Enterococcus faecalis MNH 22871 and its application

Abstract

The invention provides an Enterococcus faecalis MNH 22871 and an application thereof, and relates to the technical field of microorganisms. The Enterococcus faecalis MNH 22871 provided by the invention has a strain collection number of GDMCC NO: 62663. Experiments have confirmed that the Enterococcus faecalis strain MNH 22871 can effectively inhibit the growth rate and growth volume of liver cancer tumors through the characteristics of the strain itself, and its tumor inhibition rate is not less than 40% under a certain administration dosage, has better responsive anti-tumor efficacy, has liver cancer single-drug therapeutic efficacy, and is expected to solve the current treatment problem of "multidrug resistance" of liver cancer.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to Enterococcus faecalis MNH 22871 and applications thereof. Background Art

[0002] Primary liver cancer is the fourth most common malignant tumor in my country, with about 383,000 people dying from liver cancer each year, more than half of the global liver cancer deaths. Liver cancer in my country is different from that in foreign countries in terms of etiology, molecular biological characteristics and epidemiological characteristics, clinical manifestations and staging, and even treatment strategies and methods. It is one of the tumors with Chinese characteristics.

[0003] In addition, the liver is the most common target organ for cancer metastasis, and liver cells and immune cells together constitute a "pro-metastatic microenvironment." The occurrence and development of liver cancer are closely related to the microenvironment: microenvironmental disorders promote the occurrence of liver cancer, and the imbalance of microenvironmental immune inflammatory response is one of the most critical mechanisms of microenvironmental disorders. The liver microenvironment also determines the pathological type of liver cancer. In the apoptotic microenvironment, hepatocellular carcinoma is eventually formed through the epigenetic regulation of immune inflammation; in the necrotic microenvironment, cholangiocarcinoma is eventually formed through the epigenetic regulation of immune inflammation.

[0004] However, as a kind of tumor cell with innate drug resistance, liver cancer cells are generally insensitive to radiotherapy and chemotherapy, and their resistance mechanisms are even more complex and diverse; glutathione S-transferase (GST), DNA topoisomerase (Topo) and protein kinase C (PKC) are the main enzymes that cause liver cancer cells to produce multidrug resistance. In addition, the early symptoms of liver cancer patients lack typicality. Most patients are diagnosed in the late stage, with a short survival period and a high recurrence rate. The resulting decrease in sensitivity to chemotherapy drugs, followed by "multidrug resistance" has become one of the important reasons for the failure of primary hepatocellular carcinoma (PHC) treatment. How to effectively treat liver cancer has become a technical problem that needs to be solved urgently.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The main purpose of the present invention is to provide a specific strain of Enterococcus faecalis to at least solve one of the technical problems existing in the prior art.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] The invention provides Enterococcus faecalis MNH 22871. The Enterococcus faecalis MNH 22871 strain is preserved in Guangdong Province Microbiological Culture Collection Center, and the culture collection number is GDMCC NO: 62663.

[0009] Furthermore, the Enterococcus faecalis MNH 22871 produces low butyrate and / or valerate.

[0010] The present invention also provides a bacterial agent, which includes the above-mentioned Enterococcus faecalis MNH 22871.

[0011] The present invention also provides the use of the above-mentioned Enterococcus faecalis MNH 22871 or bacterial agent in preparing a medicine for treating liver cancer.

[0012] In addition, the present invention also provides a drug for treating liver cancer, comprising the above-mentioned Enterococcus faecalis MNH 22871 or bacterial agent.

[0013] Furthermore, the drug achieves the treatment of liver cancer by reducing tumor volume.

[0014] Furthermore, the tumor inhibition rate of the drug is ≥40%.

[0015] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0016] Furthermore, the dosage form of the drug includes oral preparations.

[0017] Furthermore, the oral preparations include tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.

[0018] Furthermore, when the drug is in liquid dosage form, the effective dosage is 8.0×10 9 ~2.5×10 10 CFU / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The inventors of the present invention have verified through a large number of experiments that the Enterococcus faecalis strain MNH 22871 can effectively inhibit the growth rate and growth volume of liver cancer tumors due to the characteristics of the strain itself. At a certain dosage, its tumor inhibition rate is not less than 40%, and it has better responsive anti-tumor efficacy and single-drug therapeutic efficacy for liver cancer, which is expected to solve the current treatment problem of "multidrug resistance" of liver cancer.

