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

Abstract

The invention discloses a lactobacillus jensenii strain and application thereof in fermentation of wall-broken ganoderma lucidum spore powder. The preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) No.31484. The strain is separated and screened from vaginal secretions of healthy women at the childbearing age, is sensitive to various common antibiotics, and has strong lactic acid production capacity; a fermentation supernatant of the bacillus subtilis shows relatively strong inhibition capability on escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, salmonella typhimurium, shigella dysenteriae, yersinia enterocolitica and gardnerella vaginalis, and has relatively strong adhesion capability, growth inhibition capability and apoptosis promotion 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 specifically relates to a strain of Lactobacillus jensenii 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, leading to imbalance of vaginal microecological balance. 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. The vagina of healthy women is rich in a variety of lactic acid bacteria. Lactobacillus jensenii is one of the dominant flora in the vagina. It produces organic acids such as lactic acid through metabolism to maintain the acidic environment of the vagina. This acidic environment has an inhibitory effect on most pathogens, thereby helping to maintain the microecological balance of the vagina. Lactobacillus jensenii helps promote the repair and regeneration of vaginal epithelial cells, enhance the barrier function of the vaginal mucosa, and reduce the invasion and infection of pathogenic microorganisms. Therefore, lactic acid bacteria have received much attention in the prevention and treatment of bacterial vaginitis (BV).

[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 fermentation to increase the content of its bioactive 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 Lactobacillus jensenii and application thereof in fermentation of broken-wall ganoderma spore powder.

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

[0006] A strain of Lactobacillus jensenii HP0411 was deposited in the General Microbiology Center of China Culture Collection Administration on July 29, 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.31484.

[0007] A biological preparation containing the above-mentioned Lactobacillus jensenii HP0411 or its fermentation liquid.

[0008] The above-mentioned Lactobacillus jensenii HP0411 or the biological preparation is used for inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica and / or Gardnerella vaginalis for non-disease treatment purposes.

[0009] The above-mentioned Lactobacillus jensenii HP0411 is used in the preparation of medicines for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica and / or Gardnerella vaginalis.

[0010] The use of the above-mentioned Lactobacillus jensenii HP0411 or the above-mentioned biological preparation in the preparation of a drug for treating or assisting in treating cervical cancer.

[0011] The application of the above-mentioned Lactobacillus jensenii HP0411 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 Lactobacillus jensenii HP0411 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 strain of Lactobacillus jensenii and its application in the fermentation of broken wall ganoderma spore powder, and the strain deposit number is CGMCC No.31484. The strain is isolated and screened from the vaginal secretions of healthy women of childbearing age, is sensitive to a variety of common antibiotics, has a strong lactic acid production ability, and its fermentation supernatant shows a strong inhibitory ability to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica and Gardnerella vaginalis, and has a strong adhesion ability, growth inhibition ability and apoptosis-promoting effect on human cervical cancer cells. The fermentation of broken wall ganoderma spore powder with the 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 mouse colitis, and significantly reduces the level of inflammatory factors. The strain not only has a significant application value in biological preparations, but also opens up a new way for the combined application of lactic acid bacteria and ganoderma spores.

[0017] Collection information:

[0018] Lactobacillus jensenii HP0411 was deposited at the General Microbiology Center of China Culture Collection Administration on July 29, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.31484. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a phylogenetic relationship diagram of Lactobacillus jensenii HP0411 in the present invention.

[0020] Figure 2 This is the effect of the fermentation supernatant of Lactobacillus jensenii HP0411 of the present invention on the reactive oxygen species (ROS) level of Hela cells.

[0021] Figure 3 This is a volcano diagram of differential metabolites of broken-wall Ganoderma lucidum spore powder fermented by Lactobacillus jensenii HP0411 strain of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0024] Experimental Example 1 Isolation, Identification and Safety of Lactobacillus jensenii

[0025] 1.1 Screening and isolation of strains

[0026] Cervical secretion samples from healthy women of childbearing age 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 medium was aspirated, diluted 10 times in a gradient, 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.

