Lactobacillus mucilaginosus and application thereof in fermentation of sporoderm-broken ganoderma lucidum spore powder

Abstract

The invention discloses lactobacillus mucilaginosus and application thereof in fermentation of sporoderm-broken ganoderma lucidum spore powder. The preservation number of the lactobacillus mucilaginosus is CGMCC (China General Microbiological Culture Collection Center) No.31453. The strain is separated and screened from vaginal secretions of healthy women at the childbearing age, is not hemolyzed, is sensitive to various common antibiotics and has strong lactic acid production capacity, and fermented supernate of the strain has strong growth inhibition capacity on escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, shigella dysenteriae and gardnerella vaginalis and can be used for producing lactic acid. 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 bioactive substances such as amino acids and derivatives thereof, fatty acids and steroids in fermentation liquor can be greatly increased, the strain has an obvious anti-inflammatory effect on DSS-induced mouse colitis, and the inflammatory factor level is remarkably reduced. The strain not only has significant application value in functional food and biological agents, but also opens up a new way for further development of ganoderma spores.

Description

Technical Field

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

[0002] Limosilactobacillus fermentum is widely present in a variety of fermented foods and the intestinal microbiota of humans and animals. It is also one of the dominant probiotics in the vagina of healthy women. It can regulate the structure of microbial flora by producing acid and inhibiting bacteria, so as to improve the intestinal and vaginal environment and promote health.

[0003] The main active ingredients of Ganoderma lucidum spore powder include triterpenes, β-glucan, protein, amino acid, trace elements and polyphenol compounds. If lactic acid bacteria can be organically combined with broken wall Ganoderma lucidum spore powder, its bioavailability can be further improved. This will provide a new perspective for the further development of Ganoderma lucidum spores. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a fermented mucus lactobacillus strain and its application in the fermentation of broken wall ganoderma spore powder.

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

[0006] A strain of Limosilactobacillus fermentum YD2-55 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.31453.

[0007] A biological preparation containing the above-mentioned fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 or its fermentation liquid.

[0008] The use of the above-mentioned fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 or the above-mentioned biological preparation in inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and / or Gardnerella vaginalis for non-disease treatment purposes.

[0009] The fermented mucus lactobacillus YD2-55 is used in the preparation of medicines for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and / or Gardnerella vaginalis.

[0010] Use of the above-mentioned fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 or the above-mentioned biological preparation in the preparation of a drug for treating or assisting in the treatment of cervical cancer.

[0011] The application of the fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 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 Limosilactobacillus fermentum YD2-55 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 invention discloses a fermented mucus lactobacillus and its application, and the strain deposit number is CGMCC No.31453. The strain is isolated and screened from the vaginal secretions of healthy women of childbearing age, is non-hemolytic, sensitive to a variety of common antibiotics, has a strong lactic acid production ability, and its fermentation supernatant has a strong growth inhibition ability on Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and Gardnerella vaginalis, and has a strong adhesion ability, growth inhibition ability and apoptosis-promoting effect on human cervical cancer cells. Fermenting the broken wall ganoderma lucidum spore powder with the strain can greatly increase the content of bioactive substances such as amino acids and their derivatives, fatty acids and steroids in the fermentation liquid, has a significant anti-inflammatory effect on DSS-induced colitis in mice, and significantly reduces the level of inflammatory factors. The strain not only has significant application value in functional foods and biological preparations, but also opens up a new way for the further development of ganoderma lucidum spores.

[0017] Collection information:

[0018] Limosilactobacillus fermentum YD2-55 was deposited at 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. 31453. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a phylogenetic relationship diagram of the fermented mucus Lactobacillus YD2-55 of the present invention.

[0020] Figure 2 The invention discloses a Hela cell growth inhibitory effect of the fermentation supernatant of the fermented Lactobacillus mucilaginosus YD2-55.

[0021] Figure 3 This is the effect of the fermentation supernatant of Lactobacillus mucilaginosus YD2-55 on the level of reactive oxygen species (ROS) in Hela cells.

[0022] Figure 4 This is a volcano diagram of differential metabolites of broken-wall Ganoderma lucidum spore powder fermented by the fermented Lactobacillus mucilaginosus YD2-55 strain of the present invention. DETAILED DESCRIPTION

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

[0024] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC53103.

