CN118652789B - Enterococcus faecalis HY0110 and application thereof in American cockroach fermentation - Google Patents
Enterococcus faecalis HY0110 and application thereof in American cockroach fermentation Download PDFInfo
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Abstract
The invention discloses enterococcus faecalis (Enterococcus faecalis) HY0110 and application thereof in American cockroach fermentation. The strain is obtained by separating and screening intestinal contents of tuna (Thunnus thynnus) with south China sea blue fins, is free of hemolysis, is sensitive to various common antibiotics, is strong in adaptability to animal gastrointestinal environment, is strong in inhibition capability to various digestive tract pathogenic bacteria, has obvious growth inhibition effect on human colon cancer cells and apoptosis promotion effect, is used for fermenting American cockroach powder, the content of various bioactive substances such as amino acid, peptides, astragaloside, eicosanoids and the like in supernatant is obviously increased, has good symptom relieving effect on a DSS (digital subscriber line) mouse colonitis model, is reduced in various inflammatory factor levels, and opens up a new way for deep development of American cockroaches.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to enterococcus faecalis with strong antibacterial and anti-inflammatory effects, and particularly relates to the field of functional lactobacillus and product production and development thereof.
Background
The American cockroach PERIPLANETA AMERICANA is commonly called cockroach and has a long medicinal history in China. Clinical researches show that the American cockroach extract for artificial feeding has various pharmacological activities of resisting tumor, protecting liver, promoting tissue repair, enhancing immunity, protecting cardiovascular and the like, and the American cockroach extract is dried into powder, has the effects of resisting tumor, resisting inflammation, easing pain, promoting tissue repair and the like, so far, various traditional Chinese medicines such as Kangfu liquid and the like are developed by taking the American cockroach as materials and are applied to clinic. We derived from wild blue fin tuna
(Thunnus thynnus) separating and screening an enterococcus faecalis (Enterococcus faecalis) HY0110 strain in intestinal tracts, and on the basis of evaluation of probiotics, the strain is used for fermentation of the traditional Chinese medicinal material periplaneta americana, and is expected to further expand the application of the periplaneta americana in biopharmaceutical.
Disclosure of Invention
The invention aims to solve the technical problem of providing the enterococcus faecalis with strong antibacterial and anticancer effects, and the enterococcus faecalis is applied to the fermentation pharmacy of periplaneta americana.
The invention has the technical scheme that enterococcus faecalis (Enterococcus faecalis) HY0110 is preserved in China general microbiological culture Collection center (CGMCC) at the address of North Star Xway No. 1, 3 in the Korean region of Beijing, and the preservation number is CGMCC No.26946.
The enterococcus faecalis (Enterococcus faecalis) HY0110 can be used for inhibiting Escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa or/and campylobacter jejuni for the purpose of non-disease treatment.
The application of the enterococcus faecalis (Enterococcus faecalis) HY0110 in preparing medicines for treating diseases caused by escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa or/and campylobacter jejuni is provided.
The enterococcus faecalis (Enterococcus faecalis) HY0110 is applied to preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.
An anti-colon cancer drug or anti-colon cancer auxiliary drug comprises the enterococcus faecalis (Enterococcus faecalis) HY0110.
A preparation contains a fermentation liquid obtained by fermenting American cockroach powder by enterococcus faecalis (Enterococcus faecalis) HY 0110.
The preparation is applied to preparing the anti-colonitis medicine.
The enterococcus faecalis HY0110 is obtained by separating and screening the intestinal tracts of tuna in blue fins collected from south China sea. Enterococcus faecalis HY0110 grows well on MRS agar culture medium, and the colony is milky white, smooth in surface, neat in edge, opaque and gram-positive. The target gene sequence consisting of 1000 base pairs (bp) is obtained by adopting the 27F/1492R of the bacterial universal primer 16S rRNA as a template for PCR amplification, and the sequence is shown as SEQ ID No. 1. The gene sequences obtained by sequencing are input into NCBI database for comparison, the similarity rate of the gene sequences with the strain Enterococcus FAECALIS STRAIN CAU:272 in GenBank reaches 99.90%, and the strain can be initially identified as Enterococcus faecalis (Enterococcus faecalis).
The enterococcus faecalis HY0110 disclosed by the invention has a certain tolerance to acid and bile salts and has a strong adaptability to human gastrointestinal environment through an acid-resistant and bile salt-resistant test.
The enterococcus faecalis HY0110 has strong inhibitory activity on various pathogenic bacteria in intestinal tracts, and can inhibit the growth of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa and campylobacter jejuni.
The fermentation supernatant of the enterococcus faecalis HY0110 strain has obvious effects of inhibiting growth and promoting apoptosis of human colon cancer cells;
after the enterococcus faecalis HY0110 strain ferments the American cockroach powder, the content of various active substances in the supernatant is obviously increased.
Animal experiments show that the enterococcus faecalis HY0110 strain of the invention ferments the American cockroach powder, and the supernatant has good prevention effect on a DSS mouse colonitis model, can relieve colonitis symptoms and reduce the level of various inflammatory factors.
Compared with the prior art, the invention has the following beneficial effects:
1. The enterococcus faecalis HY0110 disclosed by the invention has a certain tolerance to acid and bile salts and has a strong adaptability to human gastrointestinal environment through an acid-resistant and bile salt-resistant test.
