A strain of Lactobacillus paracasei with apoptosis-promoting effect on colon cancer cells and its application

Abstract

The invention discloses a strain of Lactobacillus paracasei with a colon cancer cell growth inhibitory effect and an application thereof. The strain is separated and screened from traditional fermented food in Guizhou, is non-hemolytic, is sensitive to a variety of common antibiotics, has strong adaptability to the gastrointestinal environment of animals, has a strong inhibitory ability to a variety of digestive tract pathogens, has a relieving effect on a colitis model, and its fermentation supernatant can significantly inhibit the growth of colon cancer cells, reduce the colon cancer cell colony formation ability, and promote colon cancer cell apoptosis. The strain is used to ferment broken wall ganoderma lucidum spore powder, and bioactive substances such as some fatty acids, prostaglandins and amino acids are doubled, which opens up a new way for the deep development of ganoderma lucidum spores.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a strain of Lactobacillus paracasei having a strong inhibitory effect on colon cancer cells and multiple intestinal pathogens, and particularly relates to the field of production and development of functional lactic acid bacteria and products thereof. Background Art

[0002] Colorectal cancer is a common malignant tumor of the digestive tract that occurs in the colon. The incidence of colorectal cancer has been gradually increasing in the past two decades. At the same time, the age of onset tends to be aging, and the incidence is second only to gastric cancer and esophageal cancer. Its onset is related to many factors such as dietary habits, genetic factors, and intestinal flora imbalance. In recent years, studies have shown that lactic acid bacteria have a positive effect on the prevention and treatment of colorectal cancer. For example, some lactic acid bacteria can inhibit the growth of harmful bacteria and maintain the stability of the intestinal environment. The metabolites of lactic acid bacteria can inhibit the proliferation and metastasis of colorectal cancer cells, and can also regulate the function of the immune system and improve the body's resistance to colorectal cancer. Although there are many types of lactic acid bacteria, few excellent strains that promote apoptosis of colon cancer cells have been found. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a Lactobacillus paracasei having the effects of inhibiting the growth of colon cancer cells and promoting apoptosis and the application thereof.

[0004] The technical solution of the present invention is:

[0005] A strain of Lactobacillus paracasei GS0805 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 22, 2023, with the deposit number CGMCC No.27394.

[0006] The above-mentioned Lactobacillus paracasei GS0805 is used for inhibiting Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Shigella dysenteriae for non-disease treatment purposes.

[0007] The application of the Lactobacillus paracasei GS0805 described above in the preparation of medicines for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Shigella dysenteriae.

[0008] The use of the above-mentioned Lactobacillus paracasei GS0805 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, comprising the Lactobacillus paracasei GS0805 described in claim 1.

[0010] The application of the above-mentioned Lactobacillus paracasei GS0805 in fermenting Ganoderma lucidum spore powder to increase the content of bioactive substances.

[0011] Furthermore, the bioactive substance is one or more of fatty acids, prostaglandins or amino acids.

[0012] Lactobacillus paracasei GS0805 of the present invention is obtained by separation and screening from fermented food in Guizhou. Lactobacillus paracasei GS0805 grows well on MRS agar medium, and the bacterium colony is milky white, smooth in surface, neat in edge, opaque, and Gram-positive. PCR amplification is carried out with bacterial universal primer 27F / 1492R to obtain the 16SrRNA gene sequence consisting of 1194 base pairs (bp), and the similarity with the standard strain Lactobacillus paracasei strain 46B27 in GenBank reaches 98.32%, and the bacterial strain can be preliminarily identified as Lactobacillus paracasei (Lactobacillus paracasei).

[0013] The Lactobacillus paracasei GS0805 strain of the present invention has good safety, is non-hemolytic, is sensitive to multiple common types of antibiotics, and has strong adaptability to the gastrointestinal environment of animals;

[0014] The Lactobacillus paracasei GS0805 strain of the present invention has strong inhibitory ability against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae, and has an alleviating effect on DSS-induced colitis in mice.

