Shenjing pig combined lactobacillus and application thereof

Abstract

The invention discloses a strain of Shenjing pig combined lactobacillus and application thereof. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.31452. The strain is separated and screened from vaginal secretions of healthy women at the childbearing age and is sensitive to various common antibiotics, and fermented supernate of the strain shows relatively strong inhibition capability on escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, salmonella paratyphi B and gardnerella vaginalis; the compound has strong adhesion ability, growth inhibition ability and apoptosis promoting effect on human cervical cancer cells. When the strain is used for fermenting wall-broken ganoderma lucidum spore powder, the content of amino acid and bioactive substances such as peptides, arachidonic acids, steroids and polyketones can be greatly increased, the strain has an obvious inhibition effect on DSS-induced mouse colitis, and the inflammatory factor level is remarkably reduced. The strain not only has significant application value in biological agents, but also opens up a new way for combined application of lactic acid bacteria and ganoderma spores.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Ligilactobacillus saerimneri and an application thereof. Background Art

[0002] Bacterial vaginosis (BV) is a gynecological disease caused by mixed infection of vaginal Gardnerella and various anaerobic bacteria. A large number of anaerobic bacteria replace normal lactobacilli, resulting in an imbalance of the microecological balance in the vagina. Bacterial vaginosis has a high incidence rate among women worldwide. It is estimated that more than half of women in the world will experience bacterial vaginosis at least once in their lifetime. Vaginal Gardnerella can not only cause bacterial vaginosis, but also cause cervicitis, unclean abortion, postoperative infection, urinary tract infection and other diseases. Lactobacillus is the main probiotic in the vagina and can maintain the acidic environment and microecological balance of the vagina. When the number of lactobacilli decreases, the pH value in the vagina will rise, providing favorable conditions for the growth of pathogens such as Gardnerella and anaerobic bacteria. Therefore, the application of lactic acid bacteria in bacterial vaginitis (BV) has also attracted widespread attention.

[0003] Cracked spore powder of Ganoderma lucidum has anti-inflammatory, antioxidant and immunomodulatory effects. If the cracked spore powder of Ganoderma lucidum can be organically combined with lactic acid bacteria to increase the content of its biologically active ingredients and enhance its anti-inflammatory and immunomodulatory effects, it will provide a new idea for the further development and utilization of lactic acid bacteria and Ganoderma lucidum spores. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Mythical Porcine Lactobacillus and application thereof.

[0005] The technical solution of the present invention is:

[0006] A strain of Ligilactobacillus saerimneri MV0608 was deposited in the General Microbiology Center of China Culture Collection Administration on July 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.31452.

[0007] A biological preparation containing the above-mentioned Ligilactobacillus saerimneri MV0608 or its fermentation liquid.

[0008] The above-mentioned Mythical Pig Combined Lactobacillus saerimneri MV0608 or the above-mentioned biological preparation is used to inhibit Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B or / and Gardnerella vaginalis for non-disease treatment purposes.

[0009] The above-mentioned Shenhua pig combined with Lactobacillus MV0608 is used in the preparation of medicines for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Gardnerella vaginalis.

[0010] The use of the above-mentioned Mythical Pig combined with Lactobacillus saerimneri MV0608 or the above-mentioned biological preparation in the preparation of a drug for treating or assisting in the treatment of cervical cancer.

[0011] The above-mentioned application of the mythical pig combined with Lactobacillus saerimneri MV0608 in fermenting broken-wall Ganoderma lucidum spore powder.

[0012] Furthermore, the method for fermenting the broken-wall Ganoderma lucidum spore powder is: adding water to the broken-wall Ganoderma lucidum spore powder to prepare a culture medium, inoculating the culture medium with Ligilactobacillus saerimneri MV0608 after sterilization, culturing at 37° C. for 96 hours, and collecting the supernatant by centrifugation.

[0013] A preparation comprising the supernatant described above.

[0014] The use of the above-mentioned preparation in the preparation of medicine for preventing or treating animal colitis.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The Myth Pig combined lactobacillus provided by the present invention is isolated and screened from the vaginal secretions of healthy women of childbearing age, is sensitive to a variety of common antibiotics, and its fermentation supernatant shows strong inhibitory ability against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Gardnerella vaginalis, and has strong adhesion ability, growth inhibition ability and apoptosis-promoting effect on human cervical cancer cells. Fermenting the broken wall Ganoderma lucidum spore powder with this strain can greatly increase the content of bioactive substances such as amino acids and peptides, eicosanoids, steroids and polyketides, has a significant inhibitory effect on DSS-induced colitis in mice, and significantly reduces the level of inflammatory factors. This strain not only has significant application value in biological preparations, but also opens up a new way for the combined application of lactic acid bacteria and Ganoderma lucidum spores.

[0017] Collection information:

[0018] The mythical porcine Lactobacillus (Ligilactobacillus saerimneri) MV0608 was deposited in the General Microbiology Center of the China Culture Collection Administration on July 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.31452. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the phylogenetic relationship diagram of the mythical pig-lactobacillus MV0608 in the present invention.

[0020] Figure 2 The invention discloses the inhibitory effect of the fermentation supernatant of Shenhua pig combined with Lactobacillus MV0608 on the growth of Hela cells.

[0021] Figure 3 This is the effect of the fermentation supernatant of the Myth Pig combined with Lactobacillus MV0608 on the reactive oxygen species (ROS) level of Hela cells.