[0021] Based on the above properties of the Enterococcus faecalis strain MNH 22871, the bacterial agent or medicine containing the same, as well as the application of the Enterococcus faecalis strain MNH 22871 provided by the present invention, also have the above beneficial effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 The colony morphology of strain MNH 22871 provided in the embodiment of the present invention;

[0024] Figure 2 The Gram staining result of strain MNH 22871 provided in the embodiment of the present invention;

[0025] Figure 3 Microscopic morphological photos of strain MNH 22871 provided in the embodiments of the present invention;

[0026] Figure 4 The test results of strain MNH22871 API 20A provided in the embodiments of the present invention;

[0027] Figure 5A The pH tolerance result graph of strain MNH22871 provided in the embodiment of the present invention;

[0028] Figure 5B A graph showing the NaCl tolerance results of the strain MNH22871 provided in the embodiments of the present invention;

[0029] Figure 5C A graph showing the bile salt tolerance results of strain MNH22871 provided in an embodiment of the present invention;

[0030] Figure 6 A phylogenetic tree of strain MNH22871 provided in an embodiment of the present invention;

[0031] Figure 7 The experimental results of the strain MNH22871 provided in the embodiment of the present invention in the liver cancer treatment model; wherein A is the tumor volume change curve of the Control group and the MNH22871 live bacteria drug treatment group at different time points; B is the tumor size of the Control group and the MNH22871 live bacteria drug treatment group at the endpoint; C is the tumor mass of the Control group and the MNH22871 live bacteria drug treatment group at the endpoint; D is the tumor inhibition rate of the Control group and the MNH22871 live bacteria drug treatment group at the endpoint;

[0032] Fig. 8A The volume curve of inhibiting tumor growth of different strains of the same species in the liver cancer treatment model provided by the embodiment of the present invention;

[0033] Figure 8B The tumor volume at the treatment endpoint of different strains of the same species in the liver cancer treatment model provided by the embodiment of the present invention;

[0034] Figure 8C The average tumor inhibition rate of different strains of the same species at the treatment endpoint in the liver cancer treatment model provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0035] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0036] Generally, the nomenclature and its technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods as well-known in the art, and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as generally achieved in the art, or as described herein. The nomenclature and its laboratory procedures and technology used in conjunction with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are those well-known and commonly used in the art.

[0037] 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.

[0038] According to one aspect of the present invention, a strain of Enterococcus faecalis MNH 22871 is provided, which is deposited in Guangdong Microbiological Culture Collection Center, with the deposited name Enterococcus sp. MNH 22871, the deposited number GDMCC NO: 62663, the deposited time July 28, 2022, and the deposited address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, and the proposed classification name is Enterococcus sp.

[0039] Enterococcus faecalis belongs to the normal flora in the intestines of animals. It is generally believed that Enterococcus faecalis is harmless to animals or humans and is a symbiotic bacterium. Current studies have confirmed that Enterococcus faecalis can form biofilms on intestinal epithelial cells to protect intestinal epithelial cells from invasion by harmful substances; at the same time, Enterococcus faecalis contains a variety of small peptides that can promote the absorption of amino acids and residues and increase the synthesis of tissue proteins; in addition, Enterococcus faecalis is closely related to the occurrence, development and treatment of colorectal cancer. Liver cancer is the third most common malignant tumor with a mortality rate second only to gastric cancer and esophageal cancer. It is different from colorectal cancer in etiology, symptoms, treatment options and prognosis. As an intestinal microorganism, Enterococcus faecalis cannot directly contact the liver cancer tissue structure after colonization in the intestine. In addition, after live bacterial drugs enter the intestine, they face an extremely complex intestinal environment and steady-state associations with other flora in the intestine. The present invention obtains a strain with therapeutic function for liver cancer, Enterococcus faecalis MNH 22871, by screening fecal samples. Live animal experiments confirm that when the target live bacteria drug is not in direct contact with the tumor tissue, it can still effectively act on liver cancer tumor tissue and inhibit the growth rate of liver cancer tumors. It has a high tumor inhibition rate and better responsive anti-tumor efficacy.

