Giant sphaeroides strain and its application and medicine containing the same for preventing or treating liver tumors

Abstract

The present invention provides a strain of Megasphaera elsdenii and its application and a drug containing the same for preventing or treating liver tumors, and relates to the field of microbial drug technology. The Megasphaera strain MNH 21576 can be widely used in the preparation of liver tumor inhibitors and drugs for preventing or treating liver tumors. It has been verified through experiments that the Megasphaera strain MNH 21576 of the present application can effectively inhibit the growth rate of liver cancer tumors, has a higher tumor inhibition rate and has better responsive anti-tumor efficacy than other strains of the same species.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial medicines, and in particular to a strain of Megasphaera elsdenii and an application thereof, as well as a medicine for preventing or treating liver tumors comprising the strain. Background Art

[0002] In the existing technologies, such as the field of probiotics or FMT flora transplantation, a variety of microorganisms are usually mixed to act on the intestines. There are also existing technologies that use a mixture of multiple bacteria as anti-tumor drugs. However, the mechanism of multiple bacteria acting on indications is more complicated, and there is no mature research on the mutual influence between bacteria; as live bacteria drugs, the use of multiple bacteria will disrupt the homeostasis of the intestinal flora. In comparison, a single bacterium has less effect on the homeostasis of the intestinal flora. In addition, the use of mixed bacteria requires additional consideration of whether the bacteria will affect each other's activity. At the same time, it is not clear whether it is the synergistic effect of mixed bacteria that makes it play a role in the immune process, or a single bacterium plays a role in the immune process; short-chain fatty acids are important metabolites that activate immunity. Since the presence of multiple bacteria will also affect the short-chain fatty acid pathway, it is not clear which bacteria play a role in the short-chain fatty acid pathway and effectively generate short-chain fatty acids for activating immune cells.

[0003] Under the genus Megasphaera, M.massiliensis has been extensively studied for the treatment or prevention of neurodegenerative diseases, as well as autoimmune and inflammatory diseases. For the indication of tumors, in the current existing technology, a tumor indication model has been established using a single strain of M.massiliensis, proving that M.massiliensis can enhance the anti-tumor activity of CD8+CTL by producing butyrate and valeric acid. Studies have also shown that M.elsdenii can be used for colorectal cancer, but its experimental model is to act directly on tumor-bearing mice, and its mechanism of action is actually to prevent or treat cancer caused by excessive lactic acid in the intestinal environment by converting excess lactic acid in the intestine into other products; and the strain is in the intestine and cannot come into contact with cancerous tissue structures in other parts such as liver cancer, so it is not expected that it can be derived from other cancers such as liver cancer; however, there are about 600,000 new liver cancer patients worldwide each year, ranking fifth among malignant tumors, and anti-tumor microbial drugs under the genus Megasphaera that can be effectively used for liver cancer need to be developed.

[0004] Although butyrate and valeric acid have been proven in the prior art to be metabolites of CD8+CTL anti-tumor activity, and are also important metabolites that inhibit HDAC activity and achieve anti-tumor effects, Megasphaera does not rely on butyrate and valeric acid metabolites to achieve anti-tumor effects, but rather relies on the individuality of the strain itself. That is, screening new strains that can be effectively used for anti-tumor effects from strains with low butyrate and valeric acid production means that pharmaceutical research and development companies face greater challenges.

[0005] Therefore, it is necessary and urgent to provide a Megasphaera strain that can effectively inhibit the growth rate of liver cancer tumors, improve the tumor inhibition rate, and have better responsive anti-tumor efficacy.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The present invention aims to provide a Megasphaera strain MNH 21576 and its application in preparing a liver tumor inhibitor.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The present invention provides a Megasphaera elsdenii strain, which has been deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 28, 2022, with the preservation name Megasphaerasp.MNH 21576 and the preservation number GDMCC No.62666.

[0010] It should be noted that Megasphaera strain MNH 21576 was isolated from a fecal sample of a healthy volunteer in Dali City, Yunnan Province. It has been deposited in Guangdong Microbiological Culture Collection Center, with the deposit name Megasphaera sp.MNH21576, the deposit number GDMCC 62666, the deposit date July 28, 2022, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the proposed taxonomic name is Megasphaera sp.