2. The enterococcus faecalis HY0110 has strong inhibitory activity on various pathogenic bacteria in intestinal tracts, and can inhibit the growth of escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa and campylobacter jejuni.
3. The fermentation supernatant of the enterococcus faecalis HY0110 strain has obvious effects of inhibiting growth and promoting apoptosis of human colon cancer cells.
4. After the enterococcus faecalis HY0110 strain ferments the American cockroach powder, the content of various active substances in the supernatant is obviously increased.
5. Animal experiments show that the enterococcus faecalis HY0110 strain of the invention ferments the American cockroach powder, and the supernatant has good prevention effect on a DSS mouse colonitis model, can relieve colonitis symptoms and reduce the level of various inflammatory factors.
Preservation information:
Enterococcus faecalis (Enterococcus faecalis) HY0110 is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at the date of 28 of 3.2023, and has the preservation number of CGMCC No.26946 at the institute of microbiology, china academy of sciences, north Star, beijing, kogyo.
Drawings
FIG. 1 phylogenetic relationship of enterococcus faecalis HY0110 with other strains;
FIG. 2 effect of enterococcus faecalis HY0110 strain fermentation supernatant on colon cancer cell colony growth;
FIG. 3 effect of enterococcus faecalis HY0110 strain fermentation supernatant on colon cancer cell active oxygen levels;
FIG. 4 effect of enterococcus faecalis HY0110 strain fermentation supernatant on colon cancer cell apoptosis.
Detailed Description
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from commercial sources.
EXAMPLE 1 isolation, identification and safety evaluation of enterococcus faecalis HY0110
1.1 Isolation and identification of enterococcus faecalis HY0110
The intestinal tract sample of the blue-fin tuna is taken from south China sea, a small amount of the blue-fin tuna sample is taken in 50mL MRS broth, fully and uniformly mixed in an oscillating way, and the mixture is placed in a shaking table for culturing for 24 hours at a constant temperature of 37 ℃. 10-fold gradient dilution was used, and single colonies were picked after plating on MRS agar medium, culturing at 37℃for 24 hours, and continuously purified 3 times. The purified strain was inoculated into 600. Mu.L of MRS broth medium, shake-cultured at 37℃for 18 hours, 400. Mu.L of 50% (V/V) sterile glycerol was added, and frozen in an ultra-low temperature refrigerator at-80℃for use.
After the frozen strain is activated and cultured, the strain DNA is extracted by using an MRS broth culture medium, and the amplification of the 16S rRNA is completed by adopting a colony PCR technology, wherein the PCR primers are 16S rRNA universal primers 27F and 1492R, and the sequences are as follows:
27F:5-AGAGTTTGATCMTGGCTCAG-3,1492R:5-GGTTACCTTGTTACGACTT-3
Sequencing of the PCR products was performed by biological engineering (Shanghai) Inc., and the 16S rRNA sequence is shown in SEQ ID No. 1. The sequence has a similarity of 99.90% with Enterococcus FAECALIS STRAIN CAU:272 strain in GenBank, can be initially identified as Enterococcus faecalis, and is named as Enterococcus faecalis (Enterococcus faecalis) HY0110. The phylogenetic relationship between the strain and other strains is shown in figure 1.
The strain grows well on MRS agar culture medium, and the colony forms are milky white, round convex, flat in edge and smooth in surface, and are spherical in microscopic examination and positive in gram staining.
The strain is preserved in China general microbiological culture Collection center (CGMCC) at the position of North Star Xly way No. 1, 3 in the Korean region of Beijing at the 28 th month of 2023, and the preservation number is CGMCC NO.26946.
1.2 Haemolytic and antibiotic resistance of enterococcus faecalis HY 0110:
the sensitivity of the strain to common antibiotics was tested by the paper sheet agar diffusion method. Enterococcus faecalis HY0110 strain is activated and cultured, the concentration of bacterial liquid is regulated to 1X 10 6 CFU/mL, bacterial liquid is uniformly smeared on the surface of an MRS culture medium flat plate by using a sterile cotton swab, drug sensitive paper sheets are placed after room temperature is carried out for 10min, after the culture is carried out for 24h at 37 ℃, the diameter of a bacteriostasis ring around each drug sensitive paper sheet is measured by using a vernier caliper, each antibiotic is repeated for 3 times, and the test result refers to the American clinical laboratory standards committee (NCCLS) standard to judge the drug sensitivity of the strain, and the results are expressed as sensitivity (S), intermediation (I) and drug resistance (R).
The results of the hemolysis experiments showed that no hemolysis occurred around the colonies (FIG. 3), and that the test 6 antibiotics, namely tetracycline, ampicillin, ceftriaxone, clindamycin, clarithromycin and chloramphenicol, were all sensitive (S), indicating the safety of the enterococcus faecalis HY0110 strain (Table 1).