[0040] It is worth noting that it has been disclosed that the effect of intestinal flora on cancer immunotherapy depends on its own metabolites butyrate and valeric acid. However, the SCFA measurement results of the Enterococcus faecalis strain of the present invention show that the strain of the present invention has low production of butyrate and valeric acid, from which it can be inferred that the strain MNH22871 uses its own characteristics to achieve anti-tumor effects. Among them, "low production" means that the butyrate production is not higher than 0.65μg / g, and the valeric acid production is not higher than 0.07μg / g.

[0041] The 16sDNA partial sequence of Enterococcus faecalis MNH 22871 provided by the present invention is shown in SEQ ID NO.1. By comparing SEQ ID NO.1 with the NCBI 16S ribosomal RNA sequences database, the closest species is Enterococcus faecium, with a similarity of 99.92%.

[0042] The colony characteristics of strain MNH 22871 are as follows: on MYPG solid plate medium, after anaerobic culture at 37°C for 36 hours, visible colonies are formed on the plate medium. The colonies are round, with regular and smooth edges, a diameter of about 2 mm, milky white, opaque, and no secretions are formed around the colonies. The strain is Gram-positive. Microscopic morphological observation revealed that the strain has no spores, no flagella, is non-motile, is spherical, and has a diameter of about 5 to 8 μm.

[0043] According to another aspect of the present invention, the present invention also provides a bacterial agent comprising the above-mentioned Enterococcus faecalis MNH 22871. Since the bacterial agent comprises the above-mentioned Enterococcus faecalis MNH 22871, it has all the beneficial effects of Enterococcus faecalis MNH 22871, which will not be repeated here. It can be understood that the bacterial agent can only contain Enterococcus faecalis MNH 22871, so that the bacterial agent has the function of Enterococcus faecalis MNH 22871; the bacterial agent can also contain other types of microorganisms, and Enterococcus faecalis MNH22871 can be used as the main active ingredient or auxiliary active ingredient in the bacterial agent.

[0044] Based on the ability of Enterococcus faecalis MNH 22871 to inhibit liver cancer, the present invention also provides the use of the above-mentioned Enterococcus faecalis MNH22871 or bacterial agent in the preparation of a drug for treating liver cancer. The use of Enterococcus faecalis MNH 22871 as an active ingredient to treat liver cancer can effectively inhibit the growth rate and growth volume of liver cancer tumors, and its tumor inhibition rate is not less than 40% at a certain dosage.

[0045] In addition, based on the above application, the present invention also provides a drug for treating liver cancer, which contains Enterococcus faecalis MNH 22871. The drug has better responsive anti-tumor efficacy and has the efficacy of single-drug treatment of liver cancer, and is expected to solve the current treatment problem of "multidrug resistance" of liver cancer.

[0046] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient.

[0047] Pharmaceutically acceptable excipients refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations in addition to active ingredients. The same pharmaceutical excipient can be used in pharmaceutical preparations for different routes of administration and has different effects and uses. The pharmaceutically acceptable excipients added to the drugs provided by the present invention can play a role in shaping, acting as a carrier or improving stability. In addition, they also have important functions such as solubilization, solubilization or sustained release. Typical but non-limiting pharmaceutically acceptable excipients include: one or more of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

[0048] In some optional embodiments, the dosage form of the drug includes an oral preparation. The above-mentioned drug can be made into any oral acceptable preparation form, such as, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.

[0049] In order to ensure the therapeutic effect of the drug on liver cancer, it is preferred that when the drug is in liquid dosage form, the effective dosage is 8.0×10 9 ~2.5×10 10 CFU / mL, for example, can be, but is not limited to, 8.0×10 9 CFU / mL, 9.0×10 9 CFU / mL, 1.0×10 10 CFU / mL, 2.0×10 10 CFU / mL or 2.5×10 10 CFU / mL.

[0050] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0051] Example 1 Strain Isolation

[0052] The intestinal strain MNH 22871 involved in the present invention is separated from a fecal sample of a healthy volunteer in Dali City, Yunnan Province.