[0022] Figure 4 This is a volcano diagram of differential metabolites of broken-wall Ganoderma lucidum spore powder fermented by the Shenhua pig combined with Lactobacillus MV0608 strain of the present invention. DETAILED DESCRIPTION

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0024] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0025] Experimental Example 1 Isolation, Identification and Safety of Shenhua Pig Combined Lactobacillus

[0026] 1.1 Screening and isolation of strains

[0027] Human vaginal secretion samples were collected from the gynecology clinic of West China Second Hospital, Sichuan University. The cotton swab containing the secretion sample was stirred in MRS broth and placed in a constant temperature shaker at 37°C for 24 hours. 1 mL of the culture solution was aspirated, diluted 10 times, and inoculated on MRS agar medium. After culturing at 37°C for 24 hours, a single colony was picked and purified three times in a row. The purified strain was inoculated into 600 μL MRS broth medium, cultured at 37°C for 18 hours, and 400 μL of 50% (V / V) sterile glycerol was added and frozen in an ultra-low temperature refrigerator at -80°C for later use.

[0028] The purified strain was cultured in MRS broth, and the strain DNA was extracted using the Tiangen Bacteria Genomic DNA Extraction Kit. PCR amplification was performed using 16S rRNA universal primers 27F and 1492R. The primer sequences are as follows:

[0029] 27F: 5-AGAGTTTGATCMTGGCTCAG-3,

[0030] 1492R:5-GGTTACCTTGTTACGACTT-3

[0031] The PCR product was sequenced by Sangon Biotechnology (Shanghai) Co., Ltd., and a target gene sequence consisting of 955 base pairs (bp) was obtained (SEQ ID No. 1).

[0032] The gene sequence obtained by sequencing was input into the NCBI database for comparison, and the similarity between the strain and the strain Ligiactobacillus saerimneri strain ClaCZ24 in GenBank was 98.30%, and the strain was preliminarily identified as Ligilactobacillus saerimneri. The strain was named Ligilactobacillus saerimneri MV0608.

[0033] The kit method was used to perform Gram staining on the Mythical Porcine Lactobacillus MV0608, and the bacterial morphology after staining was observed and recorded under a microscope. The Mythical Porcine Lactobacillus MV0608 grew well on MRS agar medium. The colony morphology was milky white, with round protrusions, neat edges and smooth surface. Microscopic examination showed that the bacteria were rod-shaped and purple, which was consistent with the morphological characteristics of the Mythical Porcine Lactobacillus MV0608. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 24, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 31452. The phylogenetic relationship between strain MV0608 and other strains is as follows Figure 1 shown.

[0034] 1.2 Environmental tolerance test:

[0035] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The artificial gastric juice simulation liquid contains dilute hydrochloric acid, pepsin and sodium chloride, with a final pH of 2.5; the artificial intestinal juice simulation liquid contains potassium dihydrogen phosphate and trypsin, with a final pH of 6.8. The strain Shenhua pig combined with Lactobacillus MV0608 was revived and activated for 3 generations on MRS agar plates, and the initial concentration of the bacterial solution was adjusted to 1×106 CFU / mL. 1mL of the test strain Myth Pig combined with Lactobacillus MV0608 bacterial solution was inoculated into simulated gastric fluid and cultured at 37°C for 3h. After the culture was completed, 20μL of the bacterial solution was spread on the MRS agar plate medium, set up 3 replicates, and cultured at 37°C for 24h. After the culture, observe whether there was colony growth on the surface of the MRS plate. Similarly, 1mL of the test strain Myth Pig combined with Lactobacillus MV0608 bacterial solution was inoculated into simulated intestinal fluid, cultured at 37°C for 6h, then spread on the plate and cultured at 37°C for 24h to check the colony growth.

[0036] The results of the simulated gastric fluid tolerance test showed that the Myth Pig Combined Lactobacillus MV0608 could still grow normal colonies on the MRS agar plate after being treated in simulated gastric fluid for 3 hours; the results of the simulated intestinal fluid tolerance test showed that the Myth Pig Combined Lactobacillus MV0608 could still grow normal colonies on the MRS agar plate after being treated in intestinal fluid for 6 hours, which shows that the Myth Pig Combined Lactobacillus MV0608 has a strong tolerance to gastrointestinal fluid.

[0037] 1.3 Security

[0038] The Myth Pig combined with Lactobacillus MV0608 strain was activated and purified and then streaked on Columbia blood plate medium for 24 hours, and the hemolysis was observed. Staphylococcus aureus was used as a positive control, and the experiment was repeated three times. The sensitivity of the strain to common antibiotics was detected by the paper agar diffusion method. The Myth Pig combined with Lactobacillus MV0608 strain was activated and cultured, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL, use a sterile cotton swab to evenly smear the bacterial solution on the surface of the MRS culture medium plate, place it at room temperature for 10 minutes, then put in the drug-susceptibility paper, and culture it at 37℃ for 18 hours. Use a vernier caliper to measure the diameter of the inhibition zone around each drug-susceptibility paper. Repeat each antibiotic 3 times. The test results are based on the standards of the National Committee for Clinical Laboratory Standards (NCCLS) to judge the drug sensitivity of the strain. The results are expressed as sensitive (S), intermediate (I), and resistant (R).

[0039] The results of the hemolytic experiment showed that there was no hemolysis around the colonies of Shenhua pig combined with Lactobacillus MV0608; Shenhua pig combined with Lactobacillus MV0608 was sensitive (S) to the tested tetracyclines, ampicillin, clarithromycin, and chloramphenicol, and was intermediate (I) to ceftriaxone and clindamycin. The results are shown in Table 1.