TABLE 1 sensitivity of enterococcus faecalis HY0110 Strain to 6 antibiotics
| Numbering device | Tetracycline | Ampicillin (Amoxicillin) | Ceftriaxone | Clindamycin | Clarithromycin | Chloramphenicol |
| HY0110 | S | S | S | S | S | S |
Example 2 adaptation of enterococcus faecalis HY0110 to the gastrointestinal Environment of animals
2.1, Evaluating acid resistance and cholate resistance, namely resuscitating and activating enterococcus faecalis HY0110 to be tested on an MRS agar plate for 3 generations, and regulating the initial concentration of bacterial liquid to be 1X 10 6 CFU/mL. MRS broth medium of acidity (ph=3.0) and bile salt concentration (0.3%) was prepared using hydrochloric acid and porcine bile salt, respectively. 1mL of the test strain enterococcus faecalis HY0110 bacterial solution is inoculated into a broth culture medium with pH=3.0, and is cultured for 18h at 37 ℃. After the completion of the culture, 20. Mu.L of the bacterial liquid was spread on MRS agar plate medium, and 3 replicates were set for culture at 37℃for 18 hours. After incubation, the MRS plate surface was observed for colony growth. Similarly, 1mL of the test strain enterococcus faecalis HY0110 bacterial liquid is inoculated into MRS broth culture medium with a bile salt concentration of 0.3%, and after being cultured for 18 hours at 37 ℃, the culture medium is plated and cultured for 24 hours at 37 ℃, and the colony growth condition is checked.
The test result shows that the enterococcus faecalis HY0110 can still grow normal colonies on an MRS agar plate after being treated in MRS broth culture medium with pH=3.0 for 18 hours or being tolerant in MRS broth culture medium with the concentration of bile salts of 0.3%, which indicates that the enterococcus faecalis HY0110 strain has certain acid resistance and bile salt resistance.
2.2 Simulated gastric fluid and intestinal fluid tolerance experiments simulated gastric fluid and intestinal fluid were purchased from Shanghai Yuan Yeast Biotechnology Co. The artificial gastric juice simulated liquid comprises dilute hydrochloric acid, pepsin and sodium chloride, the final pH is 2.5, and the artificial intestinal juice simulated liquid comprises potassium dihydrogen phosphate and trypsin, and the final pH is 6.8. Recovering and activating enterococcus faecalis HY0110 strain, regulating the concentration of bacterial liquid to 1X 10 8 CFU/mL, adding 1mL of bacterial liquid into 9mL of simulated gastric fluid, carrying out 10-time gradient dilution, absorbing 20 mu L of viable bacteria count of a plating plate to be used as an initial viable bacteria value of the tolerant gastric fluid, culturing the inoculated simulated gastric fluid at 37 ℃ for 3 hours, and then coating the viable bacteria count of the plating plate again to be used as an end viable bacteria value of the tolerant gastric fluid. Similarly, 1mL of the enterococcus faecalis HY0110 fermentation broth with the concentration of 1X 10 8 CFU/mL is added into 9mL of simulated intestinal fluid, viable count is carried out, the viable count is counted again after culture for 6 hours at 37 ℃, and the survival rate is calculated. Survival = number of viable bacteria at end/number of initial viable bacteria 100%.
The results show that the enterococcus faecalis HY0110 strain has better tolerance to artificial simulated stomach and intestinal juice, the survival rate of the enterococcus faecalis HY0110 strain in the artificial gastric juice after 3 hours is 69.4%, and the survival rate of the enterococcus faecalis HY0110 strain after 6 hours is 94.9% (Table 2).
TABLE 2 tolerance of enterococcus faecalis HY0110 Strain to artificial simulated gastric and intestinal fluids (%)
Example 3 acid-producing Property and bacteriostatic action of enterococcus faecalis HY0110 Strain
3.1, Determining the content of short-chain fatty acid in the fermentation liquid of enterococcus faecalis HY0110
And (3) preparing a fermentation broth, namely performing activation culture on the enterococcus faecalis HY0110 preserved strain for 24 hours, sucking 4ul of bacterial liquid, adding the bacterial liquid into 4mL of broth culture medium, and culturing at 37 ℃ for 24 hours for later use.
Detection of short chain fatty acids the detection instrument was a gas chromatograph-mass spectrometer (GCMS-QP 2010 Plus) from Shimadzu corporation, and the chromatographic column was a capillary column (30 m.times.0.25 mm.times.0.25 um) of Rtx-5 fused silica from RESTEK (Ruis Tex) corporation, U.S.A. The GC temperature program was held at an initial temperature of 40 ℃ for 5min, 5 ℃ per minute to 150 ℃, 10 ℃ per minute to 280 ℃, and 2min. The carrier gas was high purity helium (purity > 99.999%) with a flow rate of 1.0mL/min. MS conditions are that ionization mode is EI, temperature is 200 ℃, interface temperature is 220 ℃, and mass scanning range is m/z 33-500. Taking 4mL of fermentation liquor, adding 10ul of 2-ethylbutyric acid internal standard solution with the concentration of 200ug/mL, sampling 1 mu L of sample in a mode of 1:3 of a split flow mode, setting the solvent delay time to 0.1min, and setting the temperature of a sample inlet to 270 ℃. The concentration of 5 short chain fatty acids (acetic acid, n-butyric acid, isobutyric acid, isovaleric acid, isocaproic acid) was calculated using the internal standard method.
The GC-MS detection result shows that the acetic acid content in the enterococcus faecalis HY0110 fermentation broth is higher, is 16.505ug/mL, and is higher than that of the LGG standard strain. In addition, three short chain fatty acids, isobutyric acid, 2-methylbutyric acid, isovaleric acid (Table 3) were contained. Recent studies have found that short chain fatty acids have an important role in maintaining normal function of the large intestine and morphology and function of colonic epithelial cells.