[0015] The fermentation supernatant of the Lactobacillus paracasei GS0805 strain of the present invention can significantly inhibit the growth of colon cancer cells, reduce the colon cancer cell colony-forming ability, and promote the apoptosis of colon cancer cells.

[0016] The Lactobacillus paracasei GS0805 strain of the invention is used for fermenting the broken-wall ganoderma spore powder, and biologically active substances such as some fatty acids, prostaglandins and amino acids are increased by multiples.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The Lactobacillus paracasei GS0805 provided by the present invention is isolated and screened from Guizhou sauerkraut, grows well on MRS agar medium, and has good tolerance to the animal intestinal environment.

[0019] 2. The Lactobacillus paracasei GS0805 provided by the present invention has a strong antibacterial ability against common intestinal pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae, and can alleviate the symptoms of animal colitis.

[0020] 3. The fermentation supernatant of the Lactobacillus paracasei GS0805 strain of the present invention can significantly inhibit the growth of colon cancer cells, reduce the colony-forming ability of colon cancer cells, and promote the apoptosis of colon cancer cells.

[0021] 4. The Lactobacillus paracasei GS0805 strain of the present invention is used to ferment the broken-wall Ganoderma lucidum spore powder, and some biologically active substances such as fatty acids, prostaglandins and amino acids are increased exponentially.

[0022] Collection information:

[0023] Lactobacillus paracasei GS0805 was deposited on May 22, 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. 27394. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a phylogenetic relationship diagram of Lactobacillus paracasei GS0805 of the present invention and other strains;

[0025] Figure 2 The effect of the fermentation supernatant of Lactobacillus paracasei GS0805 of the present invention on the colony-forming ability of colon cancer cells;

[0026] Figure 3 The effect of the fermentation supernatant of Lactobacillus paracasei GS0805 of the present invention on the level of reactive oxygen species (ROS) in colon cancer cells;

[0027] Figure 4 The invention discloses the effect of the fermentation supernatant of Lactobacillus paracasei GS0805 on apoptosis of colon cancer cells. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1 Isolation, Screening and Molecular Biological Identification of Lactobacillus paracasei GS0805

[0030] 1. Material Preparation

[0031] The strains were derived from fermented foods in Guizhou;

[0032] The 16S rDNA universal primers 27F and 1492R were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are as follows:

[0033] 27F: 5-AGAGTTTGATCMTGGCTCAG-3,

[0034] 1492R:5-GGTTACCTTGTTACGACTT-3

[0035] The formula of MRS broth medium (per liter): 10.0 g of casein digest, 10.0 g of beef extract powder, 4.0 g of yeast extract powder, 2.0 g of triammonium citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2.0 g of dipotassium hydrogen phosphate, 20.0 g of glucose, Tween-80, and the final pH is about 5.7.

[0036] The formula of MRS agar medium (per liter): peptone 10.0g, beef extract powder 5.0g, yeast extract powder 4.0g, glucose 20.0g, Tween 80 1.0mL, dipotassium hydrogen phosphate 2.0g, sodium acetate 5.0g, triammonium citrate 2.0g, magnesium sulfate 0.2g, manganese sulfate 0.05g, agar 15.0g, the final pH is about 6.2.