[0011] Furthermore, the physiological and biochemical characteristics of the strain MNH 21576 include:

[0012] The temperature growth range of strain MNH 21576 is 30-42°C and it can grow in the pH range of 3.0-9.0;

[0013] strain MNH 21576 could tolerate up to 1% NaCl;

[0014] Strain MNH 21576 survived and grew in the bile salt concentration range of 0 to 0.40%;

[0015] The strain MNH 21576 cannot grow under aerobic conditions but grows well under anaerobic conditions, and is an obligate anaerobic bacterium.

[0016] Furthermore, the 16S ribosomal RNA sequence of the strain MNH 21576 is shown in SEQ ID NO:1.

[0017] The specific SEQ ID NO:1 sequence is as follows: "GATGAGAAGCTTGCTTCTTATCAAT TCGAGTGGCAAACGGGTGAGTAACGCGTAAGCAACCTGCCCTTCAGATGGGGACAACAGCTGGAAACGGCTGCTAATACCGAATACGTTCTTTTTGTCGCATGGCAGAGGGAAGAAAGGGAGGCTCTTCGGAGCTTTCGCTGAAGGAGGGGCTTGCGTCTGATTAGCTAGTTGGAGGGGTAACGGCCCACCAAGGCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAACGATGACGGCCTTCGGGTTGTAAAGTTCTGTTATACGGGACGAATGGCGTAGCGGTCAATACCCGTTACGAGTGACGGTACCGTAAGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCGCGCAGGCGGCGTCGTAAGT CGGTCTTAAAAGTGCGGGGCTTAACCCCGTGAGGGGACCGAAACTGCGATGCTAGAGTATCGGAGAGGAAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAAGCGGCTTTCTGGACGACAACTGACGCTGAGGCGCGAAAGCCAGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCTGGCCGTAAACGATGGATACTAGGTGTAGGAGGTATCGACCCCTTCTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGACGCAACGCGAAGAACCTTACCAAGCCTTGACATTGATTGCTATGG".

[0018] Furthermore, the ANI value of the strain MNH 21576 and the strain MNC-992 in the genus Megasphaera is ≤82.42%.

[0019] The applicant hereby declares that the technical solution of this application does not involve the biological deposit of the bacterium MNC-992 of the genus Megasphaera, and there is no need to submit a deposit certificate for the biological material sample.

[0020] Further, the Megasphaera elsdenii strain MNH 21576 is a butyrate-free or low-butyrate-producing bacterium;

[0021] Preferably, the butyrate production of Megasphaera elsdenii is less than 30.31 ug / g.

[0022] The use of the Megasphaera elsdenii strain provided by the present invention in the preparation of a liver tumor inhibitor;

[0023] Preferably, the tumor inhibition rate is ≥40%;

[0024] Preferably, the tumor suppressor is an suppressor that suppresses tumor size.

[0025] In a preferred embodiment of the present invention, the application comprises:

[0026] The number of viable bacteria was 8.0×10 9 ~2.5×10 10 CFU / mL of Megasphaera elsdenii strain solution was gavaged into the syngeneic tumor model mice at a dose of 200 μL per day.

[0027] In the above preferred embodiment, the syngeneic tumor model mice are mice in which an ectopic syngeneic tumor model is formed by subcutaneously inoculating H22 liver cancer cells.

[0028] The present invention provides a microbial agent, which comprises the above-mentioned Megasphaera elsdenii strain and / or its metabolites;

[0029] Preferably, the microbial agent is used for the prevention and / or treatment of tumor diseases;

[0030] Preferably, the microbial agent has an inhibition rate of ≥40% on tumors;

[0031] Preferably, the microbial agent inhibits tumor size.