[0040] Table 1 Sensitivity of Shenhua pig combined with Lactobacillus MV0608 strain to 6 antibiotics

[0041]

[0042] Experimental Example 2: Growth inhibition ability of Shenhua pig combined with Lactobacillus MV0608 on human cervical cancer cells

[0043] 2.1Adhesion ability of MV0608 to human cervical cancer cells Hela:

[0044] The frozen strains of Myth Pig and Lactobacillus MV0608 were revived and inoculated into MRS broth medium and cultured at 37°C for 24 hours. After the culture was completed, the suspension was centrifuged at -4°C and 5000 r / min for 10 minutes and washed several times with sterile PBS buffer. The concentration of the bacterial suspension was adjusted to 1×10 6 CFU / mL and reserve. Resuscitate human cervical cancer cells Hela, inoculate them into six-well cell culture dishes, add DMEM complete medium and culture at 37°C, 5% CO2, and change the culture medium every two days. When the cell adhesion state reaches 80%, use 0.25% trypsin-EDTA to digest and subculture. After the culture is completed, count them with a cell blood cell counting plate and adjust the cell concentration to 5×10 6 / mL. Add 1mL of cell suspension to the culture well and place it in an incubator for culture. When the cells in the culture plate grow to a monolayer, discard the DMEM culture medium and rinse each well 3 times with sterile PBS. Add 1mL of the prepared bacterial suspension to the cell well, shake the cell culture plate slightly, and aspirate a small amount of bacterial liquid in the well for plate counting. The result is used as the initial number of viable bacteria in the bacterial suspension. Incubate the cell plate at 37℃ for 2h, discard the culture medium and wash 3 times with sterile PBS buffer. Use 0.7mL 0.25% trypsin-EDTA to digest the cells for 10min. After the cells are completely detached, add 0.3mL DMEM culture medium to terminate the digestion, collect the culture medium after the adhesion experiment is completed, and count the plate. The result is used as the number of viable adherent bacteria. Adhesion rate (%) = number of lactic acid bacteria at the end of the period / initial number of lactic acid bacteria inoculated × 100%.

[0045] The experimental results showed that the adhesion rate of Shenhua pig combined with Lactobacillus MV0608 to human cervical cancer Hela cells was 40.82%±9.29%, and the colonization ability was strong (Table 2).

[0046] Table 2 Adhesion rate of Shenhua pig combined with Lactobacillus MV0608 to human cervical cancer Hela cells (%)

[0047]

[0048] 2.2 Effect of the fermentation supernatant of Shenhua pig and Lactobacillus MV0608 on the colony-forming ability of Hela cells:

[0049] The Hela colon cancer cells were revived and plated in a 6-well plate at a density of 2000 cells per well. The Mythical Porcine Lactobacillus MV0608 strain isolated and identified in Experimental Example 1 was revived and inoculated into MRS liquid culture medium and cultured at 37°C for 24 h to a bacterial concentration of 1×10 8 CFU / mL and collect the fermentation supernatant, dilute it with serum-free culture medium at a volume ratio of 1:100, culture Hela cells for about 3 weeks, and replace fresh culture medium every two days to ensure good cell growth. After visible clones are formed in the cell culture plate, discard the culture medium, wash with PBS and fix with methanol for 30min-2h. Add 1% crystal violet stain for 30min, wash off the residual crystal violet stain, and dry the culture plate. Use 1% MRS liquid culture medium supernatant as the negative control group, and set up 3 replicates in the experiment.

[0050] The results are shown in Table 3. The growth density of Hela cells in the group treated with 1% MV0608 fermentation supernatant was significantly lower than that in the control group, and the number of cell colonies decreased by 41.12%, indicating that the fermentation liquid had a significant growth inhibitory effect on human cervical cancer cells.

[0051] Table 3 Number of Hela cell clones formed after treatment with supernatant of fermentation of Shenhua pig and Lactobacillus MV0608 (individuals)

[0052]

[0053]

[0054] Experimental Example 3: Pro-apoptotic effect of Shenhua pig combined with Lactobacillus MV0608 on human cervical cancer cells

[0055] 3.1 Effect of MV0608 fermentation supernatant on reactive oxygen species (ROS) levels in Hela cells:

[0056] Cell collection: After incubating the MV0608 fermentation supernatant with adherent Hela cells for 48 hours, the fermentation supernatant was aspirated and repeatedly blown with serum-free culture medium. The bottom of the well plate (bottom of the bottle) changed from translucent (cell monolayer connected into a sheet) to transparent by naked eye observation, and almost all the cell layer was blown into the culture medium. All the cell suspension was collected into a 1.5ml centrifuge tube. Wash twice with serum-free culture medium, 1000r / min, centrifuge for 5min, aspirate the supernatant, and keep the cell pellet for determination.

[0057] Loading probe: Dilute DCFH-DA with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L. After collecting the cells, suspend them in the diluted DCFH-DA at a cell concentration of 1 million to 20 million / mL and incubate them in a 37°C cell culture incubator for 20 minutes. Invert and mix every 3-5 minutes to allow the probe and cells to fully contact. Wash the cells 3 times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells.

[0058] Detection: Use an inverted fluorescence microscope to detect the intensity of fluorescence at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Use the supernatant of MRS liquid culture medium as the negative control group, and the experiment is repeated 3 times.

[0059] The level of reactive oxygen species (ROS) reflects the state of cellular metabolic products. Under normal circumstances, the generation and removal of ROS are in a dynamic balance. If the level of ROS exceeds the normal range, it may cause oxidative damage or cell death. Figure 2 The fluorescence intensity of Hela cells treated with MV0608 fermentation supernatant was significantly higher than that of the control group, the number of fluorescently labeled cells was also greater, and the ROS expression level was significantly higher than that of the control group cells, indicating that MV0608 fermentation supernatant has a pro-apoptotic effect on colon cancer cells (Table 4).