[0025] Experimental Example 1 Isolation, Identification and Safety of Fermented Lactobacillus mucilaginosus

[0026] 1.1 Screening and isolation of strains

[0027] Vaginal secretion samples from healthy women were collected from the gynecology clinic of West China Second Hospital, Sichuan University. The cotton swabs containing the secretion samples were 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 in 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 976 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 Limosilactobacillus fermentum strain J2-9 in GenBank was 98.13%, and the strain was preliminarily identified as Limosilactobacillus fermentum. The strain was named Limosilactobacillus fermentum YD2-55.

[0033] The kit method was used to perform Gram staining on fermented mucus Lactobacillus YD2-55, and the bacterial morphology after staining was observed and recorded under a microscope. Fermented mucus Lactobacillus YD2-55 grows well on MRS agar medium. The colony morphology is round, the edges are neat, and the color is mostly milky white or light yellow. As the culture time increases, the color will deepen slightly. The colony texture is smooth, transparent or translucent, and sometimes it will appear sticky or gelatinous. YD2-55 Gram staining is positive and rod-shaped, which is consistent with the morphological characteristics of fermented mucus Lactobacillus. 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. 31453. The phylogenetic relationship between strain YD2-55 and other strains is as follows Figure 1 shown.

[0034] 1.2 Security

[0035] The fermented Lactobacillus mucosa YD2-55 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 fermented Lactobacillus mucosa YD2-55 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).

[0036] The results of the hemolytic experiment showed that there was no hemolysis around the colonies of fermented Lactobacillus mucosa YD2-55; fermented Lactobacillus mucosa YD2-55 was sensitive (S) to tetracycline, ampicillin, clarithromycin, chloramphenicol, ceftriaxone and clindamycin tested.

[0037] Table 1 Sensitivity of Lactobacillus fermentans YD2-55 strain to 6 antibiotics

[0038]

[0039] 1.3 Environmental tolerance test:

[0040] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The artificial gastric juice simulation liquid consists of dilute hydrochloric acid, pepsin and sodium chloride, with a final pH of 2.5; the artificial intestinal juice simulation liquid consists of potassium dihydrogen phosphate and trypsin, with a final pH of 6.8. The test strain Lactobacillus mucosa YD2-55 was revived and activated for 3 generations on MRS agar plates, and the initial concentration of the bacterial solution was adjusted to 1×10 6 CFU / mL. 1mL of the test strain fermented Lactobacillus mucoides YD2-55 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 fermented Lactobacillus mucoides YD2-55 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.

[0041] The results of the simulated gastric fluid tolerance test showed that fermented mucus Lactobacillus YD2-55 could still grow normal colonies on MRS agar plates after being treated in simulated gastric fluid for 3 hours; the results of the simulated intestinal fluid tolerance test showed that fermented mucus Lactobacillus YD2-55 could still grow normal colonies on MRS agar plates after being treated in intestinal fluid for 6 hours, indicating that fermented mucus Lactobacillus YD2-55 has strong acid and salt resistance.

[0042] Experimental Example 2 Acid production and antibacterial ability of fermented Lactobacillus mucilaginosus YD2-55

[0043] 2.1 Lactic acid production capacity of YD2-55 strain

[0044] 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 assay kit (Beijing Solebold Technology Co., Ltd.).

[0045] After the fermented Lactobacillus mucilaginosus YD2-55 strain was activated and cultured, it was 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 fermented Lactobacillus mucilaginosus YD2-55 fermentation liquid was 42.05±15.07μmol / mL, indicating that the fermented Lactobacillus mucilaginosus YD2-55 had a strong lactic acid production capacity (Table 2).

[0046] Table 2 Lactic acid content of fermented broth of Lactobacillus mucilaginosus YD2-55 (μmol / mL)

[0047]

[0048] 2.2 Inhibitory effect of YD2-55 strain on common vaginal harmful bacteria

[0049] Escherichia coli (CMCB 44102), Pseudomonas aeruginosa (CMCB 10104), Staphylococcus aureus (CMCB 26003), Salmonella typhimurium ATCC14028), Shigella dysenteriae (CMCB 51105), and 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 YD2-55 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.