[0032] The present invention provides a medicine for preventing or treating liver tumors, comprising the above-mentioned Megasphaera elsdenii strain and a pharmaceutically acceptable excipient or adjuvant.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The Megasphaera elsdenii strain MNH 21576 provided by the present invention can be widely used in the preparation of liver tumor inhibitors and drugs for preventing or treating liver tumors. The strain does not rely on butyrate and valeric acid metabolites to achieve anti-tumor effect, but relies on the individuality of the strain itself to achieve anti-tumor effect, that is, a new strain that can be effectively used for anti-tumor is screened from strains with low butyrate and valeric acid production. It is verified by experiments that the Megasphaera elsdenii strain MNH 21576 of the present application can effectively inhibit the growth rate of liver cancer tumors, has a higher tumor inhibition rate than other strains of the same species, and has a better responsive anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 A bar graph showing the tolerance of Megasphaera elsdenii strain MNH 21576 at different pH values ​​provided in Example 1 of the present invention;

[0037] Figure 2 A bar graph showing the tolerance of Megasphaera elsdenii strain MNH 21576 to different concentrations of NaCl provided in Example 1 of the present invention;

[0038] Figure 3 A bar graph showing the choline tolerance of Megasphaera elsdenii strain MNH 21576 provided in Example 1 of the present invention;

[0039] Figure 4 A phylogenetic tree of the Megasphaera elsdenii strain MNH 21576 provided in Example 1 of the present invention;

[0040] Figure 5This is a volume curve diagram of the inhibition of tumor growth by the Megasphaera elsdenii strain MNH 21576 provided in Example 7 of the present invention;

[0041] Figure 6 A bar graph showing that the Megasphaera elsdenii strain MNH 21576 reduces the endpoint tumor volume provided in Example 7 of the present invention;

[0042] Figure 7 A bar graph showing the tumor inhibition rate of the Megasphaera elsdenii strain MNH 21576 provided in Example 7 of the present invention;

[0043] Figure 8 A comparison graph of the inhibition of tumor growth volume curves of the Megasphaera elsdenii strain MNH 21576 and another strain B of the same species provided in Example 7 of the present invention;

[0044] Fig. 9 A comparison chart of the reduction of the tumor volume at the endpoint between the Megasphaera elsdenii strain MNH 21576 and another strain B of the same species provided in Example 7 of the present invention;

[0045] Fig.10 This is a comparison chart of the endpoint tumor inhibition rate of Megasphaera elsdenii strain MNH21576 and another strain B of the same species provided in Example 7 of the present invention. DETAILED DESCRIPTION

[0046] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0048] Example 1 Identification of physiological and biochemical characteristics of strain MNH 21576:

[0049] (I) Physiology and biochemistry of API 20A test strain:

[0050] API reagent strips (API 20A was used in this study). For specific experimental operations, see the API reagent operation guide. Culture conditions: 37°C, anaerobic.

[0051] Strain MNH21576 API 20A test results:

[0052] Test items Test results Test items Test results IND Negative ESC Negative URE Negative GLY No acid production GLU Acid production CEL No acid production MAN No acid production MNE No acid production LAC No acid production MLZ No acid production SAC No acid production RAF No acid production MAL Acid production SOR No acid production SAL No acid production RHA No acid production XY No acid production TRE No acid production ARA No acid production GEL Negative

[0053] (II) pH tolerance, NaCl tolerance, and bile salt tolerance of the strain:

[0054] In this embodiment, the pH tolerance, NaCl tolerance, and bile salt tolerance of strain MNH 21576 were detected by conventional detection methods, and strain MNH 21576 was obtained. The temperature growth range was 30-42°C, and the optimal growth was 37°C; it could grow in the pH range of 3.0 to 9.0, and the optimal growth pH was 3.0-7.0; it could tolerate up to 1% NaCl; strain MNH 21576 could survive and grow in the bile salt concentration range of 0-0.40%. Strain MNH 21576 could not grow under aerobic conditions, but grew well under anaerobic conditions, and was an obligate anaerobic bacterium.

[0055] Specific results such as Figures 1 to 3 As shown:

[0056] Figure 1 This is a bar graph showing the tolerance of strain MNH 21576 at different pH values;

[0057] Figure 2 The figure is a bar graph showing the tolerance of the strain MNH 21576 in this example to different concentrations of NaCl;

[0058] Figure 3 The figure is a bar graph showing the choline tolerance of strain MNH 21576 in this example.

[0059] Example 2 Antibiotic sensitivity test of strain MNH21576:

[0060] The antibiotic sensitivity test of strain MNH 21576 was performed using the disk diffusion method. The test results are shown in the table below.