[0037] 2. Specific methods

[0038] Take 2g of sample in 20mL MRS broth, shake and mix thoroughly, and place it in a constant temperature shaker at 37℃ for 24h. Use 10-fold gradient dilution, spread and inoculate on MRS agar medium, pick a single colony after culturing at 37℃ for 24h, and purify the single colony three times in a row. The purified strain was inoculated into 600μL MRS broth medium, cultured in a shaker at 37℃ for 18h, added 400μL of 50% (V / V) sterile glycerol, and frozen in an ultra-low temperature refrigerator at -80℃ for standby use. After the purified strain was expanded with MRS broth, the strain DNA was extracted using the Tiangen Bacteria Genomic DNA Extraction Kit, and the 16S rRNA amplification was completed using colony PCR technology. The PCR product sequencing was completed by Sangon Biotechnology (Shanghai) Co., Ltd. The target gene sequence consisting of 1194 base pairs (bp) was obtained by sequencing, and the sequence is shown in SEQ ID No.1. The sequence had a similarity of 98.32% with that of Lactobacillus paracasei strain 46B27 in NCBI. The strain was preliminarily identified as Lactobacillus paracasei and named GS0805. The phylogenetic relationship between the strain and other strains is shown in the attached Figure 1 Lactobacillus paracasei GS0805 grew well on MRS agar medium. The colony morphology was milky white, with round protrusions, flat edges and smooth surfaces. Microscopic examination showed that the bacteria were rod-shaped and purple, which was consistent with the morphological characteristics of Lactobacillus paracasei.

[0039] The strain was deposited on May 22, 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:27394.

[0040] Example 2 Adaptability and safety of Lactobacillus paracasei GS0805 to the human intestinal environment

[0041] 2.1 Tolerance to simulated gastric and intestinal fluids

[0042] 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 test strain Lactobacillus paracasei GS0805 was revived and activated for 3 generations on MRS agar plates, and the initial concentration of the bacterial solution was adjusted to 1×10 6CFU / mL. 1mL of the test strain Lactobacillus paracasei GS0805 was inoculated into simulated gastric fluid and cultured at 37°C for 3h. After the culture was completed, 20μL of the bacterial solution was spread on the MRS agar plate medium, set up 3 replicates, and cultured at 37°C for 24h. After the culture, observe whether there was colony growth on the surface of the MRS plate. Similarly, 1mL of the test strain Lactobacillus paracasei GS0805 was inoculated into simulated intestinal fluid, cultured at 37°C for 6h, then spread on the plate and cultured at 37°C for 24h to check the colony growth.

[0043] The results of the gastric simulated fluid tolerance test showed that Lactobacillus paracasei GS0805 could still grow normal colonies on MRS agar plates after being treated in simulated gastric fluid for 3 hours; the results of the intestinal simulated fluid tolerance test showed that Lactobacillus paracasei GS0805 could still grow normal colonies on MRS agar plates after being treated in simulated intestinal fluid for 6 hours, indicating that Lactobacillus paracasei GS0805 has strong acid and bile salt resistance.

[0044] 2.2 Hemolytic activity and antibiotic resistance of Lactobacillus paracasei GS0805

[0045] After the Lactobacillus paracasei GS0805 strain was revived and activated for 3 generations, it was streaked on Columbia blood agar plates and cultured at 37°C for 24 hours to observe whether a hemolytic ring appeared around the test colony, and hemolytic Staphylococcus aureus was used as a positive control. The paper agar diffusion method was used to test the drug sensitivity of the strain to common antibiotics. The Lactobacillus paracasei GS0805 strain was revived and activated for 3 generations, 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).

[0046] The results of the hemolytic test showed that no hemolytic ring phenomenon occurred around the bacterial colonies; the strain was sensitive (S) to the six antibiotics tested, indicating the safety of the Lactobacillus paracasei GS0805 strain (Table 1).

[0047] Table 1 Sensitivity of Lactobacillus paracasei GS0805 strain to 6 antibiotics

[0048] serial number tetracycline Ampicillin Gentamycin Clindamycin Clarithromycin Chloramphenicol GS0805 S S S S S I

[0049] Example 3 Antibacterial effect and antioxidant activity of fermentation broth of Lactobacillus paracasei GS0805 strain

[0050] 3.1. Antibacterial activity against common intestinal pathogens

[0051] Escherichia coli (CMCB 44102), Staphylococcus aureus (CMCB 50094), Salmonella typhimurium ATCC 14028), Pseudomonas aeruginosa (CMCB 10104), Salmonella paratyphi B (CMCB 50094) and Shigella dysenteriae (CMCB 51105) 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 8 CFU / 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.