[0053] The donor takes 2-5 grams of fresh feces and puts it into a sample collection and preservation tube. After shaking and homogenization, the processed feces sample is placed in an ice box and sent to the laboratory for strain isolation within 24 hours.

[0054] Dispense physiological saline in the biosafety cabinet, 9 ml / tube; prepare strain isolation culture medium - anaerobic blood agar plate, Columbia blood agar plate, chocolate agar plate, etc., and transfer it to the anaerobic workstation 24 hours in advance, marking the sample information, culture medium type, isolation date, etc.

[0055] Take fresh fecal samples and place them in an anaerobic operating station (Don Whitley Scientific A95) and shake them on a vortex shaker for 1 min. Pipette 1 mL of the sample into 9 mL of saline and mix for 10 min. -1 dilution, and then serially diluted to 10 -6 Dilution solution for later use.

[0056] Take 10 -6Drop the diluted solution onto the separation medium - anaerobic blood agar plate, Columbia blood agar plate, chocolate agar plate, at a drop volume of 100 μL / dish, spread evenly, wait for the surface of the plate to dry, invert the plate, and culture at 37°C for 3-5 days.

[0057] Observe the growth of the strain in the isolation medium and pick a single colony with a sterilized toothpick to purify the strain. The purified strain is placed at 37°C for anaerobically culture.

[0058] The pure culture strain was prepared into 20% glycerol / water-bacteria solution and stored at -86°C.

[0059] The strains were identified using the 16S rRNA gene sequence-based identification method.

[0060] Example 2 Morphological and physiological and biochemical characteristics of strains

[0061] 1. Morphological characteristics of strains:

[0062] The strain MNH 22871 was inoculated on MYPG solid plate medium and cultured anaerobically at 37°C for 36 hours. Visible colonies were formed on the plate medium. The colonies were round, with regular and smooth edges, about 2 mm in diameter, milky white, opaque, and no secretions formed around the colonies ( Figure 1 ); Gram-positive strains ( Figure 2 ); Microscopic morphological observation revealed that the strain had no spores, no flagella, no movement, was spherical, and had a diameter of about 5-8 μm ( Figure 3 ).

[0063] 2. Physiological and biochemical characteristics of strains:

[0064] (1) Physiological and biochemical characteristics of API 20A test strains

[0065] 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.

[0066] Strain MNH22871 API 20A test results ( Figure 4 )

[0067]

[0068]

[0069] (2) pH, NaCl, and bile salt tolerance of strains:

[0070] The strain MNH 22871 can grow in the temperature range of 30-42°C, with the optimum growth at 37°C; it can grow in the pH range of 5.0-10.0, with the optimum growth pH being 7.0-9.0 (see Figure 5A); can tolerate up to 6% NaCl (see Figure 5B ); strain MNH22871 can survive and grow in the bile salt concentration range of 0-0.40%, but cannot grow when the bile salt concentration is greater than or equal to 0.2% (see Figure 5C ). The strain MNH 22871 grows well under aerobic or anaerobic conditions and is a facultative anaerobic bacterium.

[0071] 3. Antibiotic sensitivity test of strain MNH22871:

[0072] The antibiotic sensitivity test of strain MNH 22871 was performed using the disk diffusion method. The test results are shown in Table 1. Strain MNH22871 was sensitive to antibiotics such as chloramphenicol, tetracycline, penicillin, cotrimoxazole, ampicillin, lincomycin and ceftriaxone; strain MNH 22871 was resistant to antibiotics such as erythromycin, ciprofloxacin and gentamicin.

[0073] Table 1 Antibiotic sensitivity test results of strain MNH 22871

[0074]

[0075]

[0076] Example 3 Identification of strains

[0077] 1. 16S sequence of strain MNH22871:

[0078] SEQ ID NO.1:

[0079]

[0080] 2. 16S identification results of strain MNH22871:

[0081] (1) Identification of strain MNH22871

[0082] The isolated strain (strain named MNH22871) was subjected to 16S rRNA analysis to preliminarily determine its taxonomic status. The genomic DNA of the strain was extracted, and the 16S rRNA was amplified using the extracted genomic DNA template. The amplification primers were 1492R (5'-AGAGTTTGATCATGGCTCAG-3') (SEQ ID NO.2) and 27F (5'-TAGGGTTACCTTGTTACGACTT-3') (SEQ ID NO.3). The amplified PCR product was purified and then sequenced by ABI3730XL to obtain a 1194bp 16S rRNA sequence, the sequence of which is shown in SEQ ID NO.1.