[0061] Table: Antibiotic sensitivity test results of strain MNH 21576:

[0062]

[0063] As can be seen from the above table, the strain MNH 21576 of the present application is sensitive to antibiotics such as gentamicin, erythromycin, chloramphenicol, tetracycline and ceftriaxone; the strain MNH 21576 is resistant to antibiotics such as penicillin, ciprofloxacin, co-trimoxazole, ampicillin, lincomycin, etc.

[0064] Example 3 16S identification of strain MNH21576:

[0065] (I) Identification of strain MNH21576:

[0066] The isolated strain (strain named MNH21576) was subjected to 16S rRNA analysis to preliminarily determine its taxonomic status.

[0067] The genomic DNA of the strain was extracted, and the 16S rRNA was amplified using the extracted genomic DNA template. The amplification primers were:

[0068] 1492R(5'-AGAGTTTGATCATGGCTCAG-3');

[0069] and, 27F(5′-TAGGGTTACCTTGTTACGACTT-3′);

[0070] The amplified PCR product was purified and then sequenced by ABI3730XL to obtain a 954bp 16S rRNA sequence. This sequence was compared with the NCBI 16S ribosomal RNA sequences database. The closest species was Megasphaera elsdenii with a similarity of 100%. The species classification information of this strain was preliminarily determined, that is, the species MNH21576 was preliminarily determined to be Megasphaera elsdenii.

[0071] (II) Phylogenetic tree of strain MNH21576:

[0072] This sequence was compared with the 16S rRNA sequences of related genera, and a phylogenetic tree was constructed using MEGA 5.

[0073] Figure 4 : This is the phylogenetic tree of the Megasphaera strain MNH 21576 of this example.

[0074] Example 4 ANI comparison results of strain MNH21576:

[0075] The genome of the isolated original strain MNH21576 was prepared, sequenced, assembled and analyzed.

[0076] The genome of the original strain MNH21576 was fragmented by ultrasonic method, with the fragment length ranging from 350 bp, and then the Illumina sequencing library was constructed using a standard DNA library construction kit (NEB UltraTM). The constructed sequencing library was sequenced with NovaSeq (Illumina) for double-end 150 bp. The sequencing obtained 2.80 Gbp of data, of which Q20 accounted for 97.74%.

[0077] Note: The raw sequencing data of the genome were filtered using fastp (version: 0.20.0) with filtering parameters: "--poly_g_min_len 10--poly_x_min_len 10-q 15-u 40-n 5-l 50". The filtered raw data were assembled using SPAdes (version: v3.14.0) with assembly parameters "--isolate--cov-cutoff 10". The total gene length of the genome assembly was 2.56Mbp, the N50 length was 183.0kbp, and the GC content was 52.6%.

[0078] The genomic genes were predicted and analyzed using the prokaryotic analysis software genome annotation pipeline prokka (version: 1.14.5) with the parameters "--gcode 11--evalue 1e-09". A total of 2279 CDS sequences were predicted, with an average CDS sequence length of 975 bp.

[0079] The model strain with the highest genome similarity is Megasphaera elsdenii, with an average nucleotide similarity (ANI) of 97.82% and a gene coverage of 84.99%. Therefore, it can be identified as Megasphaera elsdenii.

[0080] The results of the whole genome comparison are as follows:

[0081]

[0082] Note: MNC-992 is the bacterium MNC-992 of the genus Megasphaera in patent CN112239741A. The whole genome comparison results with the Megashpaera strain MNC-992 in patent CN112239741A show that the similarity ANI is 82.42%, AF=59%; from the genome comparison results, it can be seen that MNH21576 and MNC-992 belong to different species.

[0083] The applicant hereby declares that the technical solution of this application does not involve the biological deposit of the bacterium MNC-992 of the genus Megasphaera, and there is no need to submit a deposit certificate for the biological material sample.

[0084] Example 5 Genome analysis of strain MNH21576:

[0085] (I) Drug resistance genes:

[0086] Potential antibiotic resistance genes in the genome were analyzed using the RGI pipeline (version: 4.2.2), where the antibiotic resistance gene database was CARD (version: 3.0.0, https: / / card.mcmaster.ca / analy ze / rgi). Detailed comparison information is shown in the table below.