[0052] The fermentation broth had strong inhibitory activity against the growth of pathogenic bacteria such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and Shigella dysenteriae, which was basically equivalent to the effect of the LGG standard strain (Table 2). This shows that the bacterial agent prepared by the Lactobacillus paracasei GS0805 strain can be used for the prevention and control of the above pathogenic bacteria.

[0053] Table 2 Evaluation of antibacterial activity of Lactobacillus paracasei GS0805 strain (diameter: mm)

[0054]

[0055] 3.2 Antioxidant activity of Lactobacillus paracasei GS0805 fermentation broth

[0056] Antioxidants are any substances that can effectively inhibit the oxidation reaction of free radicals in low concentrations to offset the oxidative attack of free radicals on human cells. Its mechanism of action can be to act directly on free radicals, or to indirectly consume substances that easily generate free radicals to prevent further reactions. The stronger the body's antioxidant capacity, the healthier it is and the longer its life. More and more studies have shown that anti-oxidation is an important step in preventing aging, because free radicals or oxidants will decompose cells and tissues, affect metabolic functions, and cause different health problems. If excessive oxidative free radicals can be eliminated, many free radical-induced and aging-related diseases can be prevented. For example, common cancers, arteriosclerosis, diabetes, cataracts, cardiovascular disease, Alzheimer's disease, arthritis, etc., these diseases are all considered to be related to free radicals.

[0057] Preparation of fermentation broth: After the frozen strain of Lactobacillus paracasei GS0805 was revived and cultured for 24 hours, it was inoculated into MRS liquid culture medium at a 2% inoculum amount and cultured at 37°C for 24 hours. The bacterial solution was centrifuged at 4°C and 4000r / min for 15 minutes. The supernatant was filtered through a 0.22μm filter membrane to obtain the fermentation supernatant (CFS) and frozen for later use.

[0058] Determination method: Kit analysis method was used according to the kit instructions. Hydroxyl free radical determination kit (A018-1-1), DPPH free radical scavenging ability kit (A153-1-1) and inhibition and generation of superoxide anion free radical determination kit (colorimetric method A052-1-1) were all produced by Nanjing Jiancheng Bioengineering Research Institute, and antioxidant capacity determination was completed by Chengdu Lilai Biotechnology Co., Ltd.

[0059] Hydroxyl radical inhibition ability: Hydroxyl radical is a kind of active oxygen with strong electron-accepting ability, that is, strong oxidizing ability. Hydroxyl radical can kill red blood cells, degrade DNA, cell membrane and polysaccharide compounds. The measurement results are shown in Table 3, and the fermentation liquid of Lactobacillus paracasei GS0805 has a strong ability to inhibit hydroxyl radicals, which is 6048.80±83.24U / mL.

[0060] DPPH free radical scavenging ability: DPPH, also known as 1,1-diphenyl-2-trinitrophenylhydrazine, is a very stable nitrogen-centered free radical. Since the DPPH free radical has a single electron, it has a strong absorption at 517nm, and its alcohol solution is purple. When a free radical scavenger is present, its absorption gradually disappears due to pairing with its single electron, and the lighter the color, the more DPPH scavenging ability in the sample can be quantitatively analyzed. The results showed that the DPPH free radical scavenging ability of the fermentation liquid of Lactobacillus paracasei GS0805 was 214.81±1.32μg Trolox / mL, indicating that the fermentation liquid of Lactobacillus paracasei GS0805 has a certain scavenging ability for DPPH free radicals (Table 3).

[0061] Anti-superoxide anion ability: Superoxide anion free radicals, as a kind of free radicals produced in the metabolism of organisms, can attack biological macromolecules, such as lipids, proteins, nucleic acids and polyunsaturated fatty acids, causing cross-linking or rupture, causing damage to cell structure and function, and are closely related to aging and pathological changes of the body. The test results show that the inhibitory ability of Lactobacillus paracasei GS0805 fermentation liquid on superoxide anion free radicals is 767.32±10.78 (U / L), indicating that the inhibitory ability of Lactobacillus paracasei GS0805 fermentation liquid on superoxide anion free radicals is relatively strong (Table 3).

