A strain of Enterococcus faecalis HY0110 and its application in the fermentation of American cockroaches

Abstract

The invention discloses a strain of Enterococcus faecalis HY0110 and its application in the fermentation of American cockroaches. The strain is isolated and screened from the intestinal contents of bluefin tuna (Thunnus thynnus) collected from the South China Sea, is non-hemolytic, sensitive to a variety of common antibiotics, has strong adaptability to the gastrointestinal environment of animals, has a strong ability to inhibit a variety of digestive tract pathogens, and has a significant growth inhibition effect and a cell apoptosis-promoting effect on human colon cancer cells; the strain is used to ferment American cockroach powder, and the content of amino acids and peptides, astragaloside IV, eicosanoids and other bioactive substances in the supernatant is significantly increased, and has a good symptom relief effect on the DSS mouse colitis model, and the levels of a variety of inflammatory factors are reduced, which opens up a new way for the in-depth development of American cockroaches.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to a strain of Enterococcus faecalis with strong antibacterial and anti-inflammatory effects, and particularly to the field of production and development of functional lactic acid bacteria and products thereof. Background Art

[0002] Periplaneta americana, commonly known as cockroach, has a long history of medicinal use in my country. Clinical studies have shown that artificially raised American cockroach extracts have multiple pharmacological activities such as anti-tumor, liver protection, promotion of tissue repair, immune enhancement, and cardiovascular protection; American cockroach dried into powder has anti-tumor, anti-inflammatory, analgesic, and tissue repair effects; so far, many traditional Chinese medicines such as Kangfuxin Liquid have been developed using American cockroach as a material for clinical use. We use wild bluefin tuna

[0003] A strain of Enterococcus faecalis HY0110 was isolated and screened from the intestine of Thunnus thynnus. Based on the evaluation of its probiotic effect, it was used in the fermentation of the traditional Chinese medicine American cockroach, hoping to further expand the application of American cockroach in biopharmaceuticals. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to provide a strain of Enterococcus faecalis with strong antibacterial and anticancer effects, and apply it to American cockroach fermentation medicine.

[0005] The technical solution of the present invention is: a strain of Enterococcus faecalis HY0110, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 28, 2023, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a deposit number of CGMCC NO.26946.

[0006] The above-mentioned Enterococcus faecalis HY0110 is used for inhibiting Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and / or Campylobacter jejuni for non-disease treatment purposes.

[0007] The use of the Enterococcus faecalis HY0110 described above in the preparation of medicines for treating diseases caused by Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and / or Campylobacter jejuni.

[0008] The use of the Enterococcus faecalis HY0110 described above in the preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.

[0009] An anti-colon cancer drug or an anti-colon cancer auxiliary drug contains the above-mentioned Enterococcus faecalis HY0110.

[0010] A preparation containing a fermentation liquid obtained by fermenting American cockroach powder with Enterococcus faecalis HY0110.

[0011] Application of the above-mentioned preparation in the preparation of anti-colitis drugs.

[0012] The Enterococcus faecalis HY0110 of the present invention is obtained by separation and screening from the intestines of bluefin tuna collected from the South China Sea. The Enterococcus faecalis HY0110 grows well on MRS agar medium, and the colonies are milky white, smooth in surface, neat in edge, opaque, and Gram-positive. By using the bacterial universal primer 16S rRNA 27F / 1492R as a template for PCR amplification of its total genomic DNA, a target gene sequence consisting of 1000 base pairs (bp) is obtained, and the sequence is shown in SEQ ID No. 1. The gene sequence obtained by sequencing is input into the NCBI database for comparison, and the similarity between it and the strain Enterococcus faecalis strain CAU:272 in GenBank is 99.90%, and the strain can be preliminarily identified as Enterococcus faecalis.

[0013] The Enterococcus faecalis HY0110 of the present invention has a certain tolerance to acid and bile salts through acid resistance and bile salt resistance tests, and has a strong adaptability to the human gastrointestinal environment.

[0014] The Enterococcus faecalis HY0110 of the present invention has strong inhibitory activity against multiple intestinal pathogens, and can inhibit the growth of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa and Campylobacter jejuni.

[0015] The fermentation supernatant of the Enterococcus faecalis HY0110 strain of the present invention has obvious effects of inhibiting growth and promoting cell apoptosis on human colon cancer cells;

[0016] After the Enterococcus faecalis HY0110 strain of the present invention ferments the American cockroach powder, the contents of multiple active substances in the supernatant are significantly increased.

[0017] Animal experiments have shown that the supernatant of the Enterococcus faecalis HY0110 strain fermented with American cockroach powder has a good preventive effect on the DSS mouse colitis model, can alleviate the symptoms of colitis, and reduce the levels of multiple inflammatory factors.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The acid and bile salt resistance tests show that the Enterococcus faecalis HY0110 of the present invention has a certain tolerance to acid and bile salts and a strong adaptability to the human gastrointestinal environment.