[0060] Table 4 Effect of MV0608 fermentation supernatant on reactive oxygen species expression in human cervical cancer cells

[0061]

[0062] 3.2 Pro-apoptosis ability of MV0608 strain on human cervical cancer cell Hela

[0063] Cell culture: Human cervical cancer cells in logarithmic growth phase were digested and centrifuged and evenly plated in 96-well plates. Counting was performed to ensure 2000 cells per well. After culturing for 12 hours, 1% of GS0904 fermentation supernatant was added after cells were stably attached to the wall. Five replicates were set for each treatment, and protein was extracted after culturing for 48 hours.

[0064] Protein extraction: Wash the cultured cells twice with PBS, and remove all residual PBS on the cell surface as much as possible. Place the cells in an ice bath, add RIPA lysis buffer containing protease inhibitor cocktail (protease inhibitor diluted at a ratio of 1:100), scrape all the cells into a 1.5ml sterile centrifuge tube, and place it in an ice bath for lysis for 30 minutes. Centrifuge at 12000r / min and 4℃ for 10 minutes, collect the supernatant into a new 1.5ml sterile centrifuge tube, which is the extracted protein sample. The protein sample can be directly subjected to protein quantitative analysis or stored in a -80℃ refrigerator for standby use. For the protein sample after quantitative analysis, add protein buffer, place it in a metal bath at 98℃ for 10 minutes to denature the protein, and store it in a -20℃ refrigerator for standby use after instant centrifugation.

[0065] Protein quantification: Use the Bio-Tech Protein Quantification Kit for quantitative analysis. Dilute the protein sample 20 times and add it to the marked 96-well plate. Prepare the BCA working solution according to the ratio of BCA reagent A to B of 50:1, mix thoroughly, add 200μl of BCA working solution to each well, shake and mix for 30s, cover each sample well, and place in a constant temperature incubator at 37°C for 30min. Measure the absorbance at 562nm, read the protein concentration of each sample within the linear range of the standard curve according to the corrected absorbance of each protein sample, and calculate the amount of protein in the original sample according to the sample volume and dilution.

[0066] (1) Effect of MV0608 fermentation supernatant on cell adhesion factor β-catenin

[0067] β-catenin is a multifunctional protein involved in cell adhesion and signal transduction. β-catenin is also a core factor of the Wnt signaling pathway. When the Wnt signal is not activated, β-catenin is degraded in the cytoplasm by the GSK3β, APC and Axin complex and remains at a low level. When the Wnt signal is activated, β-catenin accumulates and transfers to the nucleus, binds to the transcription factor TCF / LEF, and activates genes related to cell proliferation, survival and stemness. In normal cells, the stability of β-catenin is strictly regulated. Abnormal expression and activation of β-catenin are closely related to the invasiveness, metastasis and poor prognosis of tumors. Studies have shown that the expression level of β-catenin in cervical cancer tissue is significantly higher than that in normal cervical tissue. This abnormal expression is usually accompanied by enhanced cell proliferation and survival signals, which promotes the growth of tumor cells. P-β-catenin is a modified form of β-catenin, and its phosphorylation usually occurs at the Ser675 site. Phosphorylated β-catenin is considered to be a response of cells to external stimuli, which can regulate its stability and function. In normal cells, the accumulation of P-β-catenin usually leads to the degradation of β-catenin, thereby inhibiting its transcriptional activity in the nucleus.

[0068] The experimental results (Table 5) show that after adding 1% MV0608 fermentation supernatant to the culture medium of human cervical cancer cells and culturing for 48 hours, the relative expression level of β-catenin in the treatment group was 0.369780731, which was significantly lower than 0.533989421 in the control group, while the result of phosphorylated β-catenin (P-β-catenin) showed a significant increase, from 1.003163 in the control group to 2.469466 in the treatment group. This result shows that MV0608 fermentation supernatant may affect cell proliferation and survival signals by inhibiting the expression of β-catenin, and has potential application value in anti-tumor.

[0069] Table 5 Effect of MV0608 strain fermentation supernatant on the content of β-catenin in human cervical cancer cells (absorbance)

[0070]

[0071]

[0072] (2) Effect of MV0608 fermentation supernatant on the protein content of apoptosis inhibitory gene

[0073] Myeloid cell leukemia 1 (MCL-1) and B cell lymphoma / leukemia-2 (BCL-2) are two important anti-apoptotic proteins that play a key role in the regulation of cell survival, proliferation and apoptosis. In the development and progression of cervical cancer, the abnormal expression of these two proteins is closely related to the occurrence and development of tumors.

[0074] MCL-1 is a member of the BCL-2 family and is mainly responsible for maintaining cell survival. Its expression is regulated by a variety of cell signaling pathways, especially those related to cell proliferation, stress response, and cell survival. Studies have shown that MCL-1 is overexpressed in a variety of cancers, including cervical cancer, which is closely related to the anti-apoptotic ability of tumor cells. The upregulation of MCL-1 can inhibit the function of pro-apoptotic proteins, such as BAX and BAK, thereby preventing cell apoptosis and promoting the survival and spread of tumor cells. The expression of MCL-1 is associated with the resistance of cervical cancer cells to chemotherapeutic drugs, and its overexpression often leads to a weakening of the therapeutic effect. Therefore, therapeutic strategies targeting MCL-1 are becoming a potential anti-tumor therapy, which may enhance the sensitivity of cervical cancer cells to chemotherapy by inhibiting its function.