[0027] 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:

[0028] 27F: 5-AGAGTTTGATCMTGGCTCAG-3,

[0029] 1492R:5-GGTTACCTTGTTACGACTT-3

[0030] The PCR product was sequenced by Sangon Biotechnology (Shanghai) Co., Ltd., and a target gene sequence consisting of 939 base pairs (bp) was obtained (SEQ ID No. 1).

[0031] The gene sequence obtained by sequencing was input into the NCBI database for comparison. The similarity between the strain and the strain Lactobacillus jensenii strain 70 (02) in GenBank was 99.25%, and the strain was preliminarily identified as Lactobacillus jensenii. The strain was named Lactobacillus jensenii HP0411. The phylogenetic relationship with other strains is as follows: Figure 1 shown.

[0032] The kit method was used to perform Gram staining on Lactobacillus jensenii HP0411, and the bacterial morphology after staining was observed and recorded under a microscope. Lactobacillus jensenii HP0411 grew well on MRS agar medium, with milky white colonies, round protrusions, neat edges, and smooth surfaces. Microscopic examination showed that the bacteria were rod-shaped and purple, which was consistent with the morphological characteristics of Lactobacillus jensenii. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on July 29, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 31484.

[0033] 1.2 Security

[0034] After Lactobacillus jensenii HP0411 was activated and purified, it was 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. Lactobacillus jensenii HP0411 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).

[0035] The results of the hemolytic experiment showed that there was no hemolysis around the colonies of Lactobacillus jensenii HP0411; Lactobacillus jensenii HP0411 was sensitive (S) to tetracycline, ampicillin, clarithromycin and chloramphenicol tested, and was intermediate (I) to ceftriaxone and clindamycin.

[0036] Table 1 Sensitivity of Lactobacillus jensenii HP0411 strain to 6 antibiotics

[0037]

[0038] 1.3 Environmental tolerance test:

[0039] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The components of artificial gastric juice simulation fluid were dilute hydrochloric acid, pepsin and sodium chloride, with a final pH of 2.5; the components of artificial intestinal juice simulation fluid were potassium dihydrogen phosphate and trypsin, with a final pH of 6.8. The test strain Lactobacillus jensenii HP0411 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 Lactobacillus jensenii HP0411 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 is colony growth on the surface of the MRS plate. Similarly, 1mL of the test strain Lactobacillus jensenii HP0411 bacterial solution was inoculated into simulated intestinal fluid, cultured at 37°C for 6h, then coated on the plate, cultured at 37°C for 24h, and the colony growth was checked.

[0040] The results of the simulated gastric fluid tolerance test showed that Lactobacillus jensenii HP0411 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 Lactobacillus jensenii HP0411 could still grow normal colonies on MRS agar plates after being treated in intestinal fluid for 6 hours, indicating that Lactobacillus jensenii HP0411 has strong tolerance to gastrointestinal fluid.

[0041] Experimental Example 2: Growth inhibition of HP0411 on human cervical cancer cells

[0042] 2.1 Adhesion ability of HP0411 to human cervical cancer cells Hela:

[0043] After the frozen strain of Lactobacillus jensenii HP0411 was revived and inoculated into MRS broth medium, the culture was cultured at 37°C for 24 hours. After the culture was completed, the culture 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%.

[0044] The experimental results showed that the adhesion rate of Lactobacillus jensenii HP0411 to human cervical cancer Hela cells was 45.18%±0.99%, and it had a strong colonization ability.

[0045] Table 2 Adhesion rate of Lactobacillus jensenii HP0411 to human cervical cancer Hela cells (%)

[0046]

[0047] 2.2 Effect of Lactobacillus jensenii HP0411 fermentation supernatant on Hela cell colony formation ability:

[0048] The Hela colon cancer cells were revived and plated in a 6-well plate at a density of 2000 cells per well. The Lactobacillus jensenii HP0411 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 the fermentation supernatant of Lactobacillus jensenii HP0411 to a concentration of 1% with serum-free culture medium at a volume ratio of 1:100. Cultivate Hela cells with 1% HP0411 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.