[0062] Table 3 Antioxidant capacity of Lactobacillus paracasei GS0805 fermentation broth

[0063]

[0064] In summary, the GS0805 fermentation broth has strong antioxidant capacity and has potential application value, including being used to improve human health.

[0065] Example 4 Growth inhibition and apoptosis-promoting effects of Lactobacillus paracasei GS0805 fermentation broth on colon cancer cells

[0066] 4.1 Adhesion of Lactobacillus paracasei GS0805 to human colon cancer cells

[0067] After the frozen strain of Lactobacillus paracasei GS0805 was revived and inoculated into MRS broth medium, the culture was cultured at 37°C for 24 hours. After the culture was completed, the culture was centrifuged at -4°C and 5000r / min for 10 minutes and washed several times with sterile PBS buffer. The concentration of the bacterial suspension was adjusted to 1×10 6CFU / 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 fluid after the adhesion experiment for plate counting, and the result is used as the number of viable adherent bacteria.

[0068] Adhesion rate (%) = number of lactic acid bacteria at the end of the period / number of lactic acid bacteria inoculated at the beginning × 100%

[0069] The results are shown in Table 4. The results show that Lactobacillus paracasei GS0805 has good adhesion to human colon cancer cells, with an average adhesion rate of 21.13%, which is conducive to the performance of the probiotic effect.

[0070] Table 4 Adhesion rate of Lactobacillus paracasei GS0805 to human colon cancer cells (%)

[0071] strain Repeat 1 Repeat 2 Repeat 3 Mean(%) GS0805 20.22 14.51 28.65 21.13±5.81

[0072] 4.2 Inhibitory effect of Lactobacillus paracasei GS0805 fermentation supernatant on colon cancer cell growth

[0073] Preparation of fermentation supernatant of Lactobacillus paracasei GS0805: After the Lactobacillus paracasei GS0805 was revived, it was inoculated into MRS broth medium and cultured at 37°C for 24 h to make the bacterial suspension concentration 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.

[0074] 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.

[0075] 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.

[0076] Cytotoxicity experiment: CCK-8 kit (Cell Counting Kit-8 cell counting reagent) was used to determine the effect of Lactobacillus paracasei GS0805 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.

[0077] The results showed that 4% Lactobacillus paracasei GS0805 fermentation supernatant showed a significant inhibitory effect on the growth of human colon cancer cells. The colon cancer cell density after 48 hours of culture was 80.32% of the control, indicating that Lactobacillus paracasei GS0805 fermentation supernatant had a significant inhibitory effect on the growth of human colon cancer cells (Table 5).

[0078] Table 5 Effect of Lactobacillus paracasei GS0805 fermentation supernatant on colon cancer cell growth (absorbance value)

[0079] 24h 48h Comparison 1.32±0.16 1.850±0.295 GS0805 1.205±0.25 1.486±0.114

[0080] At the same time, the ability of the fermentation supernatant of Lactobacillus paracasei GS0805 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% fermentation supernatant of Lactobacillus paracasei GS0805 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 clean the residual crystal violet dye, the culture plate was dried, the number of cell colonies was recorded, and the experimental results were recorded by taking pictures. The results showed that after adding 1% of the fermentation supernatant of Lactobacillus paracasei GS0805 to the culture medium, the average number of cell colonies was 793, which was only 49.78% of the number of the control group, indicating that the fermentation supernatant of Lactobacillus paracasei GS0805 can significantly reduce the colony formation ability of colon cancer cells (Table 6, Figure 2 ).

[0081] Table 6 Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on colony formation ability of colon cancer cells

[0082] Comparison 1% GS0805 fermentation supernatant Number of cell colonies 1593±374 793±216

[0083] 4.3 Pro-apoptotic effect of Lactobacillus paracasei GS0805 fermentation supernatant on human colon cancer cells

[0084] 4.3.1 Materials and methods

[0085] Cell culture: HT-29 cells in the logarithmic growth phase were digested and centrifuged and evenly plated in a 96-well plate, and 2000 cells were counted per well. After culturing for 12 hours, 4% (V / V) Lactobacillus paracasei GS0805 was added after the cells were stably attached to the wall. Five replicates were set for each treatment, and protein was extracted after culturing for 48 hours.