[0075] BCL-2 is another key anti-apoptotic protein that also plays an important role in regulating cell apoptosis. In cervical cancer cells, the expression of BCL-2 is often elevated, a phenomenon that may be related to human papillomavirus (HPV) infection. Subtypes such as HPV16 and HPV18 can interfere with the apoptosis mechanism of host cells through the E6 and E7 proteins they encode, thereby upregulating the expression of BCL-2 and further enhancing the survival ability of tumor cells. This mechanism not only promotes the growth and metastasis of cervical cancer cells, but may also affect the patient's response to radiotherapy and chemotherapy, leading to a reduction in clinical treatment efficacy.

[0076] The experimental results showed that the expression of myeloid cell leukemia 1 (MCL-1) and B cell lymphoma / leukemia-2 (BCL-2) in the MV0608 treatment group was significantly decreased by about 81.1% and 93.9%, respectively. This significant inhibitory effect indicates that the fermentation supernatant of the MV0608 strain plays an important role in regulating the mechanism of cell apoptosis, which may provide a new strategy for the treatment of cervical cancer (Table 6).

[0077] Table 6 Effect of MV0608 fermentation supernatant on the protein content of human cervical cancer cell apoptosis inhibitory gene (absorbance)

[0078]

[0079] (3) Effect of MV0608 fermentation supernatant on the content of apoptotic factor protein

[0080] Poly ADP-ribose polymerase (PARP) is a protein that plays a key role in DNA damage repair. Its main function is to sense DNA damage and promote the repair process by catalyzing the synthesis of poly ADP-ribose. During apoptosis, PARP is cleaved to form an active form (Cleaved-PARP). Studies have shown that the expression level of Cleaved-PARP is also associated with the prognosis of cervical cancer. Higher Cleaved-PARP levels may indicate a higher apoptosis rate and better treatment effect.

[0081] Caspase-3 is a key protease in the apoptosis pathway. Its activation is usually mediated by upstream Caspase-8 and Caspase-9. Activated Cleaved-caspase3 will promote the cleavage of a series of key proteins in the cell, including cytoskeletal proteins and DNA repair-related proteins, leading to changes in cell morphology, membrane damage, and DNA degradation, ultimately leading to cell death. In cervical cancer cells, the expression level of Cleaved-caspase3 is closely related to the degree of cell apoptosis. Studies have shown that the expression of Cleaved-caspase3 will increase significantly after cervical cancer cells receive chemotherapy or radiotherapy.

[0082] Cleaved-caspase-1 is the active form of caspase-1 after it is activated. Cleaved-caspase-1 mainly mediates pyroptosis by activating inflammasomes. Pyroptosis is a form of cell death characterized by inflammatory cell death, usually accompanied by the release of inflammatory factors such as IL-1 and IL-18. Cleaved-caspase-1-mediated inflammatory response can promote inflammatory response in the tumor microenvironment, thereby inhibiting tumors (Table 7).

[0083] In summary, MV0608 treatment significantly increased the expression of C-Parp, Cleaved-caspase3 and Cleaved-caspase1, suggesting that it may inhibit the growth of cervical cancer cells by promoting apoptosis and enhancing inflammatory response. This result provides an experimental basis for the potential application of MV0608 in the treatment of cervical cancer.

[0084] Table 7 Effect of MV0608 fermentation supernatant on pro-apoptotic factor proteins in cervical cancer cells

[0085]

[0086] In summary, the Myth Pig combined with Lactobacillus MV0608 strain showed good adhesion ability to cervical cancer cells, and its fermentation supernatant had a significant effect in inhibiting the proliferation of cervical cancer cells. This treatment can effectively reduce the level of β-catenin associated with cell adhesion and proliferation, and reduce the expression of apoptosis inhibitory proteins MCL-1 and Bcl-2. In addition, the strain also promoted the expression of poly (ADP-ribose) polymerase Cleaved PARP, cleaved caspase 3 (Cleaved-caspase3) and cleaved caspase 1 (Cleaved-caspase1). These results confirmed the potential of Myth Pig combined with Lactobacillus MV0608 in inhibiting the growth of cervical cancer and promoting cell apoptosis from multiple perspectives, suggesting its broad application prospects in related treatments.

[0087] Experimental Example 4 Evaluation of the acid production and antibacterial ability of Shenhua pig combined with Lactobacillus MV0608

[0088] 4.1 Lactic acid production capacity of MV0608 strain

[0089] As an important antibacterial compound in the human and animal gastrointestinal tract, lactic acid plays an important role in reducing intestinal pH and changing the permeability of Gram-positive bacterial cell membranes, thereby helping to inhibit the growth of pathogens. Under the action of lactate dehydrogenase, lactic acid generates pyruvic acid, which ultimately reduces MTT to generate a purple substance with a characteristic absorption peak at 570nm. The lactic acid content produced was determined using a lactic acid content determination kit (Beijing Solebold Technology Co., Ltd.).

[0090] After the Shenhua pig combined with Lactobacillus MV0608 strains were activated and cultured, they were inoculated into MRS broth medium, cultured at 37°C for 24h, and centrifuged at 4°C and 12000g for 15min. 100μL of the fermentation supernatant was taken and the lactic acid content was determined according to the instructions of the lactic acid kit. The results showed that the lactic acid content of the fermentation liquid of Shenhua pig combined with Lactobacillus MV0608 was 42.05±15.07μmol / mL, indicating that Shenhua pig combined with Lactobacillus MV0608 had a good lactic acid production capacity (Table 8).