[0020] 2. The Enterococcus faecalis HY0110 of the present invention has strong inhibitory activity against a variety of intestinal pathogens, and can inhibit the growth of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Campylobacter jejuni.

[0021] 3. The fermentation supernatant of the Enterococcus faecalis HY0110 strain of the present invention has obvious effects of inhibiting the growth and promoting cell apoptosis of human colon cancer cells.

[0022] 4. After the Enterococcus faecalis HY0110 strain of the present invention fermented the American cockroach powder, the contents of various active substances in the supernatant increased significantly.

[0023] 5. Animal experiments have shown that the supernatant of the Enterococcus faecalis HY0110 strain fermented with American cockroach powder has a good preventive effect on the DSS mouse colitis model, can alleviate the symptoms of colitis, and reduce the levels of multiple inflammatory factors.

[0024] Collection information:

[0025] Enterococcus faecalis HY0110 was deposited on March 28, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO.26946. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Phylogenetic relationship between Enterococcus faecalis HY0110 and other strains;

[0027] Figure 2 Effects of fermentation supernatant of Enterococcus faecalis HY0110 strain on the growth of colon cancer cell colonies;

[0028] Figure 3 Effects of fermentation supernatant of Enterococcus faecalis HY0110 strain on reactive oxygen species levels in colon cancer cells;

[0029] Figure 4Effect of fermentation supernatant of Enterococcus faecalis HY0110 strain on apoptosis of colon cancer cells. DETAILED DESCRIPTION

[0030] 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.

[0031] Example 1 Isolation, identification and safety evaluation of Enterococcus faecalis HY0110

[0032] 1.1 Isolation and identification of Enterococcus faecalis HY0110

[0033] Bluefin tuna intestinal samples were collected from the South China Sea; a small amount of bluefin tuna sample was taken in 50mL MRS broth, thoroughly shaken and mixed, and placed in a 37℃ constant temperature shaker for 24h. A 10-fold gradient dilution was used, and the strain was spread and inoculated on MRS agar medium. After culturing at 37℃ for 24h, a single colony was picked and purified three times in succession. The purified strain was inoculated in 600μL MRS broth medium, cultured at 37℃ for 18h, and 400μL of 50% (V / V) sterile glycerol was added, and frozen in a -80℃ ultra-low temperature refrigerator for later use.

[0034] After activation culture, the frozen strain was expanded with MRS broth medium, and the strain DNA was extracted with Tiangen Bacteria Genomic DNA Extraction Kit. 16S rRNA amplification was completed by colony PCR technology. The PCR primers were 16S rRNA universal primers 27F and 1492R, and the sequences were as follows:

[0035] 27F: 5-AGAGTTTGATCMTGGCTCAG-3, 1492R: 5-GGTTACCTTGTTACGACTT-3

[0036] The PCR product was sequenced by Sangon Biotech (Shanghai) Co., Ltd., and its 16S rRNA sequence is shown in SEQ ID No. 1. The sequence has a similarity of 99.90% with the Enterococcus faecalis strain CAU:272 in GenBank, and can be preliminarily identified as Enterococcus faecalis, and named Enterococcus faecalis HY0110. The phylogenetic relationship between this strain and other strains is shown in the attached Figure 1 .

[0037] The strain grows well on MRS agar medium. The colonies are milky white, round and convex, with flat edges and smooth surfaces. They are spherical under microscopic examination and Gram-staining positive.

[0038] The strain was deposited on March 28, 2023 at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCCNO.26946.

[0039] 1.2 Hemolysis and antibiotic resistance of Enterococcus faecalis HY0110:

[0040] The sensitivity of the strain to common antibiotics was tested by the paper agar diffusion method. The E. faecalis HY0110 strain was activated and cultured, and the bacterial solution concentration was adjusted to 1×10 6 CFU / mL, use a sterile cotton swab to evenly smear the bacterial solution on the surface of the MRS culture medium plate, put in the drug-susceptibility paper after 10 minutes at room temperature, and culture at 37℃ for 24 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 refer to 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).

[0041] The results of the hemolytic test showed that there was no hemolysis around the colonies ( Figure 3 ); the strain was sensitive (S) to the tested 6 antibiotics, including tetracycline, ampicillin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol, indicating the safety of the Enterococcus faecalis HY0110 strain (Table 1).