[0083] This sequence was compared with the NCBI 16S ribosomal RNA sequences database, and the closest species was Enterococcus faecium with a similarity of 99.92%. The species classification information of this strain was preliminarily determined, that is, MNH22871 was preliminarily determined to belong to Enterococcus faecium.

[0084] (2) Phylogenetic tree of strain MNH22871

[0085] Using MEGA-X software, we first retrieved the 16S rRNA gene sequences of all strains of the genus Enterococcus to which MNH22871 belonged from the LPSN database. Then, we used the MUSCLE method to perform multiple sequence alignment on the above 16S sequences and the 16S sequence of MNH22871. Finally, we used the maximum likelihood method to perform 1000 similarity repetitions to construct a phylogenetic tree ( Figure 6 ).

[0086] (3) ANI comparison results of strain MNH22871

[0087] In this example, the genome of the original strain MNH22871 isolated and obtained in Example 1 was prepared, sequenced, assembled and analyzed.

[0088] The genome of the original strain MNH22871 was fragmented by ultrasound, with a fragment length range of ~350bp, and then an 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 150bp. 2.01Gbp of data was obtained by sequencing, of which Q20 accounted for 97.54%.

[0089] The raw genome sequencing data was 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 was assembled using SPAdes (version: v3.14.0) with assembly parameters "--isolate--cov-cutoff 10". The genome assembly resulted in a total gene length of 2.44Mbp, an N50 length of 1.11kbp, and a GC content of 38.11%. The strain with the highest genome similarity is Enterococcus faecium, with an average nucleotide similarity (ANI) of 99.16% and a gene coverage of 91.47%. In summary, it can be determined that the strain of the present application is Enterococcus faecium.

[0090] Example 4 Genome analysis of strains

[0091] 1. Analysis of drug resistance genes

[0092] 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 2291 CDS sequences were predicted, with an average CDS sequence length of 912 bp.

[0093] 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 / analyze / rgi). Detailed comparison information is shown in Table 2.

[0094] Table 2 List of drug resistance gene information

[0095] Strain Gene Resistance genes Gene name Comparison consistency (%) MNH22871_01250 ARO:3003954 oeLh 99.77 MNH22871_02162 ARO:3002556 AAC(6')-Ii 99.45 MNH22871_01567 ARO:3003761 eatAv 98.4

[0096] 2. Analysis of Potentially Toxic Genes

[0097] 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 Table 3.

[0098] Table 3 List of potential toxic genes of MNH22871

[0099] Strain Gene VFDB gene Gene name Comparison consistency (%) MNH22871_00042 VFG048797 ugd 65.206 MNH22871_00044 VFG002182 cpsI 60.882 MNH22871_00088 VFG000964 hasC 75.427 MNH22871_00571 VFG002189 cpsB 73.585 MNH22871_00572 VFG002190 cpsA 78.277 MNH22871_00609 VFG002162 bsh 75.617 MNH22871_00733 VFG002158 lplA1 62.09 MNH22871_00782 VFG000077 P 81.122 MNH22871_00990 VFG006717 lap 69.641 MNH22871_01045 VFG037100 msrA / B(pilB) 66.406 MNH22871_01119 VFG002197 BpD 87.463 MNH22871_01172 VFG042979 srtC 68.86 MNH22871_01173 VFG042978 Bc 74.157 MNH22871_01175 VFG042976 ePB 63.581 MNH22871_01823 VFG000080 clpE 61.821 MNH22871_01868 VFG048830 gnd 72.281 MNH22871_01915 VFG000079 C 62.255 MNH22871_01998 VFG002165 eFA 62.987 MNH22871_02074 VFG000964 hasC 70.408 MNH22871_02338 VFG002162 bsh 77.778

[0100] 3. Analysis of potential primary metabolic gene clusters

[0101] The analysis of potential primary metabolic gene clusters in the genome was performed using gutSMASH5 (version: 1.0.0). Detailed alignment results are shown in Table 4.