TABLE 3 enterococcus faecalis HY0110 fermentation broth short chain fatty acid content (ug/mL)
| Strain number | Strain name | Acetic acid | Isobutyric acid | Isopentanoic acid | 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 |
3.2 Evaluation of bacteriostatic Activity of enterococcus faecalis HY0110 Strain
Escherichia coli (ESCHERICHIA COLI CMCCB 44102), staphylococcus aureus (Staphylococcus aureus CMCCB 50094), salmonella typhimurium (Salmonella typhimurium ATCC 14028), pseudomonas aeruginosa (Pseudomonas aeruginosa CMCCB 10104) and Campylobacter jejuni (Campylobacter jejuni ATCC 33291) were inoculated into nutrient agar medium, resuscitated and activated 3 times, respectively. Sucking a proper amount of trypticase soy peptone liquid culture medium into a centrifuge tube, inoculating the activated pathogenic bacteria into the broth culture medium, and regulating the concentration of the bacterial liquid to be 1X 10 8 CFU/mL. 1mL of the mixture of the pathogenic bacteria and the broth is sucked up and added into 500mL of nutrient agar culture medium which is not solidified temporarily after sterilization (the temperature is cooled to about 40 ℃), and the mixture is fully mixed and split-packed into culture dishes according to the amount of 20mL per dish. After the culture medium is cooled and solidified, a puncher with the diameter of 6mm is used for punching holes on a flat plate, so that a pathogenic bacteria agar plate is manufactured, each plate corresponds to one strain of bacteria, and three holes are formed as repetition. Resuscitating and activating the strain to be detected, and regulating the concentration of the cultured bacterial liquid to be 1X 10 8 CFU/mL. And (5) sucking 50 mu L of bacteria liquid to be detected, adding the bacteria liquid to the hole of the pathogenic bacteria agar plate, and culturing for 24 hours at 37 ℃. After incubation, the diameter of the zone of inhibition around the perforation point was measured using a vernier caliper and recorded. The above experimental procedure was performed simultaneously with the strain to be tested using the standard strain LGG as a control strain.
The results show that the fermentation broth of the enterococcus faecalis HY0110 strain has better inhibitory activity on the growth of pathogenic bacteria such as escherichia coli, staphylococcus aureus, salmonella typhimurium, pseudomonas aeruginosa, campylobacter jejuni and the like, and is close to or better than the antibacterial effect of the LGG standard strain (table 4).
TABLE 4 evaluation of bacteriostatic Activity of enterococcus faecalis HY0110 Strain (diameter: mm)
Example 4 Effect of enterococcus faecalis HY0110 on colon cancer cells
4.1 Cell adhesion test enterococcus faecalis HY0110 is resuscitated and inoculated into MRS broth, and cultured at 37 ℃ for 24 hours. After the cultivation, the mixture is centrifuged for 10min at-4 ℃ and 5000r/min, and washed with sterile PBS buffer solution for a plurality of times. The bacterial suspension concentration is adjusted to 1X 10 6 CFU/mL for later use. Human colon cancer cells HT-29 were resuscitated, inoculated into six well cell culture dishes, DMEM complete medium was added and incubated in 37℃5% CO 2, with medium changed once for two days. When the cell attachment state reached 80%, digestion was performed using 0.25% pancreatin-EDTA, and subcultured. After the completion of the culture, the cells were counted by a cell counting plate, and the cell concentration was adjusted to 5X 10 6 cells/mL. 1mL of the cell suspension was added to one of the culture wells of a six-well cell culture dish and placed in an incubator for culture. Cells in the plates were grown to a monolayer, DMEM medium was discarded and each well was rinsed 3 times with sterile PBS. 1mL of the prepared bacterial suspension is added into a cell hole, the cell culture plate is slightly shaken, a small amount of bacterial liquid in the hole is sucked for plate counting, and the result is taken as the initial viable bacterial count in the bacterial suspension. The cell plates were incubated at 37 ℃ for 2h, medium was discarded and washed 3 times with sterile PBS buffer. The cells were digested with 0.7mL of 0.25% trypsin-EDTA for 10min, and after the cells were completely detached, the digestion was terminated by adding 0.3mL of DMEM culture solution, and the culture solution after the end of the adhesion experiment was collected for plate counting, and the result was used as the number of adhesion viable bacteria. And the standard strain LGG was used as a control.
Adhesion (%) = number of lactic acid bacteria at end period/number of initial lactic acid bacteria inoculation x 100%
The results are shown in Table 5. The average adhesion rate of the enterococcus faecalis HY0110 to human colon cancer cells HT-29 is 61.48 percent, which is higher than the cell adhesion rate of a standard strain LGG.