[0042] Table 1 Sensitivity of Enterococcus faecalis HY0110 strain to 6 antibiotics

[0043] serial number tetracycline Ampicillin Ceftriaxone Clindamycin Clarithromycin Chloramphenicol HY0110 S S S S S S

[0044] Example 2 Adaptability of Enterococcus faecalis HY0110 to the gastrointestinal environment of animals

[0045] 2.1. Evaluation of acid resistance and bile salt resistance: The test strain Enterococcus faecalis HY0110 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. Use hydrochloric acid and porcine bile salt to prepare MRS broth culture medium with acidity (pH=3.0) and bile salt concentration (0.3%) respectively. Inoculate 1mL of the test strain Enterococcus faecalis HY0110 bacterial solution into the broth culture medium with pH=3.0 and culture at 37℃ for 18h. After the culture is completed, take 20μL of the bacterial solution and spread it on the MRS agar plate culture medium, set up 3 replicates, and culture at 37℃ for 18h. Observe whether there is colony growth on the surface of the MRS plate after culture. Similarly, inoculate 1mL of the test strain Enterococcus faecalis HY0110 bacterial solution into MRS broth culture medium with a bile salt concentration of 0.3%. After culturing at 37℃ for 18h, spread the plate and culture at 37℃ for 24h to check the colony growth.

[0046] The test results showed that E. faecalis HY0110 could still grow normal colonies on MRS agar plates after being treated in MRS broth medium with pH=3.0 for 18 hours or in MRS broth medium with bile salt concentration of 0.3% for 18 hours, indicating that E. faecalis HY0110 strain has certain acid and bile salt resistance.

[0047] 2.2. Simulated gastric juice and intestinal juice tolerance test: Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd. The artificial gastric juice simulation liquid contains dilute hydrochloric acid, pepsin and sodium chloride, with a final pH of 2.5; the artificial intestinal juice simulation liquid contains potassium dihydrogen phosphate and trypsin, with a final pH of 6.8. The Enterococcus faecalis HY0110 strain was revived and activated, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL, 1 mL of bacterial solution was added to 9 mL of simulated gastric fluid, and then diluted 10 times in a gradient, and 20 μL was taken to plate and counted as the initial viable bacteria value of tolerance to artificial gastric fluid; after the inoculated simulated gastric fluid was cultured at 37°C for 3 hours, the viable bacteria were counted again on the plate, and the final viable bacteria value of tolerance to artificial gastric fluid was calculated. Similarly, 1 mL of 1×10 8 CFU / mL The fermentation liquid of Enterococcus faecalis HY0110 was added to 9 mL of simulated intestinal fluid, and the viable bacteria were counted, and the viable bacteria were counted again after culturing at 37°C for 6 hours to calculate the survival rate. Survival rate = viable bacteria count at the end / initial viable bacteria count * 100%.

[0048] The results showed that the strain of Enterococcus faecalis HY0110 had good tolerance to artificial simulated gastric and intestinal fluids, with a survival rate of 69.4% after 3 hours in artificial gastric fluid and a survival rate of 94.9% after 6 hours in artificial intestinal fluid (Table 2).

[0049] Table 2 Tolerance of Enterococcus faecalis HY0110 strain to artificial simulated gastric and intestinal fluids (%)

[0050]

[0051] Example 3 Acid production and antibacterial effect of Enterococcus faecalis HY0110 strain

[0052] 3.1 Determination of short-chain fatty acid content in fermentation broth of Enterococcus faecalis HY0110

[0053] Preparation of fermentation broth: After activating and culturing the preserved strain of Enterococcus faecalis HY0110 for 24 hours, 4 ul of the bacterial solution was added to 4 mL of broth culture medium and cultured at 37° C. for 24 hours for use.

[0054] Detection of short-chain fatty acids: The detection instrument is a gas chromatography-mass spectrometer (GCMS-QP2010 Plus) from Shimadzu Corporation of Japan, and the chromatographic column is an Rtx-5 fused silica capillary column (30m×0.25mm×0.25um) from RESTEK Corporation of the United States. The GC heating program is an initial temperature of 40°C for 5 minutes, then increased to 150°C at 5°C per minute, then increased to 280°C at 10°C per minute, and maintained for 2 minutes. The carrier gas is high-purity helium (purity>99.999%), with a flow rate of 1.0 mL / min. MS conditions: ionization mode is EI; temperature is 200°C, interface temperature is 220°C, and mass scanning range is m / z 33-500. Take 4 mL of fermentation broth, add 10 ul of 2-ethylbutyric acid internal standard solution with a concentration of 200 ug / mL, inject 1 μL of sample in split mode 1:3, set the solvent delay time to 0.1 min, and the injection port temperature to 270 ° C. The concentrations of 5 short-chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, and isohexanoic acid) were calculated using the internal standard method.