[0050] The results of the antibacterial activity evaluation of fermented mucus lactobacillus YD2-55 are shown in Table 3 below. The fermented mucus lactobacillus YD2-55 strain has a strong inhibitory activity against the growth of pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, and Shigella dysenteriae (Table 3). Vaginal Gardnerella is one of the key pathogens of bacterial vaginitis, and its proliferation is closely related to the imbalance of vaginal microbiota. The experimental results show that YD2-55 has a strong inhibitory ability against vaginal Gardnerella.

[0051] Table 3 Evaluation of antibacterial activity of fermented Lactobacillus mucilaginosus YD2-55 strain (diameter: mm)

[0052]

[0053] Experimental Example 3 Growth inhibition and apoptosis-promoting ability of fermented supernatant of Lactobacillus mucilaginosus YD2-55 on human cervical cancer cells

[0054] 3.1 Adhesion ability of YD2-55 to human cervical cancer cells Hela:

[0055] The frozen strain of fermented Lactobacillus mucilaginosus YD2-55 was revived and inoculated into MRS broth medium and cultured at 37°C for 24 hours. After the culture was completed, it was centrifuged at -4°C and 5000r / 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%.

[0056] The experimental results showed that the adhesion rate of fermented Lactobacillus mucosa YD2-55 to human cervical cancer Hela cells was 28.96%±7.70%, which showed a strong colonization ability (Table 4).

[0057] Table 4 Adhesion rate of fermented mucus lactobacillus YD2-55 to human cervical cancer Hela cells (%)

[0058]

[0059] 3.2 Effect of fermentation supernatant of Lactobacillus mucilaginosus YD2-55 on colony formation ability of Hela cells:

[0060] The Hela cells of cervical cancer cells were revived and plated in a 6-well plate at a density of 2000 cells per well. The fermentative mucoid Lactobacillus strain YD2-55 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 8CFU / mL and collect the fermentation supernatant. Dilute the fermentation supernatant of Lactobacillus mucosa YD2-55 to a concentration of 1% with serum-free culture medium at a volume ratio of 1:100. Culture Hela cells with 1% YD2-55 fermentation broth 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 Control group, and the experiment is set up with 3 replicates.

[0061] The results are shown in Table 5 ( Figure 2 ), the growth density of Hela cells in the YD2-55 fermentation broth-treated group was significantly lower than that in the control group, and the number of cell colonies decreased by 56.17%, indicating that its fermentation broth has a significant growth inhibitory effect on human cervical cancer cells.

[0062] Table 5 Number of Hela cell clones formed after fermentation supernatant of Lactobacillus mucilaginosus YD2-55 was treated (individuals)

[0063]

[0064] 3.3 Effect of YD2-55 fermentation supernatant on the level of reactive oxygen species (ROS) in Hela cells:

[0065] Cell collection: After incubating the YD2-55 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. Collect all the cell suspension 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.

[0066] 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.

[0067] 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.

[0068] 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 3 The fluorescence intensity of Hela cells treated with YD2-55 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. This indicates that YD2-55 fermentation supernatant has a pro-apoptotic effect on cervical cancer cells (Table 6).

[0069] Table 6 Effect of fermentation supernatant of YD2-55 strain on reactive oxygen species expression in human cervical cancer cells

[0070]

[0071] 3.4 Pro-apoptotic ability of YD2-55 strain on human cervical cancer cell Hela

[0072] 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% YD2-55 fermentation supernatant was added after cells were stably attached to the wall. Five replicates were set for each treatment. Protein was extracted after culturing for 48 hours.

[0073] 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.

[0074] Protein quantification: Use the Bio-Tech Protein Quantification Kit for quantitative analysis. Dilute the protein sample 20 times and add it to the labeled 96-well plate. Prepare the BCA working solution with a ratio of 50:1 between BCA reagent A and B, 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 based on the corrected absorbance of each protein sample, and calculate the amount of protein in the original sample based on the sample volume and dilution.

