPG27 is inoculated into LB medium and cultured on a shaker at 28°C for 24 hours to obtain seed culture; the seed culture is transferred to SGTYP medium (100 mL / bottle) and cultured on a shaker at 28°C for 3 days to obtain fermentation product.
[0011] Preferably, the rotation speed of the shaker culture in step (1) is 150-180 r / min.
[0012] Preferably, the amount of seed liquid transferred in step (1) is 10%-12%.
[0013] Preferably, the amount of ethyl acetate used in step (2) is 1-2 times the volume of the fermentation broth.
[0014] This invention also provides the application of a crude extract of fermentation broth prepared from Bacillus BLLGPG27 in the preparation of an agent for controlling root and stem rot of Paris polyphylla.
[0015] Preferably, the root and stem rot of Paris polyphylla is caused by Fusarium oxysporum G1F1 and Fusarium solani G1F2.
[0016] The present invention also provides the application of a crude extract of fermentation broth prepared from Bacillus BLLGPG27 in the preparation of a drug for treating diseases caused by Gram-positive bacteria.
[0017] Preferably, the Gram-positive bacteria are one or more of methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, or Enterococcus faecium.
[0018] Beneficial effects of the present invention Attached Figure Description
[0019] Figure 1 This is a morphological image of the Bacillus genus BLLGPG27 isolated in Example 1 on a culture medium.
[0020] Figure 2Phylogenetic tree of BLLGPG27, a Bacillus bacterium isolated in Example 1, based on 16S rDNA analysis (the phylogenetic tree was constructed using MEGA 11.0 software with Neighbor-joining).
[0021] Figure 3 This is an HPLC chromatogram of the crude extract prepared in Example 1.
[0022] Figure 4 The image shows the resistance of the crude extract prepared in Example 3 to the pathogenic fungus Fusarium oxysporum, where Figure A is the experimental group and Figure B is the blank control.
[0023] Figure 5 The image shows the resistance of the crude extract prepared in Example 3 to the pathogenic fungus Fusarium solani, where Figure A is the experimental group and Figure B is the blank control. Detailed Implementation
[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are merely 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 is determined by the claims.
[0025] Example 1
[0026] Isolation, purification and analysis of Bacillus spp. BLLGPG27
[0027] The Bacillus spp. BLLGPG27 was isolated and purified from the rhizomes of healthy Paris polyphylla plants in Guanping Township, Yunlong County, Dali City, Dali Bai Autonomous Prefecture, Yunnan Province. The culture medium used for isolation and purification was R2A medium (0.5g yeast extract, 0.5g peptone, 0.5g casein hydrolysate, 0.5g glucose, 0.5g soluble starch, 0.3g dipotassium hydrogen phosphate, 0.024g anhydrous magnesium sulfate, 0.3g sodium pyruvate, 16g agar powder, and 1L distilled water).
[0028] The 16S rDNA of the isolated Bacillus strain was sequenced and analyzed by BLAST on the NCBI website. The strain BLLGPG27 showed 100% similarity to the reference strain Bacillus subtilis (GenBank PV616805.1), thus isolating a new Bacillus strain. The Bacillus strain BLLGPG27 exhibits the following characteristics: When the screened Bacillus strain BLLGPG27 was streaked on a culture medium and incubated at 28°C for 2-5 days, it was found to form round, well-defined, milky-white, opaque colonies with a dry, rough surface. Figure 1As shown. The amplified 16S rDNA sequence was 776 bp in length. Phylogenetic analysis showed that strain BLLGPG27 clustered with Bacillus subtilis (PV616805.1) with 100% homology, as shown. Figure 2 As shown.
[0029] Example 2
[0030] The specific steps for preparing the crude extract of Bacillus spp. BLLGPG27 fermentation broth are as follows:
[0031] (1) Bacillus BLLGPG27 (CGMCC No.33259) was inoculated into LB medium and cultured in a shaker at 28°C and 150 r / min for 24 h to obtain seed culture.
[0032] (2) The seed culture obtained in step (1) was transferred to SGTYP medium (inoculation amount 10%) and cultured in a shaker at 28℃ and 150r / min for 3 days to obtain the fermentation product.