TABLE 5 adhesion Rate of enterococcus faecalis HY0110 to human colon cancer cells HT-29
| Strain | Repeat 1 | Repeat 2 | Repeat 3 | Mean value of |
| Enterococcus faecalis HY0110 | 61.59% | 61.03% | 61.83% | 61.48% |
| Standard Strain LGG | 56.32% | 54.23% | 56.58% | 55.71% |
4.2 Growth inhibition of colon cancer cells by enterococcus faecalis HY0110 fermentation supernatant
Enterococcus faecalis HY0110 fermentation supernatant is prepared by resuscitating enterococcus faecalis HY0110, inoculating into MRS broth culture medium, and culturing at 37deg.C for 24 hr to obtain bacterial suspension with concentration of 1×10 9 CFU/mL. Centrifuging at-4deg.C and 5000r/min for 10min after culturing, collecting fermentation supernatant, and filtering with bacterial filter membrane.
Cancer cell resuscitation-pre-warmed DMEM medium containing 10% fbs and 1% diabody in 15mL centrifuge tubes was placed on a sterile operating table and frozen human colon cancer cells were removed from a-80 ℃ refrigerator or liquid nitrogen and quickly thawed in a 37 ℃ water bath. After thawing the cells, they were transferred on a sterile operating table to a prepared 15mL centrifuge tube containing the medium, centrifuged at 1000r/min for 3min to resuspend the cells, aspirated into a 6cm cell culture dish, and after incubation for 24h at 37 ℃ in a 5% CO 2 incubator, the fresh medium was replaced.
And (3) carrying out cell passage, namely carrying out passage when the growth state of the cells is good and the density reaches about 80%. After the medium and sterile PBS were warmed up in a 37 ℃ water bath, old medium in the cell culture dish was aspirated off on a sterile operating table, cells were washed with warmed up PBS to remove dead cells and residual medium, cells were digested with 0.25% pancreatin, pancreatin was aspirated off with a pipette after the cells became round, the digestion was stopped, and cells were collected in a sterile centrifuge tube. The supernatant was aspirated off by centrifugation at 1000r/min for 3min, and the cells were resuspended in fresh medium and then aspirated into fresh cell culture dishes and placed in a cell incubator for culture.
The cytotoxicity experiment is to measure the effect of enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell growth by using a CCK-8 kit (Cell Counting Kit-8 cell counting reagent), wherein the kit uses a tetrazolium salt WST-8 with high water solubility, and under the condition of the existence of an electron coupling reagent, the tetrazolium salt WST-8 can be reduced by some dehydrogenases in mitochondria to generate yellow formazan, the amount of formazan generated by the dehydrogenases and the number of living cells are in a direct linear relation, so that the more and faster the cancer cells proliferate, the darker the color, the more the cytotoxicity on the cancer cells, the lighter the color, the high method sensitivity and simple and convenient operation.
The measurement result shows that 4% enterococcus faecalis HY0110 fermentation supernatant has obvious growth inhibition effect on the growth of human colon cancer cells, the colon cancer cell density is obviously reduced compared with a control after 24 hours and 48 hours of culture, and the growth inhibition rates are respectively 39.12% and 35.39%, which indicates that the enterococcus faecalis HY0110 fermentation supernatant has obvious inhibition effect on the growth of human colon cancer cells (Table 6).
TABLE 6 influence of HY0110 fermentation supernatant on colon cancer cell growth (absorbance)
| 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 |
Meanwhile, the ability of enterococcus faecalis HY0110 fermentation supernatant to form human colon cancer cell populations was measured. Colon cancer cells were digested, centrifuged, resuspended, counted according to the step of cell passaging, and plated in 6-well plates at a density of 2000 cells per well. The plates with the cells spread are placed in a cell incubator for 20 days, fresh medium is changed every two days, and 1% enterococcus faecalis HY0110 fermentation supernatant is added. After the cells form macroscopic clones, the culture medium is discarded, PBS is used for washing twice, 1% crystal violet is added for dyeing for 30min, the culture plate is washed for 3-5 times by clean water, residual crystal violet dye solution is washed, the culture plate is dried, the cell population number is recorded, and the experimental result is recorded by photographing. The results showed that after 1% enterococcus faecalis HY0110 fermentation supernatant was added to the culture medium, the cell population number was 1283 on average, and the control group was 1593, which was only 80.54% of the control group number, indicating that enterococcus faecalis HY0110 fermentation supernatant was able to significantly reduce the colony forming ability of colon cancer cells (Table 7, FIG. 2).
TABLE 7 influence of enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell colony Forming Capacity
| Control | 1% HY0110 fermentation supernatant | |
| Cell population number (number) | 1593±374 | 1283±207 |
4.3 Pro-apoptotic effects of enterococcus faecalis HY0110 fermentation supernatant on human colon cancer cells
4.3.1 Materials and methods
Cell culture, namely, after digestion and centrifugation of HT-29 cells in logarithmic growth phase, uniformly spreading the HT-29 cells into a 96-well plate, and counting to ensure 2000 cells per well. After 12 hours of culture, 4% (V/V) enterococcus faecalis HY0110 strain fermentation supernatant is added after cell attachment is stable, 5 repetitions are set for each treatment, and protein is extracted after 48 hours of culture.
Protein extraction, namely washing the cultured cells twice by PBS, and sucking all residual PBS on the cell surface as much as possible. Cells were placed in an ice bath, RIPA lysate containing the protease inhibitor Cocktail (protease inhibitor diluted 1:100) was added, and the whole cells were scraped into a 1.5ml sterile centrifuge tube and placed in an ice bath for 30min. Centrifuging at 12000r/min and 4 ℃ for 10min, and collecting supernatant into a new 1.5ml sterile centrifuge tube to obtain the extracted protein sample. The protein sample can be directly subjected to protein quantitative analysis or stored in a refrigerator at-80 ℃ for later use. For the protein sample after quantitative analysis, protein buffer can be added, and the protein sample is placed in a metal bath for boiling at 98 ℃ for 10min to denature the protein, and is stored in a refrigerator at-20 ℃ for standby after instantaneous centrifugation.