[0075] (1) Effect of YD2-55 fermentation supernatant on cell adhesion factor β-catenin

[0076] β-catenin is a multifunctional protein that plays a key role in cell adhesion and signal transduction. In the Wnt signaling pathway, β-catenin, as a transcription factor, participates in regulating the expression of multiple genes and affects cell proliferation and differentiation. In cervical cancer, abnormal accumulation of β-catenin is often associated with tumor progression and malignant phenotypes. Under normal physiological conditions, β-catenin is marked for degradation through GSK3β-mediated phosphorylation, but in cervical cancer cells, inactivation of GSK3β leads to the accumulation of β-catenin. Accumulated β-catenin transfers to the nucleus, binds to the transcription factor TCF / LEF, and activates downstream genes related to cell proliferation and migration, thereby promoting tumor development. Studies have found that overexpression of β-catenin is closely related to the stage, metastasis and prognosis of cervical cancer. Targeted therapeutic strategies for β-catenin have become a research hotspot. By inhibiting its activity or preventing it from entering the nucleus, the growth and metastasis of cervical cancer can be effectively inhibited.

[0077] The experimental results are shown in Table 7. The 1% YD2-55 fermentation supernatant treatment for 48 hours reduced the β-catenin content, and the P-β-catenin factor content increased by 23.38% relative to the control. The increase in P-β-catenin may indicate that β-catenin has undergone phosphorylation, while the reduced total β-catenin content reflects the enhancement of its degradation process.

[0078] Table 7 Effect of YD2-55 strain fermentation supernatant on the β-catenin content of human cervical cancer cells (relative band intensity)

[0079]

[0080] (2) Effect of YD2-55 fermentation supernatant on the protein content of apoptosis inhibitory gene

[0081] Myeloid cell leukemia 1 (MCL-1) is a member of the BCL-2 family, and its main function is to inhibit apoptosis. Studies have shown that high expression of MCL-1 is closely related to the survival, proliferation and drug resistance of cervical cancer cells. MCL-1 prevents cells from undergoing programmed cell death by binding to pro-apoptotic proteins. This mechanism enables tumor cells to survive in adverse environments, especially during chemotherapy and radiotherapy. Overexpression of MCL-1 is usually associated with the malignancy of the tumor. Clinical studies have shown that high levels of MCL-1 are associated with poor prognosis in patients. Therapeutic strategies targeting MCL-1 have gradually become a hot topic of research. Reducing the expression of MCL-1 by small molecule inhibitors or other biotechnological means can effectively enhance the sensitivity of cervical cancer cells to chemotherapeutic drugs and promote apoptosis of tumor cells. The experimental results showed that 1% YD2-55 fermentation supernatant treatment can reduce the total amount of MCL-1 protein in cervical cancer cells by 79.39% (Table 8), indicating that the fermentation broth has the activity of promoting cancer cell apoptosis.

[0082] B cell lymphoma / leukemia-2 (BCL-2) is also a key anti-apoptotic factor. BCL-2 affects the apoptotic process of cells by regulating the permeability of the mitochondrial outer membrane, inhibiting pro-apoptotic signals. In cervical cancer, high expression of BCL-2 may enable tumor cells to resist apoptosis induced by treatment, thereby promoting tumor growth and metastasis. Studies have shown that high levels of BCL-2 are directly related to chemotherapy resistance in cervical cancer cells, which affects the therapeutic effect. Our experiments showed that adding 1% YD2-55 fermentation supernatant to the cell culture medium can reduce the total amount of BCL-2 protein in cervical cancer cells by 58.03% (Table 8), further illustrating that the fermentation supernatant has the activity of promoting cancer cell apoptosis.

[0083] Table 8 Effect of YD2-55 fermentation supernatant on the protein content of human cervical cancer cell apoptosis inhibitory gene (relative band intensity)

[0084]

[0085] (3) Effect of YD2-55 fermentation supernatant on the content of apoptotic factor protein

[0086] Poly ADP-ribose polymerase PARP is an important intranuclear enzyme involved in DNA repair and cell apoptosis. C-PARP is the cleavage form of PARP and is usually specifically cleaved during apoptosis. High levels of C-PARP are usually associated with apoptosis of tumor cells, and studies have shown that the expression of C-PARP can be used as a biomarker to assess treatment response. For example, in cervical cancer patients treated with paclitaxel and cisplatin, the increase in C-PARP is positively correlated with the effectiveness of treatment.