[0049] The results are shown in Table 3. The growth density of Hela cells in the HP0411 fermentation liquid treatment group was significantly lower than that in the control group, and the number of cell colonies decreased by 66.3%, indicating that the fermentation liquid had a significant growth inhibitory effect on human cervical cancer cells.

[0050] Table 3 Number of Hela cell clones formed after Lactobacillus jensenii HP0411 fermentation supernatant treatment (individuals)

[0051]

[0052] Experimental Example 3: Pro-apoptotic effect of Lactobacillus jensenii HP0411 on human cervical cancer cells

[0053] 3.1 Effect of HP0411 fermentation supernatant on reactive oxygen species (ROS) levels in Hela cells:

[0054] Cell collection: After incubating the HP0411 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.

[0055] 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.

[0056] 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.

[0057] 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 HP0411 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 HP0411 fermentation supernatant has a pro-apoptotic effect on colon cancer cells (Table 4).

[0058] Table 4 Effect of the fermentation supernatant of strain HP0411 on the expression of reactive oxygen species in human cervical cancer cells

[0059]

[0060] 3.2 Pro-apoptotic ability of HP0411 strain on human cervical cancer cell Hela

[0061] Cell culture: Human cervical cancer cells in the logarithmic growth phase were digested and centrifuged and evenly plated in a 96-well plate. Counting was performed to ensure that there were 2,000 cells per well. After culturing for 12 hours until the cells were stably attached to the wall, 1% of the fermentation supernatant of the HP0411 strain was added. Five replicates were set for each treatment, and protein was extracted after culturing for 48 hours.

[0062] 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.

[0063] 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.

[0064] (1) Effect of HP0411 fermentation supernatant on cell adhesion factor β-catenin

[0065] β-Catenin is a key multifunctional protein in the Wnt / β-Catenin signaling pathway, involved in cell adhesion, signal transduction and transcriptional regulation. In normal cells, the stability of β-Catenin is strictly regulated. Activation of the Wnt signaling pathway usually leads to the accumulation of β-Catenin, which in turn transcribes downstream target genes and promotes cell proliferation. However, in many cancers, including cervical cancer, abnormal activation or mutation of the β-Catenin signaling pathway can lead to the accumulation of β-Catenin in the cytoplasm or abnormal transfer to the nucleus, thereby promoting the progression of cancer. The phosphorylation of β-Catenin promotes its degradation and prevents its accumulation and activation in the nucleus. Our experimental results (Table 5) show that after adding 1% HP0411 fermentation supernatant to the culture medium of human cervical cancer cells and culturing for 48 hours, the β-catenin content was reduced by 61.72% compared with the control without fermentation medium. At the same time, the content of phosphorylated β-catenin (P-β-catenin) increased by 78% relative to the control group (Table 5). The results showed that HP0411 fermentation supernatant can reduce the expression of cell adhesion factor β-catenin and increase the expression of phosphorylated β-catenin, thereby reducing the adhesion and spread of cancer cells.

[0066] Table 5 Effect of the fermentation supernatant of strain HP0411 on the content of β-catenin in human cervical cancer cells (absorbance)

[0067]

[0068] Effect of apoptosis inhibitory gene protein content

[0069] Mcl-1 (Myeloid Cell Leukemia 1) is an anti-apoptotic protein belonging to the Bcl-2 family. It plays an important role in cell survival and apoptosis. Overexpression or amplification of Mcl-1 is common in many types of cancer, so it is considered to be one of the most relevant oncoproteins. Our study showed that the addition of 1% HP0411 fermentation supernatant to the culture medium can reduce the total amount of MCL-1 protein in cervical cancer cells by 44.47% (Table 6), indicating that the fermentation broth has the activity of promoting cancer cell apoptosis. Studies have shown that the expression of mRNA and protein levels of mcl1 gene subtype 1 in cervical tumor tissues and corresponding adjacent tissues are higher than those in the normal group. mcl1 subtype 1 and subtype 2 are positively correlated with the malignancy of colon cancer tumors, and can be used as one of the effective diagnostic indicators for clinical evaluation of the malignancy of colon cancer.