[0087] Table: Drug resistance gene information list:

[0088]

[0089]

[0090] (II) Analysis of Potentially Toxic Genes:

[0091] The analysis of potential virulence factors and related genes in the genome was performed using NCBI blastp (version: 2.7.1+) to compare the virulence factor database VFDB (virulence factor database, http: / / www.mgc.ac.cn / cgi-bin / VFs / v5 / main.cgi, updated on September 19, 2019). The detailed comparison results are shown in the table below.

[0092] Table: List of potential toxic genes of MNH21576:

[0093] Strain Gene VFDB gene Gene name Comparison consistency (%) MNH21576_01002 VFG011430 acpXL 66.216 MNH21576_01600 VFG001967 glf 60.331 MNH21576_01667 VFG000964 hasC 65.672 MNH21576_01839 VFG000077 P 68.449 MNH21576_01999 VFG001855 htb 61.143

[0094] (III) Analysis of potential primary metabolic gene clusters:

[0095] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). The detailed comparison results are shown in the table below:

[0096] Table: List of potential primary metabolic gene clusters of MNH21576:

[0097]

[0098]

[0099] (IV) Potential secondary metabolism gene clusters:

[0100] The analysis of potential secondary metabolism gene clusters in the genome was performed using antiSMASH6 (version: 6.0.1). Detailed comparison results are shown in the table:

[0101] Table: List of potential secondary metabolism gene clusters of MNH21576.

[0102]

[0103] Example 6 Determination of short-chain fatty acids (SCFA) of strain MNH21576:

[0104] (1) Preparation of bacterial cells: The strain MNH21576 was inoculated into a liquid culture medium and cultured anaerobically at 37°C for 48 hours. The bacterial cells were collected by centrifugation and stored at -86°C for later use.

[0105] (2) Preparation of standards: Weigh acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid standards and prepare eight mixed standard concentration gradients of 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL and 100 μg / mL with ethyl acetate.

[0106] Take 600 μL of standard, add 25 μL of 4-methylvaleric acid with a final concentration of 500 μM as internal standard, mix well and add to the injection bottle, enter GC-MS detection, injection volume 1 μL, split ratio 10:1, split injection.

[0107] (3) Metabolite extraction: Thaw the sample on ice, take 80 mg of the sample and put it into a 2 mL glass centrifuge tube, add 900 μL of 0.5% phosphoric acid to resuspend, shake and mix for 2 min, centrifuge at 14000 g for 10 min, take 800 μL of the supernatant, add an equal amount of ethyl acetate to extract, shake and mix for 2 min, centrifuge at 14000 g for 10 min, take 600 μL of the upper organic phase, add 4-methylpentanoic acid with a final concentration of 500 μM as an internal standard, mix well and add to the injection bottle, enter GC-MS detection, injection volume 1 μL, split ratio 10:1, split injection.

[0108] (4) Sample detection and analysis: Agilent DB-WAX capillary column (30m×0.25mm ID×0.25μm) gas chromatography system was used to separate the samples. Program temperature: initial temperature 90℃, temperature increased to 120℃ at 10℃ / min, then to 150℃ at 5℃ / min, and finally to 250℃ at 25℃ / min and maintained for 2min. The carrier gas was helium with a flow rate of 1.0mL / min.

[0109] Agilent 7890A / 5975C gas chromatograph was used for mass spectrometry analysis. The injection port temperature was 250°C, the ion source temperature was 230°C, the transfer line temperature was 250°C, and the quadrupole temperature was 150°C. Electron impact ionization (EI) source, full scan and SIM scanning mode, electron energy 70 eV.

[0110] The chromatographic peak area and retention time were extracted using MSD ChemStation software. The calibration curve was drawn to calculate the content of short-chain fatty acids in the sample.

[0111] Table: Short chain fatty acid (SCFA) production results of strain MNH21576:

[0112]

[0113] It can be seen from the test results that strain MNH21576 produces low butyrate and valeric acid during growth. Although butyrate and valeric acid have been proven in the prior art to be metabolites of CD8+CTL anti-tumor activity; they are also important metabolites that inhibit HDAC activity and achieve anti-tumor effects, the M. elsdenii strain of the present invention, through the measurement results of SCFA, shows that the strain of the present invention produces low butyrate and valeric acid, and the therapeutic effect on liver cancer does not rely on the known butyrate, valeric acid and other metabolites in the genus Megasphaera to achieve anti-tumor effects.