[0091] Table 8 Lactic acid content of the fermentation liquid of Shenhua pig combined with Lactobacillus MV0608 (μmol / mL)

[0092]

[0093] 4.2 Inhibitory effect of MV0608 on common intestinal pathogens and Gardnerella vaginalis

[0094] Escherichia coli (CMCB 44102), Pseudomonas aeruginosa (CMCB 10104), Staphylococcus aureus (CMCB 26003), Salmonella typhimurium ATCC14028), Salmonella paratyphi B (CMCB 50094), Gardnerella vaginalis ATCC 14018) were revived and purified three times, inoculated into liquid culture medium, and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL. Pipette the above pathogenic bacteria solution at 0.01% and add it to the solid agar medium cooled to 40-45°C after sterilization. Mix thoroughly and dispense into culture dishes at a rate of about 20 mL per dish. After the culture medium cools and solidifies, use a 6mm diameter puncher to punch holes on the plate. Adjust the concentration of the MV0608 strain solution to 1×10 8 CFU / mL, 50 μL of bacterial solution was added to the agar plate wells of the pathogenic bacteria, and the diameter of the inhibition zone around the punch point was measured with a vernier caliper after incubation at 37°C for 18 h. The experiment was repeated three times, with LGG as the control.

[0095] The results of the antibacterial activity evaluation of Shenhua pig combined with Lactobacillus MV0608 are shown in Table 10 below. Shenhua pig combined with Lactobacillus MV0608 strains showed inhibitory activity against the growth of pathogenic bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium and Salmonella paratyphi B (Table 9). Vaginal Gardnerella is considered to be one of the key pathogens of bacterial vaginosis (BV). MV0608 has a significant inhibitory ability against vaginal Gardnerella, suggesting its potential application prospects in regulating vaginal microbial balance and antibacterial treatment.

[0096] Table 9 Evaluation of antibacterial activity of Myth Pig combined with Lactobacillus MV0608 strain (diameter: mm)

[0097]

[0098] Example 5 Analysis of the wall-broken spore powder of Ganoderma lucidum fermented by MV0608 strain and its anti-inflammatory effect

[0099] 5.1 Fermentation increases the content of active substances

[0100] Fermentation method: 3 grams of broken wall Ganoderma lucidum spore powder and 20 mL of water were prepared into a culture medium. After sterilization and cooling, the culture medium was inoculated with Myth Pig and Lactobacillus MV0608 and cultured at 37°C for 96 hours. The fermentation broth was centrifuged at 4000 rpm and 4°C for 10 minutes, and the supernatant was filtered through a bacterial filter membrane and sent to the company for non-targeted metabolomics analysis by liquid chromatography-mass spectrometry (LC-MS). The control group was not inoculated with MV0608, and the culture and analysis methods were the same as those of the treatment group.

[0101] Analytical methods: Based on high-resolution mass spectrometry (HRMS) detection technology, the high-quality mzCloud database constructed by standards is combined with the mzVault and MassList databases to match and identify molecular characteristic peaks. The raw data is preprocessed using CD3.3 data processing software. First, simple screening is performed by parameters such as retention time and mass-to-charge ratio, and the peak area is quantified at the same time. Then, the high-resolution secondary spectrum databases mzCloud and mzVault and the MassList primary database are compared (searched) to identify metabolites. Using CD3.3 data processing software, the chromatographic peaks detected in the sample are integrated, where the peak area of ​​each characteristic peak represents the relative quantitative value of a metabolite. The total peak area is used to standardize the quantitative results, and finally the quantitative results of the metabolites are obtained. The results are expressed as the multiple increase in the content of the corresponding component in the sample compared with the control.

[0102] (1) Amino acids and peptide compounds:

[0103] Amino acids are the basic building blocks of proteins and play an important role in human health and normal functions, such as protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance. Metabolomics results showed that the content of various amino acids and peptide compounds increased significantly in the fermentation supernatant of broken wall Ganoderma lucidum spore powder fermented by Myth Pig and Lactobacillus MV0608 strain. Among them, the content of 8-aminooctanoic acid (8-Aminooctanoic Acid) was 31.28 times that of the control (FC=31.2836, P<0.0001). As a ω-amino fatty acid, 8-aminooctanoic acid is widely involved in metabolic processes in organisms. Studies have shown that it can also be used as a precursor for the synthesis of oleanolic acid derivatives, which have significant anti-HIV activity.

[0104] (5-L-Glutamyl)-L-Amino Acid and 4-Hydroxy-L-Glutamic Acid also showed significant increases, FC=11.5117 and FC=8.8784, respectively, with P values ​​below 0.005. (5-L-Glutamyl)-L-Amino Acid and 4-Hydroxy-L-Glutamic Acid are derivatives of glutamic acid, which have important biological functions in protein modification and neurotransmission, respectively. (5-L-Glutamyl)-L-Amino Acid participates in the glutamylation process, regulates protein stability and function, and has antioxidant, immunomodulatory and potential anti-cancer effects. 4-Hydroxy-L-Glutamic Acid affects glutamate receptors in the central nervous system, regulates neuronal excitability, and may play a role in neuroprotection and antioxidant processes. Gamma-Glu-Leu is an intermediate product of glutamyl transpeptidase reaction and has antioxidant effects. It functions through the glutathione metabolic pathway, helping cells cope with oxidative stress and maintaining the cell's antioxidant capacity. In addition, it may play a certain regulatory role in peptide signal transduction, participating in immune response and cell metabolic balance.