[0087] Caspase-3 is an important executor of apoptosis. It belongs to the caspase family and is mainly responsible for cleaving a variety of intracellular substrates. It is usually activated when cells are exposed to pro-apoptotic signals, thereby triggering a series of cell death signals. In tumor biology, the expression of Cleaved-caspase3 is closely related to the progression, prognosis and treatment response of cancer. In the study of cervical cancer, Cleaved-caspase3, as an important biomarker, is widely used to evaluate the degree of apoptosis and the therapeutic effect.

[0088] Cleaved-caspase 1 is a key regulator in apoptosis and inflammatory responses. It is mainly responsible for activating inflammasomes and participating in the process of apoptosis. Inflammasomes are polymers composed of a series of proteins, mainly including NLRP3, ASC and pro-caspase-1. When cells are infected by pathogens or other stress stimuli, these components aggregate and activate pro-caspase-1, leading to its cleavage and activation. Activated Cleaved-caspase1 is then able to cleave precursor cytokines (such as IL-1 and IL-18) to produce biologically active inflammatory factors. In cervical cancer cells, the expression of Cleaved-caspase1 is closely related to apoptosis. Studies have shown that high expression of Cleaved-caspase1 in cervical cancer cells is associated with increased cell death rate.

[0089] The results showed that the levels of C-Parp, Cleaved-caspase-1 and Cleaved-caspase3 proteins were significant after 24 h of treatment with 1% fermentation supernatant of YD2-55 strain, indicating that YD2-55 has good activity in promoting cancer cell apoptosis (Table 9).

[0090] Table 9 Effect of YD2-55 fermentation supernatant on cervical cancer cell apoptosis factor protein (relative band intensity)

[0091]

[0092] In summary, the fermented Lactobacillus mucosa YD2-55 strain showed good adhesion to cervical cancer cells, and its fermentation supernatant significantly inhibited the growth of cervical cancer cells. Studies have shown that the supernatant reduced the content of β-catenin associated with cell adhesion and proliferation, and at the same time decreased the protein levels of apoptosis inhibitory genes MCL-1 and Bcl-2. In addition, the expression levels of pro-apoptotic factors such as poly (ADP-ribose) polymerase (Cleaved PARP), cleaved caspase 3 (Cleaved-caspase3) and cleaved caspase 1 (Cleaved-caspase1) were significantly increased. These results confirmed the inhibitory effect of the fermented Lactobacillus mucosa YD2-55 strain on the growth of cervical cancer and the promotion of apoptosis from multiple aspects, showing its wide application potential.

[0093] Experimental Example 4 Application of fermented mucus lactobacillus strain YD2-55 in fermentation of broken wall Ganoderma lucidum spore powder

[0094] 4.1 Fermentation and analytical methods

[0095] 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 fermented mucus lactobacillus YD2-55 was inoculated and cultured at 37 degrees 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 YD2-55, and the culture and analysis methods were the same as those of the treatment group.

[0096] 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.

[0097] 4.2 Analysis results

[0098] (1) Amino acids and peptide compounds:

[0099] Amino acids are the basic units that make up proteins and are essential for human health and normal function, involving protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance. The results showed that the content of various amino acids and peptide compounds increased significantly in the fermentation supernatant of the fermented Lactobacillus mucilaginosus YD2-55 strain fermented broken wall Ganoderma lucidum spore powder (Table 10). For example, the content of 8-aminooctanoic acid was 6.6027 times that of the control. 8-aminooctanoic acid is an amino acid derivative that is mainly involved in fatty acid metabolism and the regulation of the nervous system. It can serve as a precursor for fatty acid synthesis, promote energy generation and storage, and thus affect overall energy metabolism. Studies have shown that 8-aminooctanoic acid can play a positive role in weight management and metabolic health. In the nervous system, 8-aminooctanoic acid, as a precursor for the synthesis of neurotransmitters such as norepinephrine and dopamine, can regulate neural signal transmission and thus affect cognitive functions such as emotions, learning and memory. Additionally, 8-aminooctanoic acid supports nerve cell function by enhancing the energy supply to neurons and improving nerve cell survival, especially in response to oxidative stress and other cellular damage.