[0070] B-cell lymphoma / leukemia-2 gene (Bcl-2) is an important gene that regulates cell apoptosis. It can prevent programmed cell death by inhibiting the mitochondrial-mediated apoptosis pathway. Studies have found that in cervical cancer cells, the expression of BCL-2 is often higher than that in normal cervical epithelial cells. This overexpression can inhibit the apoptosis of cancer cells, promote their survival and proliferation, and thus promote the development of cancer. In early cervical cancer, the high expression of BCL-2 may be related to the malignant progression of tumors. Our experiments showed that adding 1% HP0411 fermentation supernatant to the cell culture medium can reduce the total amount of BCL-2 protein in cervical cancer cells by 50.11% (Table 6), indicating that the fermentation supernatant has the activity of promoting cancer cell apoptosis.

[0071] Table 6 Effect of HP0411 fermentation supernatant on the protein content of human cervical cancer cell apoptosis inhibitory gene (absorbance)

[0072]

[0073] (3) Effect of HP0411 fermentation supernatant on the content of apoptotic factor protein

[0074] Poly ADP-ribose polymerase PARP (poly ADP-ribose polymerase) is a DNA repair enzyme, a key enzyme family involved in DNA damage repair, and is involved in DNA repair. C-Parp (i.e. cleaved PARP) is the form of PARP produced by cleavage during cell apoptosis, indicating that the cell has undergone programmed cell death. C-Parp is considered an important indicator of cell apoptosis and is also generally considered an indicator of Caspase 3 activation.

[0075] Caspase-3 is a key executor protease in the apoptosis pathway. When cells are subjected to stress, DNA damage or other stimuli, Caspase-3 is activated and initiates a series of proteolytic reactions, leading to the degradation of the cytoskeleton and other important structures, and ultimately inducing apoptosis. The activation of Caspase-3 is the final step of cell death and an important mechanism for clearing damaged or abnormal cells. Cleaved-caspase-3 is the activated form of Caspase-3 during apoptosis. Our results showed that the addition of 1% HP0411 fermentation supernatant to the culture medium can increase the total amount of Cleaved-caspase-3 in cervical cancer cells by 124.5% compared with the control (Table 7), indicating the pro-apoptotic activity of HP0411 fermentation supernatant.

[0076] Cleaved-caspase-1 is the active form of caspase-1 after activation, which is responsible for initiating cell pyroptosis and activating proinflammatory cytokines. Caspase-1 is part of the inflammasome. When cells are infected or stimulated by other inflammation, Caspase-1 is activated and cleaved into Cleaved-caspase-1. Cleaved-caspase-1 can activate proinflammatory cytokines such as IL-1β and IL-18, playing an important role in the inflammatory response. Cleaved-caspase-1 is also a key executor of cell pyroptosis. Cell pyroptosis is an inflammatory cell death mode that usually occurs during infection and inflammation. Cleaved-caspase-1 cleaves gasdermin D protein, causing the cell membrane to form holes, causing the cell to rupture and release cell contents, further stimulating a strong inflammatory response. Studies have shown that the inflammatory response mediated by Cleaved-caspase-1 can promote the inflammatory response in the tumor microenvironment, thereby inhibiting tumors. The results showed that the cleaved-caspase-1 protein increased by 101.18% after treatment with HP0411 fermentation supernatant (Table 7), indicating that HP0411 has good anti-cervical cancer activity.

[0077] Table 7 Effect of HP0411 fermentation supernatant on pro-apoptotic factor proteins in cervical cancer cells

[0078]

[0079] In summary, Lactobacillus jensenii HP0411 strain has good adhesion to cervical cancer cells, and its fermentation supernatant has a significant inhibitory effect on the growth of cervical cancer cells, can reduce the content of β-catenin related to adhesion and diffusion, can reduce the content of MCL-1 and Bcl-2 proteins representing apoptosis inhibitory genes, and can increase the content of pro-apoptotic factors poly (ADP-ribose) polymerase Cleaved parp, cleaved caspase 3 (Cleaved-caspase3) and cleaved caspase 1 (Cleaved-caspase1). This shows from many aspects that Lactobacillus jensenii HP0411 strain has a growth inhibitory effect on cervical cancer and apoptosis-promoting effect, and has broad application prospects.