[0105] Palmitoyl ethanolamide (PEA) is an endogenous fatty acid amide with biological functions such as anti-inflammatory, analgesic and neuroprotective. PEA reduces cytokine release by regulating the activity of immune cells in allergic and inflammatory reactions, and inhibits the expression of inflammation-related genes by binding to the nuclear factor PPAR-α. It has shown a wide range of application potential in neurodegenerative diseases, pain management and metabolic syndrome. N-Oleoyl Glycine (NOG) is a fatty acid amino acid complex that is mainly involved in regulating energy metabolism and signal transduction. NOG may participate in cell metabolism and immune regulation by acting on G protein-coupled receptors (GPCRs), and has the potential for anti-inflammatory and neuromodulatory effects. Its application in improving insulin sensitivity and metabolic diseases has attracted much attention, and it may also be involved in regulating neuroprotective mechanisms.

[0106] Proline is an important non-essential amino acid that is widely involved in protein synthesis and structural stability. The presence of proline in proteins is essential for the formation of secondary structures, especially helices and folds, and plays a key role in the synthesis of extracellular matrices such as collagen. Proline is also involved in cell osmotic pressure regulation and cell metabolic pathways, and has antioxidant, cell growth and repair effects. L-aspartic acid is an important amino acid involved in energy metabolism. It can also participate in the synthesis of neurotransmitters, such as N-methyl-D-aspartate (NMDA), which plays an important role in signal transduction in the central nervous system. Ergothioneine is a potent antioxidant that helps protect cells from oxidative stress and may support mitochondrial function (Table 10).

[0107] Table 10 Effect of MV0608 strain fermentation of Ganoderma lucidum spore powder on the content of amino acids and peptide compounds

[0108]

[0109]

[0110] (2) Fatty acids:

[0111] The analysis results showed (Table 11) that after 96 hours of fermentation of broken wall Ganoderma lucidum spore powder by Mythology pig combined with Lactobacillus MV0608 strain, a variety of eicosanoid compounds were detected in the supernatant, and the FC values ​​were relatively high, especially 11-deoxy prostaglandin F2β and 15-deoxy-oxy-Deoxy prostaglandin D2, reaching 34.78 and 33.53 respectively, and the P values ​​were both lower than 0.001. 11-Deoxy prostaglandin F2β is an important prostaglandin derivative with strong anti-inflammatory and antioxidant properties. Studies have shown that it can reduce inflammatory responses by inhibiting the release of inflammatory cytokines, and also shows potential in regulating vasodilation and reproductive system health, and can inhibit the proliferation of tumor cells. 15-Deoxy-Δ12,14-prostaglandin D2 is also an important member of the eicosanoid class, and its functions are mainly concentrated in immunomodulation and anti-inflammatory effects. Studies have shown that this compound can inhibit the production of inflammatory mediators and play an important role in regulating allergic reactions and respiratory diseases. It also has a positive effect on neuroprotection and promoting nerve regeneration, showing its potential for application in the treatment of neurodegenerative diseases. Arachidonic acid (Arachidonic acid), as an important component of cell membrane phospholipids, has an FC value of 3.15 and a P value of 0.0204. It is a precursor for the synthesis of bioactive molecules such as prostaglandins, thrombin and leukotrienes, and therefore plays a key role in inflammation and immune responses. 15-Deoxy-oxy-synthesized prostaglandin J2-2-glyceride (15-Deoxy-thrombin and leukotrienes) is a precursor for bioactive molecules such as prostaglandins, thrombin and leukotrienes, and therefore plays a key role in inflammation and immune responses. It plays an anti-inflammatory and immunomodulatory role in neurodegenerative diseases by inhibiting the NF-κF pathway.

[0112] 10-Hydroxydecanoic acid is a medium-chain fatty acid extracted from royal jelly that has antibacterial, anti-inflammatory and immunomodulatory effects. 10-Hydroxydecanoic acid can also promote lipid metabolism by activating the AMPK pathway and may play a role in energy balance and weight management. It is currently widely studied for skin health, helping to improve skin barrier function and having antioxidant properties.

[0113] Acetylcarnitine is an important intermediate compound in the metabolism and transport of fatty acids, which can promote energy metabolism. Studies have shown that acetylcarnitine has a significant effect on neuroprotection, can improve nerve function, and alleviate the symptoms of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Oleamide is an endogenous hypnotic factor that can promote sleep. It affects the sleep cycle by regulating the release of neurotransmitters in the brain. In addition, oleamide exhibits anti-inflammatory properties, can reduce chronic inflammatory responses, and may play a role in the treatment of chronic pain and neuropathic pain.

[0114] Table 11 Effect of MV0608 strain fermentation of Ganoderma lucidum spore powder on the content of fatty acid compounds

[0115]

[0116]

[0117] (3) Steroids and polyketides

[0118] Trigoneoside Xb is a natural compound extracted from the plant Trigonella foenum-graecum (fenugreek), and studies have shown that it has antioxidant, anti-inflammatory and hypoglycemic effects. Trigoneoside Xb may have a potential protective effect on diabetes-related complications by inhibiting oxidative stress and inflammatory pathways. Cymarin is a steroid compound extracted from plants, mainly used to treat heart disease, with cardiotonic, diuretic and anti-inflammatory effects. Cymarin increases the concentration of calcium ions in myocardial cells by inhibiting the activity of sodium-potassium ATPase, thereby enhancing myocardial contractility and improving cardiac function. Ergosterol, as a precursor of vitamin D, helps regulate calcium and phosphorus metabolism and promote bone health after being converted into active forms in the body. In addition, ergosterol has antioxidant properties and can protect cells from oxidative stress. Guggulsterone is a bioactive ingredient extracted from Commiphora mukul, which has a significant lipid-lowering effect and can reduce cholesterol and triglyceride levels in the blood, thereby improving cardiovascular health.