[0102] Table 4 List of potential primary metabolic gene clusters of MNH22871

[0103]

[0104]

[0105] 4. Potential secondary metabolism gene clusters

[0106] The analysis of potential secondary metabolism gene clusters in the genome was performed using antiSMASH6 (version: 6.0.1). Detailed alignment results are shown in Table 5.

[0107] Table 5 List of potential secondary metabolism gene clusters of MNH22871

[0108] Gene cluster range type from arrive Most similar known gene cluster Similarity Region 7.1 cyclic-lactone-autoinducer 102773 118399 - - Region 14.1 T3PKS 1 37667 - - Region 39.1 RiPP-like 1 1539 - -

[0109] Example 5 Determination of short-chain fatty acids (SCFA) of strains

[0110] 1. Preparation of Bacteria

[0111] The strain MNH22871 was inoculated into a liquid culture medium and cultured anaerobically at 37°C for 48 hours. The bacteria were collected by centrifugation and stored at -86°C for later use.

[0112] 2. Preparation of standard products

[0113] Acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid standards were weighed and prepared with ethyl acetate into 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.

[0114] 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.

[0115] 3. Metabolite Extraction

[0116] The sample was thawed on ice, 80 mg of the sample was taken into a 2 mL glass centrifuge tube, and 900 μL of 0.5% phosphoric acid was added to resuspend, and the mixture was shaken for 2 min, and centrifuged at 14000 g for 10 min. 800 μL of the supernatant was taken, and an equal amount of ethyl acetate was added for extraction, and the mixture was shaken for 2 min, and centrifuged at 14000 g for 10 min. 600 μL of the upper organic phase was taken, and 4-methylpentanoic acid with a final concentration of 500 μM was added as an internal standard. The mixture was mixed and added to the injection bottle for GC-MS detection. The injection volume was 1 μL, the split ratio was 10:1, and split injection was performed.

[0117] 4. Sample testing and analysis

[0118] The samples were separated using an Agilent DB-WAX capillary column (30m×0.25mm ID×0.25μm) gas chromatography system. The temperature was programmed as follows: the initial temperature was 90°C, then the temperature was raised to 120°C at 10°C / min, then to 150°C at 5°C / min, and finally to 250°C at 25°C / min and maintained for 2min. The carrier gas was helium with a flow rate of 1.0mL / min.

[0119] 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.

[0120] 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.

[0121] Table 6. Short chain fatty acid (SCFA) production results of strain MNH22871

[0122]

[0123] It can be seen from the test results that strain MNH22871 produces low butyrate and valeric acid during its 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 SCFA measurement results of the Enterococcus faecalis strain of the present invention show 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 metabolites of butyrate, valeric acid, etc. to achieve anti-tumor effects.

[0124] Example 6 Experimental verification of the therapeutic effect of the strain on tumors

[0125] 1. Experimental methods

[0126] Through the H22 homologous subcutaneous transplant tumor therapeutic liver cancer model in BALB / C mice, the animals were housed in independent ventilation cages (IVC). The cages were made of polyphenylene sulfone resin (PPSU), with cage specifications of 372mm×205mm×172mm, and 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 communicating areas. The environmental conditions were controlled at a temperature of 20.0℃~26.0℃, relative humidity of 40%~70%, and lighting was alternating between light and dark for 12h / 12h day and night. The animals were free to eat qualified SPF-grade mouse and mouse maintenance feed (produced by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.). After the quarantine of male BALB / C mice, 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 At the same time, mice with abnormal tumor growth were removed and randomly divided into groups according to tumor volume. Drug administration began on the day of grouping. The control group and the MNH22871 group were given 200 μL of control substance (PBS-Cys(glycine)) or MNH22871 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, and tumor volume (mm 3) measurement, after reaching the end point of the experiment, all surviving mice were dissected and sampled, tumor weight and tumor volume were measured, and finally statistical analysis and comparison were performed on the change of tumor volume curve (Tumor volume), tumor volume at endpoint, tumor weight at endpoint and tumor inhibition rate at endpoint. Tumor inhibition rate: (average volume of control group - volume of experimental group) / average volume of control group × 100%, evaluation criteria: tumor inhibition rate <40% represents low / no response to antitumor efficacy; tumor inhibition rate ≥40% represents responsive antitumor efficacy, all data are expressed in mean ± SD form, and GraphPadPrism 8.0.2 software was used for plotting and statistical analysis. For pairwise comparison, t test (Student's t test) analysis method was used, and for two-factor investigation, two-way variance with Sidak multiple comparison analysis was used for analysis, and significant differences were indicated by *, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0127] Test strain: MNH22871 live bacterial suspension (8.0×10 9 CFU / mL≤viable count≤2.5×10 10 CFU / mL).