[0080] Experimental Example 4 Acid production and antibacterial ability of Lactobacillus jensenii HP0411

[0081] 4.1 Lactic acid production capacity of strain HP0411

[0082] 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.).

[0083] After the Lactobacillus jensenii HP0411 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. 100 μL of 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 Lactobacillus jensenii HP0411 was 42.05±15.07 μmol / mL, indicating that the lactic acid production capacity of Lactobacillus jensenii HP0411 was strong (Table 8).

[0084] Table 8 Lactobacillus jensenii HP0411 fermentation broth lactic acid content (μmol / mL)

[0085]

[0086] 4.2 Inhibitory effect of HP0411 strain on common intestinal pathogens and Gardnerella vaginalis

[0087] Escherichia coli (CMCB 44102), Pseudomonas aeruginosa (CMCB 10104), Staphylococcus aureus (CMCB 26003), Salmonella typhimurium (ATCC14028), Shigella dysenteriae (CMCB 51105), Yersinia enterocolitica subspenterocolitica (CMCB 52204), 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 the 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 HP0411 strain solution to 1×108 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.

[0088] The results of the antibacterial activity evaluation of Lactobacillus jensenii HP0411 are shown in Table 4 below. The strain of Lactobacillus jensenii HP0411 has a strong inhibitory activity against the growth of pathogenic bacteria of Escherichia coli, Salmonella paratyphi B, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica (Table 9). 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 HP0411 has a strong inhibitory ability against vaginal Gardnerella.

[0089] Table 9 Evaluation of antibacterial activity of Lactobacillus jensenii HP0411 strain (diameter: mm)

[0090]

[0091] Experimental Example 5 Application of Lactobacillus jensenii HP0411 strain in fermentation of broken wall Ganoderma lucidum spore powder

[0092] 5.1 Fermentation and analytical methods

[0093] 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, Lactobacillus jensenii HP0411 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 HP0411, and the culture and analysis methods were the same as those of the treatment group.

[0094] 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.

[0095] 5.2 Analysis results

[0096] (1) Amino acids and peptide compounds:

[0097] Amino acids are the basic building blocks of proteins and play an important role in the health and normal functions of the human body, such as 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 broken wall Ganoderma lucidum spore powder fermented by Lactobacillus jensenii HP0411 strain, such as 4-Hydroxy-L-Glutamic Acid (4-HG), which was 15.06 times that of the control. 4-Hydroxy-L-Glutamic Acid is a derivative of glutamate and is widely present in plants and animals. As a derivative of glutamate, 4-HG may affect neurotransmission by regulating the metabolism of glutamate. 4-HG has been found to reduce oxidative stress in nerve cells and protect nerve cells from damage. For example, 4-HG can reduce the level of reactive oxygen species (ROS) in cells, thereby protecting neurons from hypoxia and toxic substances. Studies have shown that 4-HG may also support the growth and survival of nerve cells by promoting the release of nerve growth factor. These effects make it a candidate for investigation as a potential therapeutic strategy for neurodegenerative diseases such as Alzheimer's and Parkinson's diseases.

[0098] In the fermentation supernatant, the content of N-oleoylglycine (N-Oleoyl Glycine) is 3.8 times that of the control. It is a fatty acid derivative 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 the compound may exert neuroprotective effects by reducing oxidative stress, improving cellular energy metabolism, and promoting neuronal survival.

[0099] Octopine was three times higher than the control, and is involved in cellular energy metabolism and stress response, especially in some plants and microorganisms. N-Acetyl-L-methionine, as an antioxidant, is involved in amino acid metabolism and may protect cells from oxidative damage. Ergothioneine is a potent antioxidant that helps protect cells from oxidative stress and may support mitochondrial function. D-Proline is involved in protein synthesis, can regulate the cell's response to stress, and helps stabilize protein and cell structure (Table 10).