[0055] The results of GC-MS test showed that the acetic acid content in the fermentation broth of Enterococcus faecalis HY0110 was high, at 16.505ug / mL, which was higher than that of the LGG standard strain. In addition, it also contained three short-chain fatty acids, namely isobutyric acid, 2-methylbutyric acid and isovaleric acid (Table 3). The latest research found that short-chain fatty acids play an important role in maintaining the normal function of the large intestine and the morphology and function of colonic epithelial cells.

[0056] Table 3 Short-chain fatty acid content of Enterococcus faecalis HY0110 fermentation broth (ug / mL)

[0057] Strain No. Species name Acetic acid Isobutyric acid Isovalerate 2-Methylbutyric acid HY0110 Enterococcus faecalis 16.505 0.288 0.283 0.115 LGG Lactobacillus rhamnosus 13.08 0.18 0.41 0.23

[0058] 3.2 Evaluation of antibacterial activity of Enterococcus faecalis HY0110 strain

[0059] Escherichia coli (CMCB 44102), Staphylococcus aureus (CMCB 50094), Salmonella typhimurium ATCC14028), Pseudomonas aeruginosa (CMCB 10104), and Campylobacter jejuni ATCC 33291 were inoculated into nutrient agar medium, revived, and activated three times. An appropriate amount of tryptic soy bean liquid medium was drawn into a centrifuge tube, and the activated pathogenic bacteria were inoculated into the broth medium, and the bacterial solution concentration was adjusted to 1×10 8CFU / mL. Take 1mL of the mixture of the above pathogenic bacteria and broth and add it to 500mL of sterilized nutrient agar medium that has not yet solidified (the temperature has been cooled to about 40°C), mix thoroughly and dispense into culture dishes at a volume of 20mL per dish. After the culture medium has cooled and solidified, use a 6mm diameter puncher to punch holes on the plate to make pathogenic bacteria agar plates. Each plate corresponds to one strain of bacteria, and three holes are set as replicates. Resuscitate and activate the strain to be tested, and adjust the concentration of the cultured bacterial solution to 1×10 8 CFU / mL. Pipette 50 μL of the test bacterial solution into the wells of the pathogenic bacteria agar plate and culture at 37°C for 24 hours. After culture, use a vernier caliper to measure the diameter of the inhibition zone around the punch point and record it. Use the standard strain LGG as the control strain and perform the above experimental operations simultaneously with the test strain.

[0060] The results showed that the fermentation broth of Enterococcus faecalis HY0110 strain had good inhibitory activity against the growth of pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, and Campylobacter jejuni, which was close to or better than the antibacterial effect of the LGG standard strain (Table 4).

[0061] Table 4 Evaluation of antibacterial activity of Enterococcus faecalis HY0110 strain (diameter: mm)

[0062]

[0063] Example 4 Effect of Enterococcus faecalis HY0110 on Colon Cancer Cells

[0064] 4.1. Cell adhesion test: After the resuscitation of Enterococcus faecalis HY0110, it was 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 set aside. Resuscitate human colon cancer cells HT-29, 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 for digestion 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 one of the culture wells of a six-well cell culture dish 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 for plate counting. The result is used as the number of viable adherent bacteria. The standard strain LGG is used as a control.

[0065] Adhesion rate (%) = number of lactic acid bacteria at the end of the period / number of lactic acid bacteria inoculated at the beginning × 100%

[0066] The results are shown in Table 5. The average adhesion rate of the Enterococcus faecalis HY0110 to human colon cancer cell HT-29 was 61.48%, which was higher than the cell adhesion of the standard strain LGG.

[0067] Table 5 Adhesion rate of Enterococcus faecalis HY0110 to human colon cancer cell HT-29

[0068] strain Repeat 1 Repeat 2 Repeat 3 Mean Enterococcus faecalis HY0110 61.59％ 61.03％ 61.83％ 61.48％ Standard strain LGG 56.32％ 54.23％ 56.58％ 55.71％

[0069] 4.2 Inhibitory effect of Enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell growth

[0070] Preparation of fermentation supernatant of Enterococcus faecalis HY0110: After resuscitation, Enterococcus faecalis HY0110 was inoculated into MRS broth medium and cultured at 37°C for 24 h to make the concentration of bacterial suspension reach 1×10 9 After the culture is completed, centrifuge at -4°C and 5000r / min for 10 min, collect the fermentation supernatant, filter it with a bacterial filter membrane and set aside.

[0071] Cancer cell recovery: Prepare preheated DMEM culture medium containing 10% FBS and 1% double antibody in a 15mL centrifuge tube, place it on a sterile operating table, take out the frozen human colon cancer cells from the -80℃ refrigerator or liquid nitrogen and thaw them quickly in a 37℃ water bath. After the cells are thawed, transfer them to the prepared 15mL centrifuge tube containing culture medium on the sterile operating table, centrifuge at 1000r / min for 3min to resuspend the cells, aspirate them into a 6cm cell culture dish, culture them at 37℃ in a 5% CO2 incubator for 24h, and then replace with fresh culture medium.