[0100] 10-Hydroxydecanoic acid has potential clinical application value in improving metabolic health, promoting antimicrobial defense and regulating immune response, especially in the treatment of obesity and related metabolic diseases. 10-Hydroxydecanoic acid can inhibit the proliferation and differentiation of adipocytes by activating specific signaling pathways, thus playing a key role in regulating body weight and preventing obesity. In addition, 10-Hydroxydecanoic acid can also regulate the immune system and reduce chronic inflammation associated with obesity and metabolic syndrome by inhibiting the release of pro-inflammatory cytokines. The content of 10-Hydroxydecanoic acid after fermentation is 4.5733 times that of the control, indicating that broken wall Ganoderma lucidum spores have good health potential.

[0101] N-Acetyl-D-alloisoleucine is an important derivative of amino acid metabolism, which can regulate energy metabolism and protect the nervous system. In terms of energy metabolism, N-acetyl-D-isoleucine can help maintain stable blood sugar levels by affecting fat and carbohydrate metabolism pathways. The specific mechanism may be related to its enhancement of insulin signaling, which promotes the effective use of insulin in cells, thereby increasing glucose uptake, reducing fat accumulation, and improving the overall metabolic state. In terms of neuroprotection, N-acetyl-D-isoleucine reduces oxidative stress damage to nerve cells through its antioxidant properties. This mechanism can be achieved by enhancing the expression of endogenous antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase; in addition, N-acetyl-D-isoleucine can also promote the release of dopamine and norepinephrine, thereby promoting the health and function of neurons.

[0102] N-Oleoyl Glycine is a fatty acid derivative, which is composed of oleic acid and glycine. In recent years, N-Oleoyl Glycine has attracted widespread attention due to its potential in metabolic regulation, neuroprotection and anti-inflammatory effects. Studies have shown that N-Oleoyl Glycine has the function of improving insulin sensitivity and promoting fat metabolism. In addition, N-Oleoyl Glycine can enhance satiety by activating specific neurotransmitters (such as peptide YY and glucagon-like peptide-1), thereby playing an important role in appetite regulation. This makes N-Oleoyl Glycine of great significance in the study of obesity and metabolic syndrome, and may become a potential candidate for the development of new anti-obesity drugs. In addition, N-Oleoyl Glycine can inhibit the production of inflammatory mediators and reduce the inflammatory response of cells and tissues. The role of N-Oleoyl Glycine in neuroprotection has also attracted attention. Studies have shown that this compound may play a neuroprotective role by reducing oxidative stress, improving cellular energy metabolism and promoting neuronal survival.

[0103] 12-Hydroxydodecanoic acid has antibacterial and anti-inflammatory properties. 12-Hydroxydodecanoic acid can embed into bacterial cell membranes, change the fluidity and integrity of the membrane, lead to damage of cell function, and inhibit the growth of pathogens. Studies have shown that this compound can also inhibit the production of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6), thereby reducing the occurrence of chronic inflammation-related diseases, especially in inflammatory states associated with metabolic syndrome. Cyclohexaneacetic acid also has potential anti-inflammatory and analgesic properties, which may relieve pain by inhibiting the release of inflammatory mediators. In addition, it may have an effect on the central nervous system, regulating pain perception and response. In pharmacological studies, cyclohexaneacetic acid has shown good therapeutic potential, especially in chronic pain management.

[0104] Table 10 Effect of fermentation of Ganoderma lucidum spore powder with strain YD2-55 on the content of amino acids and their derivative compounds

[0105]

[0106] (2) Fatty acids:

[0107] The fatty acids in Ganoderma lucidum mainly include unsaturated fatty acids, such as linoleic acid, oleic acid and palmitic acid. These fatty acids are not only important components of cell membranes, but also play a key role in signal transduction, energy metabolism and cell function regulation. After fermentation, lactic acid bacteria can break down fat and release short-chain fatty acids (SCFAs), which have multiple biological functions, including promoting intestinal health, regulating immune response and improving metabolic function.