[0100] Table 10 Effect of HP0411 strain fermentation of Ganoderma lucidum spore powder on the content of amino acids and peptide compounds

[0101]

[0102] (2) Fatty acids:

[0103] Eicosanoids, also known as eicosanoids, are a large class of biologically active unsaturated fatty acids produced by the oxidation of twenty-carbon polyunsaturated fatty acids, including prostaglandins, thromboxanes and leucotriene. They are important inflammatory factors that are widely present in body fluids and tissues, regulating many physiological and pathological processes in the body.

[0104] The results of the analysis showed that after the broken wall Ganoderma lucidum spore powder was fermented by Lactobacillus jensenii HP0411 strain for 96 hours, a variety of eicosanoid compounds were detected in the supernatant, and the content was greatly increased compared with the control. The content of deoxyprostaglandin (15-Deoxy-Δ12,14-prostaglandin D2, referred to as 15d-PGD2) was 31.02 times that of the control (Table 11). 15d-PGD2 is an important bioactive prostaglandin that plays a key role in inflammation and immune response. Studies have shown that 15d-PGD2 has the ability to inhibit inflammation and can reduce the inflammatory response caused by cytokines by regulating some key molecules and signaling pathways in the inflammatory process. In addition, 15d-PGD2 can also affect the activity, proliferation and differentiation of immune cells, thereby regulating the intensity and direction of the immune response. 19(R)-Hydroxy prostaglandin F1α (19(R)-H-PGF1α) is an important metabolite of prostaglandins, which mainly plays a physiological role by dilating blood vessels and promoting blood circulation. This compound can effectively improve hemodynamics, help lower blood pressure and reduce the burden on the heart. In addition, 19(R)-H-PGF1α also shows significant anti-inflammatory effects, can inhibit the release of inflammatory mediators, and reduce the intensity of local inflammatory responses. In the fermentation broth, the effective content of 19(R)-H-PGF1 is 2.08 times that of the control. 2-Arachidonoyl glycerol (2-AG) is an endogenous cannabinoid that mainly plays a role in the nervous system. It regulates neurotransmission and pain perception by activating CB1 and CB2 cannabinoid receptors, and also plays an anti-inflammatory role in the immune system. 2-AG is also related to appetite regulation and energy metabolism, and affects the function of fat cells.

[0105] (9cis)-Retinal is a form of vitamin A that 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. In addition, (9cis)-Retinal also plays an important role in cell proliferation and differentiation. It participates in a variety of physiological processes by regulating the expression of specific genes. Recent studies have found that (9cis)-Retinal also exhibits potential biological activity in anti-oxidation and anti-inflammatory aspects, which may provide new ideas for the treatment of retinal diseases and other inflammatory diseases. (+)-ar-Turmerone has significant anti-inflammatory, antioxidant and anti-tumor activities. It can reduce inflammatory responses and promote the body's self-repair by inhibiting the production of inflammatory mediators. In addition, (+)-ar-Turmerone also plays an important role in the regulation of cell proliferation and apoptosis, and may inhibit the growth of tumor cells by regulating the cell cycle and apoptosis signaling pathways. The experimental results showed that the contents of (9cis)-Retinal and (+)-ar-Turmerone increased by 1.54 and 1.52 times respectively after fermentation.

[0106] Oleoyl ethylamide is an endogenous fatty acid derivative that has similar functions of regulating appetite and energy metabolism as N-Oleoyl Glycine mentioned above. Studies have shown that Oleoyl ethylamide may have 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 HP0411 fermentation is 2.29 times that of the control, indicating that the fermentation broth of broken Ganoderma lucidum spores and lactic acid bacteria has great potential in improving metabolic syndrome and related inflammation.