[0072] Cell passaging: When the cells are growing well and the density reaches about 80%, passaging is performed. Preheat the culture medium and sterile PBS in a 37°C water bath and place on a sterile operating table. Aspirate the old culture medium in the cell culture dish and wash the cells with preheated PBS to remove dead cells and residual culture medium. Digest the cells with 0.25% trypsin. After the cells become round, aspirate the trypsin with a pipette. The culture medium stops digestion and the cells are collected in a sterile centrifuge tube. Centrifuge at 1000r / min for 3min, aspirate the supernatant, resuspend the cells with fresh culture medium, aspirate them into a new cell culture dish, and culture them in a cell culture incubator.

[0073] Cytotoxicity experiment: CCK-8 kit (Cell Counting Kit-8 cell counting reagent) was used to determine the effect of Enterococcus faecalis HY0110 fermentation supernatant on the growth of colon cancer cells. The kit uses a highly water-soluble tetrazolium salt WST-8, which can be reduced by some dehydrogenases in the mitochondria to generate orange-yellow formazan in the presence of an electron coupling reagent. The amount of formazan produced by the dehydrogenase is directly linearly related to the number of living cells. The more and faster the cancer cells proliferate, the darker the color; the greater the toxicity to the cancer cells, the lighter the color. The method has high sensitivity and is easy to operate.

[0074] The results showed that 4% Enterococcus faecalis HY0110 fermentation supernatant exhibited a significant growth inhibitory effect on the growth of human colon cancer cells. After 24h and 48h of culture, the density of colon cancer cells was significantly lower than that of the control, and the growth inhibition rates were 39.12% and 35.39%, respectively, indicating that Enterococcus faecalis HY0110 fermentation supernatant had a significant inhibitory effect on the growth of human colon cancer cells (Table 6).

[0075] Table 6 Effect of HY0110 fermentation supernatant on colon cancer cell growth (absorbance value)

[0076] 24h 48h Control 1.32±0.16 1.850±0.295 HY0110 0.8036±0.1302 1.1953±0.153 Inhibition rate % 39.12 35.39

[0077] At the same time, the ability of Enterococcus faecalis HY0110 fermentation supernatant to form colonies of human colon cancer cells was determined. The method is to digest, centrifuge, resuspend, and count colon cancer cells according to the steps of cell passage, and plate them in a 6-well plate at a density of 2000 cells per well. The culture plate with cells was placed in a cell culture incubator for 20 days, and fresh culture medium was replaced every two days, and 1% Enterococcus faecalis HY0110 fermentation supernatant was added. After the cells form visible clones, the culture medium was discarded, washed twice with PBS, 1% crystal violet was added for 30 minutes, and the culture plate was washed 3-5 times with clean water to wash off the residual crystal violet stain, the culture plate was dried, the number of cell colonies was recorded, and the experimental results were recorded by taking photos. The results showed that after adding 1% of Enterococcus faecalis HY0110 fermentation supernatant to the culture medium, the average number of cell colonies was 1283, while the control group was 1593, which was only 80.54% of the control group, indicating that Enterococcus faecalis HY0110 fermentation supernatant can significantly reduce the colony formation ability of colon cancer cells (Table 7, Figure 2 ).

[0078] Table 7 Effect of Enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell colony formation ability

[0079] Comparison 1% HY0110 fermentation supernatant Number of cell colonies 1593±374 1283±207

[0080] 4.3 Pro-apoptotic effect of Enterococcus faecalis HY0110 fermentation supernatant on human colon cancer cells

[0081] 4.3.1 Materials and methods

[0082] Cell culture: HT-29 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, 4% (V / V) fermentation supernatant of Enterococcus faecalis HY0110 strain was added. Five replicates were set for each treatment, and protein was extracted after culturing for 48 hours.

[0083] 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.

[0084] 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.

[0085] 4.3.2 Experimental Results

[0086] (1) Effect of Enterococcus faecalis HY0110 fermentation supernatant on cell adhesion factor β-catenin

[0087] The spread of cancer cells is characterized by disordered cell-cell interactions and cell adhesion. β-catenin is a multifunctional protein and an important cell adhesion molecule that participates in cell growth and repair and plays an important role in the process of tumorigenesis and metastasis. The postoperative recurrence rate of colorectal cancer is high and the survival time of patients is short. Almost all mutations in the Wnt / β-catenin signaling pathway in colorectal cancer will eventually lead to the accumulation of β-catenin. Therefore, the content of β-catenin can be used as an auxiliary factor in clinical evaluation of the development and prognosis of colorectal cancer. The nuclear accumulation of β-catenin may be a malignant-related marker for colorectal cancer invasion, metastasis and poor prognosis. Our experimental results showed that after adding the fermentation supernatant of Enterococcus faecalis HY0110 to the culture medium of colon cancer cells, the content of β-catenin was greatly reduced after 48 hours of culture, and was only 37.24% of the control without fermentation medium. Our results indicate that the fermentation supernatant of Enterococcus faecalis HY0110 can reduce the expression of cell adhesion factor β-catenin, thereby reducing the adhesion and spread of cancer cells (Table 8).