And (3) protein quantification, namely carrying out quantitative analysis by using a Biyundin protein quantification kit. Protein samples were diluted 20-fold and added to labeled 96-well plates. BCA working solution is prepared according to the ratio of BCA reagent A to BCA reagent B of 50:1, fully and uniformly mixed, 200 μl of BCA working solution is added into each hole, and then uniformly mixed for 30s by shaking, each sample hole is covered, and the mixture is placed in a constant temperature incubator at 37 ℃ for 30min. The absorbance at 562nm was measured, the protein concentration of each sample was read out in a linear range of the standard curve based on the corrected absorbance for each protein sample, and the amount of protein in the original sample was calculated based on the sample volume and dilution.
4.3.2 Experimental results
(1) Effect of enterococcus faecalis HY0110 fermentation supernatant on cell adhesion factor beta-catenin
Cancer cell spread is characterized by disordered interactions between cells and cell adhesion. Beta-catenin (beta-catenin) is a multifunctional protein, is an important cell adhesion molecule, participates in cell growth and repair, and plays an important role in the processes of tumorigenesis and metastasis. The postoperative recurrence rate of colorectal cancer is high, the survival time of patients is short, and mutations of Wnt/beta-catenin signal channels in almost all colorectal cancers finally lead to accumulation of beta-catenin. Therefore, the content of β -catenin can be used as an auxiliary factor for clinically judging the development and prognosis of colorectal cancer, and nuclear accumulation of β -catenin can be a malignant-related marker for invasion, metastasis, poor prognosis and the like of colorectal cancer. Our experimental results show that the content of beta-catenin (beta-catenin) is greatly reduced after 48 hours of culture by adding enterococcus faecalis HY0110 fermentation supernatant into colon cancer cell culture solution, which is only 37.24% of that of a control without fermentation solution. Our results demonstrate that enterococcus faecalis HY0110 fermentation supernatant can reduce the expression of cell adhesion factor beta-catenin, thereby reducing the adhesion and diffusion of cancer cells (Table 8).
TABLE 8 influence of enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell beta-catenin content (absorbance)
| Protein of interest/reference protein | Control | HY0110 |
| Beta-catenin (beta-catenin) | 37781 | 14071 |
(2) Effect of enterococcus faecalis HY0110 fermentation supernatant on apoptosis inhibitor gene protein content
The B cell lymphoma/leukemia-2 gene (bcl-2) also has obvious effect of inhibiting apoptosis, can inhibit cell death caused by various cytotoxins, is closely related to various cancers such as colon cancer, ovarian cancer and the like, and can enhance the resistance of cells to most cytotoxins through the overexpression of the gene. Our experiments show that after enterococcus faecalis HY0110 is added into the cell culture medium to ferment supernatant, the total amount of colon cancer cell Bcl-2 protein is 24.14% of that of the control (Table 9), which shows that the ferment supernatant has the activity of promoting cancer cell apoptosis.
TABLE 9 Effect of enterococcus faecalis HY0110 fermentation supernatant on protein content of apoptosis inhibitor gene of colon cancer cells
| Protein of interest/reference protein | Control | HY0110 |
| B cell lymphoma/leukemia-2 (BCL-2) | 35004 | 8449 |
(3) Effect of enterococcus faecalis HY0110 fermentation supernatant on apoptosis factor protein content
The P62 protein is an oncogene protein, and a target gene for transcriptional regulation is involved in the processes of regulation of cell cycle, autophagy, cell proliferation, apoptosis, immortalization and the like, and plays an important role in the occurrence of a plurality of tumors. The P62 gene can help the human body to better recognize and resist exogenous antigens, thereby reducing the harm caused by infection. The P62 protein can also exert a cancer suppressing effect by promoting selective autophagy to prevent the accumulation of genotoxic and oncogenic mutations. When the P62 gene binds to an antigen, it triggers antigen-specific T cell and B cell functions. T cells secrete specific Cytokine to kill foreign pathogens, while B cells secrete specific antibodies to inhibit pathogen replication. Thus, the P62 gene can bind to an antigen, promote an immune response, and help the human body resist the foreign antigen, thereby reducing injury 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 by about 1.47 times of that of a control (Table 9), which shows the pro-apoptosis activity of enterococcus faecalis HY0110 fermentation supernatant.
Poly ADP-ribose polymerase, a poly a-p polymerase, is a DNA repair enzyme, and is a cleavage substrate for caspases (caspases) that are the core members of apoptosis, and thus it plays an important role in DNA damage repair and apoptosis. PARP cleavage is considered an important indicator of apoptosis and is also commonly considered an indicator of Caspase 3 activation. PARP plays an important role in colon cancer growth and metastasis. Our results show that after adding enterococcus faecalis HY0110 fermentation supernatant to the culture medium, the total amount of poly (adenosine diphosphate) ribose polymerase C-Parp of colon cancer cells is 5.57 times that of the control (Table 10), which shows the pro-apoptotic activity of enterococcus faecalis HY0110 fermentation supernatant.