[0114] Example 7 Experiment to verify the therapeutic effect of strain MNH21576 on tumors:

[0115] (I) Experimental methods:

[0116] Through the BALB / C mouse H22 homologous subcutaneous transplant tumor therapeutic liver cancer model, the animals were housed in independent ventilation cages (IVCs). The cages were made of polyphenylene sulfone resin (PPSU) and the cages were 372mm×205mm×172mm, with 3-4 animals per cage. The barrier environment animal room was well ventilated, with a minimum air change rate of not less than 15 times / hour and a minimum static pressure difference of ≥10Pa in the intercommunication area.

[0117] Environmental conditions were controlled at a temperature of 20.0°C to 26.0°C, relative humidity of 40% to 70%, and lighting was alternating between 12h / 12h day and night. Animals were free to eat qualified SPF-grade mouse and rat maintenance feed (produced by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.). After the quarantine of male BALB / C mice, H22 mouse liver cancer cells were subcutaneously injected into the right shoulder blade (2×105 cells / mouse). When the tumor grew to an average volume of 80-100mm 3 At the same time, mice with abnormal tumor growth were eliminated and randomly stratified into groups according to tumor volume.

[0118] The control group and the MNH21576 group were given oral administration of 200 μL of the control substance (PBS-Cys(glycine)), MNH21576 bacterial resuspension (1-5×10 9 CFU), once a day, the average tumor volume of any group of mice exceeded 2000mm 3 The overall endpoint of the trial was general clinical observation, body weight monitoring, tumor volume (mm 3) measurement, and after reaching the end point of the experiment, all surviving mice were dissected to measure tumor weight and calculate tumor volume. Finally, statistical analysis and comparison of tumor volume curve change (Tumor volume), endpoint tumor volume (Tumor volume at endpoint) and endpoint tumor inhibition rate (Tumor inhibition rate at endpoint) were performed:

[0119] 1. Tumor inhibition rate: (average volume of control group minus the volume of experimental group) / average volume of control group × 100%;

[0120] 2. Evaluation criteria: Tumor inhibition rate <40% represents low / no response antitumor efficacy; tumor inhibition rate ≥40% represents responsive antitumor efficacy. All data were expressed in the form of mean±SD and were plotted and statistically analyzed using GraphPad Prism8.0.2 software.

[0121] For pairwise comparison, Student's t test was used for analysis. For two-factor analysis, two-way ANOVA with Sidak's multiple comparison was used for analysis. Significant differences are indicated by *, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0122] 3. Test strain: MNH21576 live bacterial suspension (8.0×109CFU / mL≤live bacterial count≤2.5×1010CFU / mL);

[0123] 4. Tumor cells: H22 mouse liver cancer cells. The size of the seed tumor was measured every two days and the volume of the seed tumor was calculated using the formula: tumor volume = (1 / 2) × tumor long diameter 2 × tumor wide diameter.

[0124] (II) Experimental results of strain MNH21576 on tumor treatment:

[0125] Figure 5 This is a volume curve diagram of the inhibition of tumor growth by the strain MNH21576 in this example;

[0126] Figure 6 This is a bar graph showing that strain MNH21576 of this example reduces the endpoint tumor volume;

[0127] Figure 7 The figure is a bar graph showing the tumor inhibition rate of the strain MNH21576 in this example;

[0128] In the H22 liver cancer therapeutic model, the tumor was about 80-100mm 3Through intervention, MNH21576 can inhibit the tumor growth volume curve, achieving statistically significant differences, and can reduce the tumor volume size at the endpoint. Further analysis of the endpoint tumor inhibition rate showed that the endpoint average tumor inhibition rate of MNH21576 was close to 40%, and half of the mice vaccinated with MNH21576 had a tumor inhibition rate ≥40%, with better responsive anti-tumor efficacy, suggesting that MNH21576 has potential as a single-agent therapeutic for liver cancer.