[0108] The results of the analysis showed that after the broken wall Ganoderma lucidum spore powder was fermented by Lactobacillus mucilaginosus YD2-55 strain for 96 hours, a variety of bioactive fatty acid compounds were detected in the supernatant. 2-Hydroxycaproic acid was 32.68 times that of the control, which can be used as an intermediate product of energy metabolism to promote the β-oxidation process, thereby enhancing the energy supply of cells. In addition, studies have shown that 2-hydroxycaproic acid has antioxidant properties, can reduce cell damage caused by oxidative stress, improve the overall health of cells, and may play a protective role in certain metabolic diseases. The content of acetylcarnitine was 3.8701 times that of the control (Table 11). Acetylcarnitine is a key energy metabolism factor that can promote the transport of long-chain fatty acids into mitochondria for oxidation, thereby improving the efficiency of cellular energy production. It is also of great significance in neuroprotection. By promoting the synthesis of neurotransmitters and improving cognitive function, it may have a positive effect on neurodegenerative diseases (such as Alzheimer's disease).

[0109] (S)-Leucic acid ((S)-Leucic acid) is an important amino acid derivative that plays an important role in muscle growth and metabolic regulation. It mainly plays an important role in the retina. As a light-sensitive molecule, it participates in key reactions in the visual process and is a component of rhodopsin. Studies have shown that it can activate the mTOR (mammalian target of rapamycin) signaling pathway, which is a key mechanism for regulating cell growth, proliferation and protein synthesis. By activating the mTOR pathway, (S)-Leucic acid can increase the activity of anabolic metabolism in muscle cells, thereby promoting muscle protein synthesis and improving muscle mass. In addition, (S)-Leucic acid can promote the oxidation of fatty acids, thereby reducing fat accumulation, thus showing potential in weight management and obesity prevention. (S)-Leucic acid can also have a positive effect on metabolic diseases such as diabetes by improving insulin sensitivity and regulating blood sugar levels. Propionyl-L-carnitine has biological activities such as promoting fatty acid metabolism, neuroprotection, improving cardiovascular function and regulating sugar metabolism, and has important value in the research and application of sports nutrition, cardiovascular health and metabolic diseases. The experimental results showed that after fermentation, the contents of (S)-Leucic acid and propionyl-L-carnitine increased by 3.0530 and 3.0031 times, respectively.

[0110] Oleoyl ethylamide is an endogenous fatty acid derivative. Studies have shown that oleoyl ethylamide has a positive effect on weight management and metabolic syndrome by affecting the endocannabinoid system, regulating appetite and energy balance. In addition, oleoyl ethylamide also shows potential biological activity in neuroprotection, and may alleviate the effects of neurodegenerative diseases through antioxidant and anti-inflammatory effects. The content of oleoyl ethylamide after YD2-55 fermentation is 2.29 times that of the control, indicating the great potential of the broken wall Ganoderma lucidum spore lactic acid bacteria fermentation broth in improving metabolic syndrome and related inflammation.

[0111] The experimental group also had a significant increase in eicosanoid compounds. 15-OxoEDE (15-oxido-eicosatrienoic acid) is a derivative of the twenty-carbon series of fatty acids, which is mainly involved in the regulation of inflammation and immune responses in the body. 15-OxoEDE can act as a precursor of prostaglandins, and by binding to specific receptors, it promotes the release of inflammatory mediators, thereby enhancing the body's resistance to pathogens. In addition, it may also reduce the risk of chronic inflammation by inhibiting the synthesis of proinflammatory factors, thereby having a positive effect on cardiovascular disease and metabolic syndrome. Studies have shown that 15-OxoEDE may also play a role in regulating vasodilation, improving microcirculation, and lowering blood pressure. 19(R)-hydroxyprostaglandin F1 is a biologically active prostaglandin derivative that is mainly involved in inflammatory responses and vasodilation. It can regulate vascular contraction and dilation by activating specific prostaglandin receptors, thereby affecting blood pressure and blood flow. In addition, 19(R)-hydroxyprostaglandin F1 also has anti-inflammatory effects and can inhibit the release of proinflammatory cytokines, thus playing an important role in the prevention and treatment of various inflammatory diseases. Its role in regulating physiological functions makes it of great research value in cardiovascular diseases, metabolic diseases and related pathological conditions.