TABLE 10 influence of enterococcus faecalis HY0110 fermentation supernatant on colon cancer cell pro-apoptosis factor protein (gray value)
| Protein of interest/reference protein | Control | HY0110 |
| P62 | 21348 | 31317 |
| Poly (apyrase) C-Parp | 7701 | 42882 |
(4) Effect of enterococcus faecalis HY0110 fermentation supernatant on human colon cancer cell Reactive Oxygen Species (ROS) levels
Cell collection, namely sucking off colon cancer cell culture solution which is subjected to adherence culture, repeatedly blowing by using serum-free culture solution, visually observing the bottom (bottle bottom) of the pore plate, converting the semitransparent (cell monolayer is connected into a sheet) into the transparent, almost completely blowing the cell layer into the culture solution, completely collecting the cell suspension into a 1.5ml centrifuge tube, washing 2 times by using the serum-free culture solution, 1000r/min, centrifuging for 5min, sucking the supernatant, and reserving cell sediment for measurement.
The probe was loaded by diluting DCFH-DA with serum-free medium at a ratio of 1:1000 to a final concentration of 10. Mu. Mol/l. After cell collection, the cells were suspended in diluted DCFH-DA at a cell concentration of one to two million/ml and incubated in a 37℃cell incubator for 20 minutes. The mixture was inverted and mixed every 3-5 minutes to allow the probe and the cells to be in sufficient contact. The cells were washed three times with serum-free cell culture medium to remove sufficiently DCFH-DA that did not enter the cells, and cells were stimulated with enterococcus faecalis HY0110 fermentation supernatant. Fluorescence intensity before and after stimulation was detected in real time at 488nm excitation wavelength and 525nm emission wavelength using a fluorescence spectrophotometer, and photographed.
Reactive oxygen species (Reactive Oxygen Species, ROS) levels reflect the status of cellular metabolites. Normally, ROS production and clearance are in dynamic equilibrium, and if ROS levels exceed normal ranges, oxidative damage or cell death may result. Colon cancer cells treated with enterococcus faecalis HY0110 fermentation supernatant had significantly higher fluorescence intensity than control, and the number of fluorescently labeled cells was also greater, with ROS levels significantly higher than control cells (table 11, fig. 3).
TABLE 11 cell number of ROS-expressing cells after treatment of enterococcus faecalis HY0110 fermentation supernatant
(5) One-step TUNEL apoptosis assay
When cells undergo apoptosis, some DNA endonucleases are activated, which cleave the genomic DNA between nucleosomes. The apoptosis can be detected by fluorescence microscopy by adding a fluorescent probe dUTP after catalysis of terminal deoxynucleotidyl transferase (Terminal Deoxynucleotidyl Transferase, tdT). The specific operation steps are carried out according to the instructions of the one-step TUNEL apoptosis detection kit. The detection result shows 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 intensity of red fluorescence of the treatment group is 287+/-42, and the average intensity of red fluorescence of the control group is only 38+/-4 (FIG. 4).
In conclusion, the enterococcus faecalis HY0110 strain has good adhesion to colon cancer cells, the fermentation supernatant has obvious effect of inhibiting the growth of colon cancer cells, the content of beta-catenin related to adhesion diffusion can be reduced, the content of Bcl-2 protein representing apoptosis inhibition genes can be reduced, the content of pro-apoptosis factors poly (adenosine diphosphate) -ribose polymerase Cleaved-parp and P62 protein representing autophagy can be increased, and the effects of the enterococcus faecalis HY0110 on the growth inhibition of colon cancer cells and the effect of promoting apoptosis are demonstrated in multiple aspects.
Example 5 application of enterococcus faecalis HY0110 strain in fermentation of American cockroach powder
5.1 Metabolic analysis of the fermentation of American cockroach powder by enterococcus faecalis HY0110 Strain
The fermentation method comprises preparing a culture medium from 10 g of American cockroach powder, 3 g of fructo-oligosaccharide and 100mL of water, sterilizing, cooling, inoculating enterococcus faecalis HY0110, and culturing at 37 ℃ for 96 hours. The fermentation broth was centrifuged at 4000rpm at 4 ℃ for 10min, and the supernatant was filtered through a bacterial filter membrane and sent to the company for liquid chromatography-mass spectrometry (LC-MS) non-targeted metabonomics analysis. The experiment was repeated three times, without probiotic inoculation as control, and the culture and analysis method was the same as the treatment group.
The analysis method is based on a high-resolution mass spectrum (HRMS) detection technology, and is combined with a high-quality mzCloud database built by a standard substance to match mzVault and MassList databases, so that the molecular characteristic peaks are identified in a matching way, and the original data of the machine is subjected to data preprocessing by using CD3.3 data processing software. Firstly, parameters such as retention time, mass-to-charge ratio and the like are simply screened, and meanwhile, the peak area is quantified. And then, the high-resolution secondary spectrogram databases mzCloud and mzVault and the MassList primary database are compared for searching (searching) to identify the metabolites. The chromatographic peaks detected in the samples were integrated using CD3.3 data processing software, wherein the peak area of each characteristic peak represents a relative quantitative value of the metabolite, the quantitative result was normalized using the total peak area, and finally the quantitative result of the metabolite was obtained, the result being expressed as a multiple of the increase in the content of the corresponding component in the sample compared to the control.