[0033] (3) Separate the fermentation broth and cells from the fermentation product obtained in step (2). The fermentation broth is extracted twice with 1.5 times the volume of ethyl acetate. The extracts are combined, concentrated under reduced pressure, and dried to obtain the crude extract.
[0034] (4) The crude extract obtained in step (3) was analyzed by HPLC, with LB blank medium as a control. The HPLC conditions were as follows: High performance liquid chromatograph: Agilent 1260; chromatographic column: Agilent ZORBAX Eclipse XDS-C18 (5μm, 4.6mm×250mm); chromatographic conditions: flow rate 1.0mL / min, injection volume 5μL; detection wavelength: 210, 254 and 330nm; mobile phase: water (A)-methanol (B), gradient elution program: 0-35min, 5%-100% B; 35-40min, 100%-100% B; 40-41min, 100%-5% B; 41-46min, 5%-5% B. The results are as follows. Figure 3 Secondary metabolites of the crude extract of Bacillus BLLGPG27 fermentation broth appeared between 5 min and 35 min and were of moderate polarity.
[0035] Example 3
[0036] Antibacterial activity test of crude extract of Bacillus spp. BLLGPG27 fermentation broth
[0037] Indicator pathogens tested: methicillin-resistant Staphylococcus aureus (ATCC 43300, MRSA ATCC 43300), Staphylococcus aureus (S. aureus ATCC 25923), Staphylococcus epidermidis (S. epidermidis ATCC 12228), Enterococcus faecalis (Enterococcus faecalis ATCC 51299), and E. faecalis (E. faecalis ATCC 35667).
[0038] The antibacterial activity was detected using the "micro-broth dilution method," as described in the reference [Microbiology China, 2022, 49(9):3813-3836, Acta Microbiologica Sinica, 2021, 61(4):862-874]. The specific steps are as follows:
[0039] (1) Inoculate the pathogen indicator bacteria into 5 ml of liquid LB medium and incubate at 37°C and 160 r / min for 12 h.
[0040] (2) Dilute the above-cultured bacterial solution to 5×10⁻⁶. 5 CFU / mL, add 1 mL of diluted bacterial solution to each well of a 48-well plate, then add 2 μL of DMSO solution of the 25 mg / mL crude extract prepared in Example 1 to make the initial concentration of the test 100 μg / mL, and then dilute it 2 / 3 times (100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL). The negative control is 1 mL of diluted bacterial solution + 2 μL of DMSO. When the pathogen indicator is Gram-positive, the positive control is vancomycin at a final concentration of 2.0 μg / mL. When the pathogen indicator is Gram-negative, the positive control is ciprofloxacin at a final concentration of 2.0 μg / mL. The blank control is the same volume of LB medium without bacterial solution and sample. Six replicates are set for each concentration. (The first six wells of the 48-well plate contain 100 μg / mL samples, and the second to eighth wells contain 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, negative, blank, and positive samples, respectively.)
[0041] (3) After incubating the 48-well plate in a 37℃ incubator for 24 hours, the absorbance of each well was measured using an ELISA reader. The results are shown in the table below.
[0042]
[0043] The results showed that the crude extract prepared in Example 1 had broad-spectrum antibacterial activity. It exhibited moderate to strong antibacterial activity against both Gram-positive pathogens, S. aureus ATCC 25923 and S. epidermidis ATCC 12228 (MIC range of 6.25-75.00 μg / mL).
[0044] No significant antibacterial activity was detected for Enterococcus faecalis ATCC 51299, E. faecium ATCC 35667, and methicillin-resistant S. aureus (MRSA) ATCC 43300, with MIC values >100 μg / mL.
[0045] Example 4
[0046] Inhibitory effect of crude extract of Bacillus BLLGPG27 fermentation broth on Fusarium oxysporu, the pathogenic fungus causing root and stem rot of Paris polyphylla.