[0088] Table 8 Effect of Enterococcus faecalis HY0110 fermentation supernatant on β-catenin content in colon cancer cells (absorbance value)

[0089] Target protein / reference protein Comparison HY0110 β-catenin 37781 14071

[0090] (2) Effect of Enterococcus faecalis HY0110 fermentation supernatant on the content of apoptosis inhibitory gene protein

[0091] B cell lymphoma / leukemia-2 gene (bcl-2) also has a significant effect of inhibiting cell apoptosis, can inhibit cell death caused by a variety of cytotoxins, and is closely related to a variety of cancers such as colon cancer and ovarian cancer. Overexpression of this gene can enhance the resistance of cells to most cytotoxins. Our experiments showed that after adding the fermentation supernatant of Enterococcus faecalis HY0110 to the cell culture medium, the total amount of Bcl-2 protein in colon cancer cells was 24.14% of the control (Table 9), indicating that the fermentation supernatant has the activity of promoting cancer cell apoptosis.

[0092] Table 9 Effect of Enterococcus faecalis HY0110 fermentation supernatant on the content of apoptosis inhibitory gene protein in colon cancer cells

[0093] Target protein / reference protein Comparison HY0110 B-cell lymphoma / leukemia-2 (BCL-2) 35004 8449

[0094] (3) Effect of Enterococcus faecalis HY0110 fermentation supernatant on the content of apoptosis factor protein

[0095] The P62 protein is an oncogene protein. The target gene of transcriptional regulation participates in the regulation of the cell cycle, autophagy, cell proliferation, apoptosis and immortalization, and plays an important role in the occurrence of many tumors. The P62 gene can help the human body better identify and resist exogenous antigens, thereby reducing the harm caused by infection. The P62 protein can also prevent the accumulation of genotoxicity and carcinogenic mutations by promoting selective autophagy, thereby playing a tumor suppressor role. When the P62 gene binds to the antigen, it triggers antigen-specific T cell and B cell functions. T cells secrete specific Cytokines to kill foreign pathogens, while B cells secrete specific antibodies to inhibit the replication of pathogens. Therefore, the P62 gene can bind to antigens, promote immune responses, and help the human body resist foreign antigens, thereby reducing the damage caused by viral infection or bacterial infection. Our experiments show that the addition of Enterococcus faecalis HY0110 fermentation supernatant to the cell culture medium can greatly increase the total amount of P62 protein in colon cancer cells, which is about 1.47 times that of the control (Table 9), indicating the pro-apoptotic activity of Enterococcus faecalis HY0110 fermentation supernatant.

[0096] Poly ADP-ribose polymerase PARP (poly ADP-ribose polymerase) is a DNA repair enzyme and a cleavage substrate of caspase, a core member of apoptosis. Therefore, it plays an important role in DNA damage repair and apoptosis. PARP cleavage is considered an important indicator of apoptosis and is also generally considered an indicator of Caspase3 activation. PARP plays an important role in the growth and metastasis of colon cancer. Our results showed that after the fermentation supernatant of Enterococcus faecalis HY0110 was added to the culture medium, the total amount of poly ADP-ribose polymerase C-Parp in colon cancer cells was 5.57 times that of the control (Table 10), indicating the pro-apoptotic activity of the fermentation supernatant of Enterococcus faecalis HY0110.

[0097] Table 10 Effect of Enterococcus faecalis HY0110 fermentation supernatant on pro-apoptotic factor proteins in colon cancer cells (gray value)

[0098] Target protein / reference protein Control HY0110 P62 21348 31317 Poly ADP-ribose polymerase C-Parp 7701 42882

[0099] (4) Effect of Enterococcus faecalis HY0110 fermentation supernatant on reactive oxygen species (ROS) levels in human colon cancer cells

[0100] Cell collection: Remove the culture medium of adherent colon cancer cells and repeatedly blow and vortex with serum-free culture medium. Observe with naked eyes that the bottom of the well plate (bottom of the bottle) changes from translucent (cell monolayer connected into a sheet) to transparent, and almost all the cell layers are blown into the culture medium. Collect all the cell suspension into a 1.5ml centrifuge tube. Wash twice with serum-free culture medium, centrifuge at 1000r / min for 5min, aspirate the supernatant, and keep the cell pellet for determination.