[0128] Tumor cells: H22 mouse hepatoma cells.

[0129] 2. Experimental results of strain MNH22871 on tumor treatment

[0130] In the H22 liver cancer therapeutic model, when the tumor is about 80-100mm 3 Interventional intervention, MNH22871 can show inhibition of tumor growth volume curve ( Figure 7 A), achieved statistically significant differences and reduced the tumor volume at the endpoint ( Figure 7 B) and tumor mass ( Figure 7 C), further analysis of the endpoint tumor inhibition rate showed that the endpoint average tumor inhibition rate of MNH22871 was close to 40% ( Figure 7D), and 42.86% (3 / 7) were tumor inhibition rates (tumor inhibition rate <40% represents low / no response to antitumor efficacy; tumor inhibition rate ≥40% represents responsive antitumor efficacy); indicating that MNH22871 has better responsive antitumor efficacy and has the efficacy of single-agent treatment of liver cancer. All the above data are expressed in the form of mean±SD, and GraphPad Prism 8.0.2 software was used for graphics and statistical analysis. For two-factor investigation, two-way variance with Sidak multiple comparison was used for analysis (A); for pairwise comparison (BD), t-test (Student's t test) analysis method was used, and the significant difference was indicated by *, ***p<0.001.

[0131] 3. Experimental comparison of different strains of Enterococcus faecium in liver cancer treatment models

[0132] The inventors also isolated another strain C (MNH09296) of the same species. This experiment compared the therapeutic effects of MNH22871 (B) and another strain C of the same species in the liver cancer treatment model. The experimental method used was the same as above; 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.

[0133] Comparison of the inhibition curve of tumor growth volume between MNH22871 and another strain C of the same species ( Fig. 8A ), achieved statistically significant differences, and could reduce the tumor volume of the endpoint ( Figure 8B ), further analysis of the endpoint tumor inhibition rate showed that the endpoint average tumor inhibition rate of MNH22871 was greater than that of another strain of the same species, C( Figure 8C ), among the same strains, the MNH22871 of the present invention has a better responsive anti-tumor efficacy than another strain.

[0134] 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 with 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. Enterococcus faecalis ( Enterococcus faecium ), characterized in that The Enterococcus faecalis strain is deposited in Guangdong Microbiological Culture Collection Center, and the culture collection number is GDMCC NO: 62663.

2. A bacterial agent, characterized in that: The bacterial agent includes the Enterococcus faecalis according to claim 1.

3. Use of the Enterococcus faecalis according to claim 1 or the bacterial agent according to claim 2 in the preparation of a drug for treating or preventing liver cancer.

4. A drug for treating liver cancer, characterized in that: It includes the Enterococcus faecalis according to claim 1 or the bacterial agent according to claim 2.

5. The drug according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

6. Use of the drug according to claim 4 or 5 in the preparation of a drug for treating or preventing liver cancer.

7. The use according to claim 3 or 6, characterized in that The drug achieves treatment or prevention of liver cancer by reducing tumor volume or inhibiting tumor growth.

8. The use according to claim 3 or 6, characterized in that: The tumor inhibition rate of the drug is ≥40%.

9. The use according to claim 3 or 6, characterized in that: The dosage form of the drug includes oral preparations.

10. The use according to claim 3 or 6, characterized in that: When the drug is in liquid dosage form, the effective dosage is 8.0×10 9 ~2.5×10 10 CFU / mL.

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