[0047] This embodiment of the experiment used the "filter paper disc method". The pathogenic fungus Fusarium oxysporum G1F1 was isolated, purified and identified from the rhizomes of diseased Paris polyphylla plants suffering from rhizome rot in Guanping Township, Yunlong County, Yunnan Province. BLAST comparison analysis on the NCBI website showed a 100% similarity to the reference strain Fusarium oxysporum (GenBank JK989157.1). The specific steps are as follows:
[0048] The fungal disc was collected using a 5mm perforator. Using sterile forceps, the disc was placed in the center of a PDA plate. Four 6mm diameter filter paper discs were placed 2.5cm away from the disc, and 4μL of ethyl acetate containing the crude extract (25mg crude extract per mL of ethyl acetate) was added to each disc. A control was prepared by inoculating only the tested pathogen. Each treatment was repeated in triplicate, and the plates were incubated upside down at 28°C for 7 days. The diameter of the fungal colonies was measured using the cross-hatching method, and the inhibition rate of mycelial growth was calculated using the following formula. The calculated inhibition rate of the crude extract of this invention against *Fusarium oxysporum* was 59.48%.
[0049]
[0050] Antibacterial results showed that the crude extract prepared in Example 1 had antibacterial activity against the root and stem rot pathogen (Fusarium oxysporum). Figure 4 ).
[0051] Example 5
[0052] Inhibitory effect of crude extract of Bacillus BLLGPG27 fermentation broth on Fusarium solani, the pathogenic fungus causing root and stem rot in Paris polyphylla.
[0053] This embodiment of the experiment used the "filter paper disc method". The pathogenic fungus Fusarium solani G1F2 was isolated, purified and identified from the rhizomes of diseased Paris polyphylla plants in Guanping Township, Yunlong County, Yunnan Province, which caused root and stem rot. BLAST comparison analysis on the NCBI website showed a 100% similarity to the reference strain Fusarium solani (GenBank MH857319.1). The specific steps are as follows:
[0054] The fungal disc was collected using a 5mm perforator. Using sterile forceps, the disc was placed in the center of a PDA plate. Four 6mm diameter filter paper discs were placed 2.5cm away from the disc, and 4μL of ethyl acetate containing the crude extract (25mg crude extract per mL of ethyl acetate) was added to each disc. A control was prepared by inoculating only the tested pathogen. Each treatment was repeated in triplicate, and the plates were incubated upside down at 28°C for 7 days. The diameter of the fungal colony was measured using the cross-hatching method, and the inhibition rate of mycelial growth was calculated using the following formula. The calculated inhibition rate of the crude extract described in this invention against *Fusarium solani* was 33.90%.
[0055]
[0056] Antibacterial results showed that the crude extract prepared in Example 1 had antibacterial activity against Fusarium solani, the pathogen causing root and stem rot. Figure 5 ).
Claims
1. A Bacillus species, specifically Bacillus BLLGPG27, characterized in that: The Bacillus species BLLGPG27 has the following accession number: CGMCC No. 33259, and its taxonomic name is: Bacillus subtilis .
2. The crude extract of fermentation broth prepared using the Bacillus BLLGPG27 bacterium described in claim 1.
3. The application of the crude extract of fermentation broth prepared from Bacillus BLLGPG27 as described in claim 2 in the preparation of an agent for preventing and controlling root and stem rot of Paris polyphylla.
4. The application of the crude extract of fermentation broth obtained from Bacillus BLLGPG27 as described in claim 3 in the preparation of an agent for controlling root and stem rot of Paris polyphylla, characterized in that: The root and stem rot disease of Paris polyphylla is caused by the pathogen Fusarium oxysporum. Fusarium oxysporum Fusarium solani Fusarium solani cause.
5. The use of the crude extract of fermentation broth obtained from Bacillus BLLGPG27 as described in claim 2 in the preparation of a medicament for treating diseases caused by Gram-positive bacteria.
6. The use of the crude extract of fermentation broth obtained from Bacillus BLLGPG27 according to claim 5 in the preparation of drugs for treating diseases caused by Gram-positive bacteria, characterized in that: The Gram-positive bacteria are one or more of the following: methicillin-resistant Staphylococcus aureus, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, or Enterococcus faecium.
7. The method for preparing the crude extract of fermentation broth obtained from Bacillus BLLGPG27 according to claim 2, characterized in that: The preparation method specifically includes the following steps: (1) The fermentation broth of Bacillus spp. BLLGPG27 was obtained by culturing; (2) Separate the fermentation broth and the cells, extract with ethyl acetate, combine the extracts, concentrate under reduced pressure and dry to obtain the crude extract.