[0101] Loading probe: Dilute DCFH-DA with serum-free culture medium at 1:1000 to a final concentration of 10 micromol / 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 three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells, and stimulate the cells with the fermentation supernatant of Enterococcus faecalis HY0110. Use a fluorescence spectrophotometer at an excitation wavelength of 488nm and an emission wavelength of 525nm to detect the intensity of fluorescence before and after stimulation in real time, and take pictures.

[0102] The level of reactive oxygen species (ROS) reflects the state of cellular metabolites. Under normal circumstances, the generation and removal of ROS are in a dynamic balance. If the ROS level exceeds the normal range, it may cause oxidative damage or cell death. The fluorescence intensity of colon cancer cells treated with the fermentation supernatant of Enterococcus faecalis HY0110 was significantly higher than that of the control group, the number of fluorescently labeled cells was also greater, and the ROS level was significantly higher than that of the control cells (Table 11, Figure 3 ).

[0103] Table 11 Number of cells expressing ROS after treatment with Enterococcus faecalis HY0110 fermentation supernatant

[0104]

[0105] (5) One-step TUNEL cell apoptosis detection

[0106] When cells undergo apoptosis, some DNA endonucleases are activated, which cut the genomic DNA between nucleosomes. After catalysis by terminal deoxynucleotidyl transferase (TdT), the fluorescent probe dUTP can be added, and the occurrence of cell apoptosis can be detected with the help of a fluorescence microscope. The specific operation steps are carried out according to the instructions of the one-step TUNEL cell apoptosis detection kit. The test results show that after adding 4% (V / V) of HY0110 strain fermentation supernatant (sterile) to the culture medium, a strong apoptosis signal can be detected. The average red fluorescence intensity of the treatment group is 287±42, while the average red fluorescence intensity of the control group is only 38±4 ( Figure 4 ).

[0107] In summary, the Enterococcus faecalis HY0110 strain has good adhesion to colon cancer cells, and its fermentation supernatant has a significant inhibitory effect on the growth of colon cancer cells. It can reduce the content of β-catenin related to adhesion and diffusion, and can reduce the content of Bcl-2 protein representing apoptosis inhibition gene, and can increase the content of pro-apoptotic factor poly (ADP-ribose) polymerase Cleaved-parp and P62 protein representing autophagy. These results illustrate the growth inhibition and apoptosis-promoting effects of Enterococcus faecalis HY0110 on colon cancer cells from multiple aspects.

[0108] Example 5 Application of Enterococcus faecalis HY0110 strain in fermentation of American cockroach powder

[0109] 5.1 Metabolomics analysis of Enterococcus faecalis HY0110 fermented American cockroach powder

[0110] Fermentation method: 10 grams of American cockroach powder, 3 grams of oligofructose, and 100 mL of water were prepared into a culture medium. After sterilization and cooling, Enterococcus faecalis HY0110 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 liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics analysis. The experiment was set up in three replicates, and the control group without probiotics was used. The culture and analysis methods were the same as those of the treatment group.

[0111] 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.

[0112] The results showed that after 96 hours of fermentation of American cockroach powder with Enterococcus faecalis HY0110, the content of various amino acids in the supernatant increased significantly, especially N-arachidonic acid-L-serine, succinyl-homoserine, valine and homocysteine, which increased by more than 10 times compared with the control without fermentation (Table 12). Amino acids are the basic building blocks of proteins and play an important role in human health and normal functions, such as protein synthesis, physiological regulation, immune function, energy supply, and maintaining acid-base balance.

[0113] Table 12 Growth multiples of amino acid and peptide content in American cockroach powder fermented by HY0110 strain

[0114]

[0115]

[0116] Many active substances in the human body exist in the form of peptides. Peptides are involved in the human body's hormones, nerves, cell growth and reproduction. Their importance lies in regulating the physiological functions of various systems and cells in the body, activating relevant enzyme systems in the body, promoting the permeability of intermediate metabolic membranes, or controlling DNA transcription or affecting specific protein synthesis, ultimately producing specific physiological effects. The analysis results show that after the American cockroach powder was fermented by Enterococcus faecalis HY0110 for 96 hours, the content of various peptide substances in the supernatant also increased significantly, especially the content of the polypeptide (L-Leucyl-L-Alanine) composed of L-leucine and L-alanine was compared with the control without fermentation, the increase was 16.89 times, and S-lactoylglutathione (S-lactoylglutathione) also increased by 7.95 times (Table 12).

[0117] It is worth noting that the content of Astragaloside A (also known as Cucurbitacin glycosides) in the fermentation supernatant increased by 668.73 times compared with the unfermented control. Previous studies have shown that this substance has a variety of important biological activities. For plants, cucurbitacin is a natural green pesticide that can resist the invasion of pests and diseases. For humans, cucurbitacin has extremely high medicinal value. It can not only be used to treat various inflammations, but also to protect the liver and fight tumors. For example, cucurbitacin B has been developed into an anti-liver cancer drug-cucurbitacin tablets, and new drug dosage forms centered on cucurbitacin are constantly developing.