[0107] Table 11 Effect of Ganoderma lucidum spore powder fermented by strain HP0411 on the content of fatty acid compounds

[0108]

[0109] (3) Phenylpropanoids and polyketides

[0110] Phenylpropanoids and polyketones are widely present in plants and have many important biological functions. Phenylpropanoids, derived from phenylalanine, have significant antioxidant, anti-inflammatory and anti-tumor effects. Polyketones, such as resveratrol and flavonoids, exhibit strong antioxidant capacity due to their rich phenolic hydroxyl structure, which can effectively scavenge free radicals and slow down the aging process. At the same time, they play an anti-inflammatory role by inhibiting pro-inflammatory 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 flavonoid compound with a unique structure and has multiple biological activities. It is mainly found in certain plants, especially in some medicinal plants and spices. Studies have shown that 3-Methoxyflavone exhibits significant antioxidant capacity, can effectively scavenge free radicals and protect cells from oxidative damage. 3-Methoxyflavone can also reduce inflammatory responses by inhibiting the release of pro-inflammatory factors and their signaling pathways, thereby playing a protective role in chronic inflammation-related diseases such as arthritis and cardiovascular disease. At the same time, 3-Methoxyflavone has also attracted attention in tumor research. It may inhibit tumor cell proliferation and induce cell apoptosis by regulating the cell cycle and affecting related signal pathways.

[0111] (+ / -)-Equol is a metabolite of soy isoflavones with estrogen-like activity, which can regulate hormone levels and improve bone health. Equol also has antioxidant and anti-inflammatory effects, can reduce oxidative stress and inflammatory response, and promote cardiovascular health. In addition, Equol also shows potential in preventing hormone-related tumors such as breast and prostate tumors, and has good prospects for health care and clinical application.

[0112] Trans-Cinnamic acid reduces cellular oxidative damage through its antioxidant and anti-inflammatory properties and shows potential in inhibiting tumor cell proliferation. 3-Phenyllactic acid mainly exhibits good antibacterial activity by inhibiting the growth of pathogenic microorganisms and enhancing immune responses, and has application value in the fields of food preservation and health products. D(+)-Phenyllactic acid is its enantiomer, which also has antioxidant and immunomodulatory functions and may improve intestinal health by regulating the intestinal microbiome. Overall, these compounds have important application prospects in disease prevention and treatment, and it is worth in-depth study of their mechanism of action and clinical application potential (Table 12).

[0113] Table 12 Effect of HP0411 strain fermentation of Ganoderma lucidum spore powder on the content of phenylpropanoids and polyketides

[0114]

[0115] Experimental Example 6 Effect of Ganoderma lucidum spore fermentation supernatant on mouse colitis model

[0116] 6.1 Preparation of fermentation supernatant: See Experimental Example 5. 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.

[0117] 6.2 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 (HP0411). The experimental treatments are shown in Table 13.

[0118] Table 13DSS-induced colitis experiment in mice

[0119]

[0120] 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.

[0121] 5.3 Results Analysis

[0122] 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 model group were significantly higher than those in the control group, and the levels of IL-6 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 also increased compared with the DSS model group. This shows that the supernatant of broken Ganoderma lucidum spore fermentation of Lactobacillus jensenii HP0411 has a significant alleviating effect on DSS-induced colon inflammation in mice (Table 14).

[0123] Table 14 Changes in the concentration of inflammatory factors in the serum of mice in each group (Unit: pg / mL)

[0124]

[0125]

[0126] 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 jensenii HP0411, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No.31484.

2. A biological preparation containing the Lactobacillus jensenii HP0411 or its fermentation liquid according to claim 1.

3. Use of Lactobacillus jensenii HP0411 according to claim 1 or the biological preparation according to claim 2 for non-disease treatment purposes to inhibit Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae, Yersinia enterocolitica and / or Gardnerella vaginalis.

4. Use of the Lactobacillus jensenii HP0411 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, Yersinia enterocolitica and / or Gardnerella vaginalis.

5. Use of Lactobacillus jensenii HP0411 according to claim 1 or the biological preparation according to claim 2 in the preparation of a drug for the treatment or adjuvant treatment of cervical cancer.

6. Use of Lactobacillus jensenii HP0411 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 Lactobacillus jensenii HP0411 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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