[0129] By the above Figure 5 , Figure 6 , Figure 7 It can be seen that MNH21576 inhibits the growth of H22 liver cancer tumors and shows a good tumor inhibition rate in the H22 liver cancer treatment model. The tumor volume change curves, endpoint tumor size and tumor inhibition rate of the Control group and the MNH21576 live bacteria drug treatment group at different time points.

[0130] Among them, the tumor inhibition rate <40% represents low / no response antitumor efficacy; the tumor inhibition rate ≥40% represents responsive antitumor efficacy. All data are expressed in the form of mean±SD and were plotted and statistically analyzed using GraphPad Prism 8.0.2 software.

[0131] Note: For two-factor investigation, two-factor variance analysis with Sidak multiple comparisons was used ( Figure 5 ); for pairwise comparisons ( Figure 6 , Figure 7 ), Student's t test was used for analysis, and significant differences were indicated by *, *p<0.05.

[0132] (III) Experimental comparison results of different strains of Megasphaera elsdenii in liver cancer treatment model:

[0133] The applicant also isolated another strain B (MNH22008) of the same species. This experiment compared the therapeutic effects of MNH21576 and another strain B of the same species in the liver cancer treatment model. The experimental method (I) of this embodiment was used; H22 mouse liver cancer cells were inoculated subcutaneously at the right scapula (2×10 5 cells / cell), when the tumor grows to an average volume of 80-100mm 3 When the tumor size was about 80-100 mm, mice with abnormal tumor growth were removed and randomly stratified and grouped according to tumor volume. 3 Intervention

[0134] Note: The applicant hereby declares that the technical solution of this application does not involve the biological deposit of the bacterium MNH22008 of the genus Megasphaera, and there is no need to submit a deposit certificate for biological material samples.

[0135] Figure 8 This is a comparison of the tumor growth volume inhibition curves of MNH21576 in this example and another strain B of the same species;

[0136] Fig. 9 This is a comparison chart of MNH21576 in this example and another strain B of the same species in reducing the tumor volume at the endpoint;

[0137] Fig.10 , Comparison of endpoint tumor inhibition rates of MNH21576 of this example and another strain B of the same species.

[0138] From the above, it can be seen that MNH21576 and another strain B of the same species have statistically significant differences in the inhibition of tumor growth volume curve, and can reduce the tumor volume size at the endpoint. Further analysis of the endpoint tumor inhibition rate shows that the endpoint average tumor inhibition rate of MNH21576 is greater than that of another strain B of the same species. Among the same strains, MNH21576 of the present invention has better responsive anti-tumor efficacy.

[0139] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A species of Giant sphaerocephala ( Megasphaera elsdenii ) strain MNH 21576, characterized in that The Escherichia coli strain MNH 21576 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 28, 2022, with the deposit number GDMCC No.62666.

2. A microbial agent, comprising the Megasphaera elsdenii strain MNH21576 according to claim 1.

3. A microbial agent, comprising the Megasphaera elsdenii strain MNH 21576 according to claim 1 and metabolites thereof, wherein the metabolites include short-chain fatty acids.

4. The microbial agent according to claim 3, characterized in that: The metabolites include at least one of acetic acid, isovaleric acid, caproic acid, propionic acid, and isobutyric acid.

5. A drug for treating liver tumors, characterized in that: The medicine comprises the microbial agent according to any one of claims 2 to 4 and a pharmaceutically acceptable excipient.

6. The drug for treating liver tumors according to claim 5, characterized in that: The auxiliary material is an excipient.

7. Use of the Megasphaera elsdenii strain MNH 21576 according to claim 1, the microbial agent according to any one of claims 2 to 4, or the drug according to claim 5 or 6 in the preparation of a liver tumor inhibitor.

8. The use according to claim 7, characterized in that: The tumor inhibition rate of the liver tumor inhibitor is ≥40%.

9. The use according to claim 7, characterized in that: The liver tumor inhibitor is an inhibitor that inhibits the size of liver tumors.

10. The use according to claim 7, characterized in that: The viable count of the Megasphaera elsdenii MNH 21576 was 8.0×10 9 ~2.5×10 10 CFU / mL.

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