Analysis results show that after the American cockroach powder is fermented for 96 hours by enterococcus faecalis HY0110, the content of various amino acid substances in supernatant is obviously increased, and particularly the content of N-arachidonic acid-L-serine, succinyl-homoserine, valine and homocysteine is increased by more than 10 times compared with a control without fermentation (Table 12). Amino acids are the basic constituent units 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, maintenance of acid-base balance, etc.
Table 12 increase in amino acid and peptide content of American cockroach powder fermented by HY0110 strain
Many active substances of the human body exist in the form of peptides. Peptides are involved in the hormone, nerve, cell growth and reproductive fields of the human body and are important in regulating physiological functions of various systems and cells in the body, activating related enzymes in the body, promoting permeability of the intermediary metabolic membrane, or ultimately producing specific physiological effects by controlling DNA transcription or affecting specific protein synthesis. Analysis results show that after the American cockroach powder is fermented for 96 hours by enterococcus faecalis HY0110, the content of various peptide substances in supernatant is obviously increased, and particularly, the content of polypeptide (L-Leucyl-L-Alanine) consisting of L-leucine and L-alanine is increased by 16.89 times compared with a control without fermentation, and the content of S-lactoyl glutathione (S-lactoylglutathione) is also increased by 7.95 times (Table 12).
Notably, the content of astragaloside IV (Astragaloside A, also known as cucurbitacin Cucurbitacin glycosides) in the fermentation supernatant increased by up to 668.73 fold compared to the control without fermentation. Previous studies have shown that this material has a variety of important biological activities. For plants, cucurbitacin is a natural green pesticide, and can resist invasion of plant diseases and insect pests. For human beings, cucurbitacin has extremely high medicinal value, not only can be used for treating various inflammations, but also can be used for protecting liver, resisting tumors and the like. For example, cucurbitacin B has been developed as an anti-hepatoma drug-cucurbitacin tablet, and new formulations of drugs that are developed around cucurbitacin are being developed.
The eicosanoid (Eicosanoids) compound, dimethyl prostaglandin (16, 16-Dimethyl prostaglandin A1), was also significantly increased in the fermentation supernatant, at a level of 116.15 times that of the non-fermented control. The compound has effects on endocrine, reproductive, digestive, blood respiratory, cardiovascular, urinary and nervous systems, and is an active substance with various physiological effects. Has effects in contracting smooth muscle, inhibiting gastric acid secretion, preventing gastric mucosa erosion caused by strong acid, strong alkali, alcohol, etc., and protecting cells. Also has protective effect on small intestine, colon, pancreas, etc. Can also stimulate intestinal secretion, bile secretion, and cholecyst muscle contraction.
5.2 Effect of fermentation supernatant on DSS-induced mouse colitis model
Experimental method
Experimental animals the experimental mice were randomly divided into 6 cages, 3 per cage. The animals were randomized into 3 groups of 2 cages each, including a placebo group (CK), a DSS model group (DSS), and an enterococcus faecalis HY0110 treated group. The experimental treatments are shown in table 13.
Table 13 American cockroach powder HY0110 strain fermentation supernatant versus DSS induced mouse colitis model animal experiment
Dextran sulfate sodium salt solution (DSS) A4% (w/v) aqueous DSS solution was prepared with dextran sulfate sodium salt.
Preparation of fermentation supernatant the American cockroach powder fermentation broth prepared according to the above 5.1 is centrifuged at 4000rpm and 4 ℃ for 10min, and the supernatant is filtered by a bacterial filter membrane and stored in a refrigerator for later use.
Inflammatory factor analysis, during the experimental period, the activity status, the fecal status and the bloody stool phenomenon are observed every day. At the end of the experiment (14 days), 3 mice were sacrificed at random and blood was collected for serum inflammatory factor detection. Fresh blood samples were collected with a sterile centrifuge tube and centrifuged at 3000r/min for 10min to obtain serum. Serum inflammatory factors (IL-1. Beta., IL-6, IL-8, IL-10 and TNF-. Alpha.) were measured using ELISA kit (Jiangsu Jingmei Biotech Co., ltd.) and the procedure was followed according to the kit instructions.
Analysis of results
Observations show that the DSS induced colonitis mice generally have symptoms of listlessness, dark hair color, reduced activity, emaciation, hematochezia and the like, and the symptoms of the fermentation supernatant group of the enterococcus faecalis HY0110 strain are obviously reduced. The serum inflammatory factor measurement results show that the indexes of the IL-1 beta, IL-6, IL-8, IL-10 and TNF-alpha of the enterococcus faecalis HY0110 strain treatment group are obviously lower than those of a DSS model group (Table 14), which shows that the American cockroach powder fermentation supernatant of the enterococcus faecalis HY0110 strain has obvious alleviation effect on DSS-induced mouse colonitis.
TABLE 14 Effect of enterococcus faecalis HY0110 fermentation broth on mouse serum inflammatory factors(pg/ml)
| Inflammatory factor | 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 processing | 39.930±13.324 | 36.185±7.604 | 20.795±3.896 | 104.845±39.929 | 86.714±17.385 |
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