[0119] Polyketides exhibit strong antioxidant capacity due to their rich phenolic hydroxyl structure, which can effectively scavenge free radicals, thereby slowing down the aging process. At the same time, they exert anti-inflammatory effects by inhibiting proinflammatory factors and regulating signaling pathways, reducing the risk of chronic inflammation and related diseases. These compounds not only help maintain the health of the body, but also show broad application prospects in the prevention and treatment of various diseases. 3-Methoxyflavone is a unique flavonoid compound that is mainly found in some medicinal plants and spices. It has significant antioxidant capacity and can effectively scavenge free radicals and protect cells from oxidative damage. In addition, 3-Methoxyflavone can also reduce inflammatory responses by inhibiting the release of proinflammatory factors and their signaling pathways, protecting against chronic inflammatory-related diseases such as arthritis and cardiovascular disease. Trans-Cinnamic acid also has antioxidant and anti-inflammatory properties, which can reduce cellular oxidative damage and inhibit tumor cell proliferation (Table 12).

[0120] Table 12 Effect of MV0608 strain fermentation of Ganoderma lucidum spore powder on the content of phenylpropanoids and polyketides

[0121]

[0122]

[0123] In summary, after the broken-wall Ganoderma lucidum spore powder was fermented with Shenhua pig and Lactobacillus MV0608 strain, the contents of various active ingredients in the fermentation supernatant increased significantly, and many ingredients were closely related to immunity and anti-inflammation.

[0124] 5.2 Effect of MV0608 Ganoderma lucidum spore powder fermentation supernatant on colitis in mice

[0125] (1) Animal experiment: The fermentation liquid was centrifuged at 4000 rpm and 4°C for 10 min, and the supernatant was filtered through a bacterial filter to obtain a sterile supernatant. The mouse direct enteritis model was induced with a 4% (w / v) dextran sulfate sodium salt (DSS) aqueous solution. The experimental mice were provided by Chengdu Dashuo Experimental Animal Co., Ltd. 27 4-week-old SPF male Kunming mice were randomly divided into 9 cages, 3 mice per cage, and after adaptive feeding, they were randomly divided into 3 groups, 3 cages per group, including a blank control group (CK), a DSS model group (DSS), and a Ganoderma lucidum spore fermentation supernatant treatment group (MV0608 strain). The experimental treatments are shown in Table 13.

[0126] Table 13DSS-induced colitis experiment in mice

[0127]

[0128] Serum inflammatory factor detection: At the end of the experiment, three mice were randomly killed in each group, and blood was collected from the eye sockets. The blood was centrifuged at 3000 r / min for 15 min to obtain serum. The inflammatory factors (IL-10, IL-6, IL-8 and TNF-α) in the serum were determined using ELISA kits according to the instructions of the kit.

[0129] (2) Analysis of anti-inflammatory effects

[0130] At the end of the experiment, three pro-inflammatory factors, TNF-α, IL-6 and IL-8, and the anti-inflammatory factor IL-10 were measured. The results showed that the levels of TNF-α, IL-6 and IL-8 in the serum of mice in the DSS model group were significantly higher than those in the blank control group, and the levels of IL-6, IL-8 and TNF-α in the serum of mice in the Ganoderma lucidum spore fermentation supernatant group were significantly lower than those in the DSS model group, and closer to the blank control group, and the anti-inflammatory factor IL-10 was also significantly higher than that in the DSS model group. This shows that the supernatant of broken Ganoderma lucidum spore fermentation of Shenhua pig combined with Lactobacillus MV0608 has a significant alleviating effect on DSS-induced colon inflammation in mice (Table 14).

[0131] Table 14 Changes in the concentration of inflammatory factors in the serum of mice in each group (Unit: pg / mL)

[0132]

[0133] Note: Compared with the blank control group, **p<0.01; compared with the DSS model group, ##p<0.01.

Claims

1. A strain of Ligilactobacillus saerimneri MV0608, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31452.

2. A biological preparation containing the Ligilactobacillus saerimneri MV0608 or its fermentation liquid as described in claim 1.

3. Use of the Ligilactobacillus saerimneri MV0608 described in claim 1 or the biological preparation described in claim 2 for inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B or / and Gardnerella vaginalis for non-disease treatment purposes.

4. Use of the Shenhua pig combined with Lactobacillus MV0608 described in claim 1 in the preparation of drugs for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Gardnerella vaginalis.

5. Use of the Ligilactobacillus saerimneri MV0608 described in claim 1 or the biological preparation described in claim 2 in the preparation of a drug for treating or assisting in the treatment of cervical cancer.

6. Use of the Shenhua pig combined with Lactobacillus saerimneri MV0608 described in claim 1 in fermenting broken-wall Ganoderma lucidum spore powder.

7. The use according to claim 6, characterized in that: The method for fermenting the broken wall ganoderma spore powder is as follows: adding water to the broken wall ganoderma spore powder to prepare a culture medium, inoculating the culture medium with Ligilactobacillus saerimne ri MV0608 after sterilization, culturing at 37°C for 96 hours, and collecting the supernatant by centrifugation.

8. A preparation, characterized in that Containing the supernatant according to claim 7.

9. Use of the preparation according to claim 8 in the preparation of a medicament for preventing or treating colitis in animals.

Images