[0112] Table 11 Effect of fermentation of Ganoderma lucidum spore powder with strain YD2-55 on the content of fatty acid compounds (fold)

[0113]

[0114] (3) Steroid compounds

[0115] Steroids and their derivatives refer to a class of organic compounds with a specific four-ring structure, which play a variety of important biological functions in organisms, involving hormone regulation, anti-inflammatory effects, metabolic regulation, cell function regulation, neuroprotection, muscle growth and cardiovascular health. Ergosterol, as a precursor of vitamin D2, affects cell growth, differentiation and function by regulating the fluidity and stability of cell membranes. Under light, ergosterol can be converted into vitamin D2, which promotes calcium absorption and is essential for bone health. In addition, ergosterol has also been studied for use in anti-tumor and anti-microbial fields, and its mechanism may be related to its effect on cell membrane structure and its interaction with other biological molecules. Guggulsterones are plant sterols extracted from the guggul tree and are widely used in traditional medicine. They have shown the potential for lipid-lowering, anti-tumor and anti-inflammatory effects through multiple mechanisms such as activating the PPAR-γ pathway, inducing cell apoptosis, inhibiting the synthesis of pro-inflammatory factors and regulating immune responses.

[0116] Deoxycortisol is a corticosteroid that is mainly synthesized in the adrenal glands and has the function of regulating electrolyte balance. It can stimulate the reabsorption of sodium by the renal tubules and promote water retention, thereby affecting the maintenance of blood pressure. In addition, deoxycortisol plays a role in coping with stress and inflammatory responses and participates in the regulation of the body's stress response. It is clinically used to treat specific types of adrenal insufficiency and hypotension (Table 12).

[0117] Table 12 Effect of fermentation of Ganoderma lucidum spore powder with strain YD2-55 on the content of sterol compounds

[0118]

[0119] Experimental Example 5 Effect of Ganoderma lucidum spore fermentation supernatant on mouse colitis model

[0120] 5.1 Preparation of fermentation supernatant: See Experimental Example 4. The fermentation broth 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. At the same time, a 4% (w / v) aqueous solution of dextran sulfate sodium salt (DSS) was prepared for use.

[0121] 5.2 Animal experiment: The experimental mice were provided by Chengdu Dashuo Experimental Animal Co., Ltd. Twenty-seven 4-week-old SPF-grade male Kunming mice were randomly divided into 9 cages, with 3 mice in each cage. After adaptive feeding, they were randomly divided into 3 groups, with 3 cages in each group, including a blank control group (CK), a DSS model group (DSS), and a Ganoderma lucidum spore fermentation supernatant treatment group (YD2-55). The experimental treatments are shown in Table 13.

[0122] Table 13DSS-induced colitis experiment in mice

[0123]

[0124] 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.

[0125] 5.3 Results Analysis

[0126] 5.3.1 Detection of blood inflammatory factors: 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 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 YD2-55 strain treatment group were significantly lower than those in the DSS model group, and closer to the blank control group. The anti-inflammatory factor IL-10 also increased compared with the DSS group. This shows that the fermentation supernatant of broken wall Ganoderma lucidum spores fermented with Lactobacillus mucosa YD2-55 has a significant alleviating effect on DSS-induced colon inflammation in mice (Table 14).

[0127] Table 14 Changes in the concentration of inflammatory factors in the serum of mice in each group (Unit: pg / mL)

[0128]

[0129] Note: Compared with the blank control group, **p<0.01; compared with the DSS model group, ##p<0.01.

Claims

1. A strain of Lactobacillus fermentum YD2-55 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No.31453.

2. A biological preparation containing the fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 or its fermentation liquid according to claim 1.

3. Use of the fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 according to claim 1 or the biological preparation according to claim 2 for the purpose of inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and / or Gardnerella vaginalis for non-disease treatment purposes.

4. Use of the fermented mucus lactobacillus YD2-55 according to claim 1 in the preparation of a medicament for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and / or Gardnerella vaginalis.

5. Use of the fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 according to claim 1 or the biological preparation according to claim 2 in the preparation of a drug for treating or assisting in the treatment of cervical cancer.

6. Use of the fermented mucus lactobacillus (Limosilactobacillus fermentum) YD2-55 according to 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 Lactobacillus fermentum YD2-55 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.

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