[0118] The eicosanoid compound dimethyl prostaglandin (16,16-Dimethyl prostaglandin A1) in the fermentation supernatant also increased significantly, and the content was 116.15 times that of the non-fermented control. This compound has effects on the endocrine, reproductive, digestive, blood respiratory, cardiovascular, urinary and nervous systems, and is an active substance with multiple physiological effects. Effects on the gastrointestinal tract: It can cause smooth muscle contraction, inhibit gastric acid secretion, prevent strong acid, strong alkali, alcohol-free, etc. from eroding the gastric mucosa, and has a cell-protective effect. It also has a protective effect on the small intestine, colon, pancreas, etc. It can also stimulate intestinal fluid secretion, bile secretion, and gallbladder muscle contraction.

[0119] 5.2 Effect of fermentation supernatant on DSS-induced colitis model in mice

[0120] Experimental methods

[0121] Experimental animals: The experimental mice were randomly divided into 6 cages, 3 mice in each cage. They were randomly divided into 3 groups, 2 cages in each group, including a blank control group (CK), a DSS model group (DSS), and an Enterococcus faecalis HY0110 treatment group. The experimental treatments are shown in Table 13.

[0122] Table 13 Animal experiment on DSS-induced colitis model in mice using the fermentation supernatant of American cockroach powder HY0110 strain

[0123]

[0124] Dextran sulfate sodium salt solution (DSS): Dextran sulfate sodium salt was used to prepare a 4% (w / v) DSS aqueous solution.

[0125] Preparation of fermentation supernatant: The American cockroach powder fermentation liquid prepared according to the above 5.1 was centrifuged at 4000 rpm and 4°C for 10 min. The supernatant was filtered through a bacterial filter membrane and stored in a refrigerator for later use.

[0126] Analysis of inflammatory factors: During the experiment, their activity status, fecal status, and bloody stool were observed every day. At the end of the experiment (14 days), 3 mice were randomly killed and blood was taken for serum inflammatory factor detection. Fresh blood samples were collected in sterile centrifuge tubes and centrifuged at 3000r / min for 10 minutes to obtain serum. The ELISA kit (Jiangsu Jingmei Biotechnology Co., Ltd.) was used to determine the inflammatory factors (IL-1β, IL-6, IL-8, IL-10 and TNF-α) in the serum, and the operation steps were carried out according to the instructions of the kit.

[0127] Results Analysis

[0128] Observations showed that mice with DSS-induced colitis generally showed symptoms such as mental depression, dull fur, reduced activity, emaciation, and blood in the stool, while the symptoms of the group that was gavaged with the fermentation supernatant of the Enterococcus faecalis HY0110 strain were significantly alleviated. The results of serum inflammatory factor determination showed that the five inflammatory factor indicators of IL-1β, IL-6, IL-8, IL-10 and TNF-α in the Enterococcus faecalis HY0110 strain treatment group were significantly lower than those in the DSS model group (Table 14), indicating that the fermentation supernatant of the American cockroach powder of the Enterococcus faecalis HY0110 strain had a significant alleviating effect on DSS-induced colitis in mice.

[0129] Table 14 Effects of Enterococcus faecalis HY0110 fermentation broth on serum inflammatory factors in mice (pg / ml)

[0130] Inflammatory factors IL-1β IL-6 IL-8 TNF-α IL-10 CK blank control 18.168±1.961 17.260±8.195 8.579±4.239 69.721±12.897 55.029±11.398 DSS Model Group 47.576±10.537 55.687±12.220 32.784±12.504 153.431±48.932 123.966±26.444 HY0110 treatment 39.930±13.324 36.185±7.604 20.795±3.896 104.845±39.929 86.714±17.385

Claims

1. A strain of Enterococcus faecalis ( Enterococcus faecalis )HY0110, deposited on March 28, 2023 in the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with the deposit number CGMCC NO.26946.

2. The Enterococcus faecalis according to claim 1 ( Enterococcus faecalis ) Application of HY0110 in the preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.

3. An anti-colon cancer drug or an anti-colon cancer auxiliary drug, characterized in that: Containing the Enterococcus faecalis according to claim 1 ( Enterococcus faecalis )HY0110.

4. A preparation, characterized in that The preparation contains the Enterococcus faecalis according to claim 1 ( Enterococcus faecalis ) HY0110 is the fermentation liquid obtained by fermenting American cockroach powder.

5. Use of the preparation according to claim 4 in the preparation of anti-colitis drugs.

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