[0086] 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.

[0087] 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.

[0088] 4.3.2 Experimental Results

[0089] (1) Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on cell adhesion factor β-catenin

[0090] 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 recurrence rate of colorectal cancer after surgery 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 marker related to colorectal cancer invasion, metastasis and poor prognosis. Our experimental results showed that after adding GS0805 fermentation supernatant to the culture medium of colon cancer cells and culturing for 48 hours, the content of β-catenin was greatly reduced to only 42.10% of the control without fermentation medium. Our results showed that the fermentation supernatant of Lactobacillus paracasei GS0805 could reduce the expression of cell adhesion factor β-catenin, thereby reducing the adhesion and spread of cancer cells (Table 7).

[0091] Table 7 Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on β-catenin content in colon cancer cells (absorbance value)

[0092] Target protein / reference protein Comparison GS0805 β-catenin 40263 16952

[0093] (2) Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on the content of apoptosis inhibitory gene protein

[0094] Myeloid cell leukemia 1 (Mcl-1) is a member of the anti-apoptotic bcl2 family. Overexpression or amplification of MCL1 is common in many types of cancer, so it is considered to be one of the most relevant oncoproteins. Our study showed that after adding GS0805 fermentation supernatant to the culture medium, the total amount of MCL-1 protein in colon cancer cells was 71.74% of the control (Table 8), indicating that the fermentation broth has the activity of promoting cancer cell apoptosis. Studies have shown that the expression of mRNA and protein levels of mcl1 gene subtype 1 in colon tumor tissue and corresponding adjacent tissues are higher than those in the normal group. mcl1 subtype 1 and subtype 2 are positively correlated with the malignancy of colon cancer tumors, which can be used as one of the effective diagnostic indicators for clinical evaluation of the malignancy of colon cancer.

[0095] 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 GS0805 fermentation supernatant to the cell culture medium, the total amount of Bcl-2 protein in colon cancer cells was 46.09% of the control (Table 8), indicating that the fermentation supernatant has the activity of promoting cancer cell apoptosis.

[0096] Table 8 Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on the content of apoptosis inhibitory gene protein in colon cancer cells

[0097] Target protein / reference protein Comparison GS0805 Myeloid cell leukemia 1 (MCL-1) 30619 21966 B-cell lymphoma / leukemia-2 (BCL-2) 37799 17420

[0098] (3) Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on the content of apoptotic factor protein

[0099] P62 protein is an oncogene protein. The target gene of transcriptional regulation participates in the regulation of cell cycle, autophagy, cell proliferation, apoptosis and immortalization, and plays an important role in the occurrence of many tumors. P62 gene can help the human body better identify and resist exogenous antigens, thereby reducing the harm caused by infection. P62 protein can also prevent the accumulation of genotoxicity and carcinogenic mutations by promoting selective autophagy, thereby playing a tumor suppressor role. When P62 gene binds to 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, P62 gene can bind to antigens, promote immune response, help the human body resist foreign antigens, and thus reduce the damage caused by viral infection or bacterial infection. Our experiments show that the addition of Lactobacillus paracasei GS0805 fermentation supernatant to the cell culture medium can greatly increase the total amount of P62 protein in colon cancer cells, which is about 1.64 times that of the control (Table 9), indicating the pro-apoptotic activity of GS0805 fermentation supernatant.

[0100] 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 adding GS0805 fermentation supernatant to the culture medium, the total amount of poly ADP-ribose polymerase C-Parp in colon cancer cells was 2.55 times that of the control (Table 9), indicating the pro-apoptotic activity of Lactobacillus paracasei GS0805 fermentation supernatant.

[0101] Table 9 Effect of fermentation supernatant of Lactobacillus paracasei GS0805 on apoptosis-promoting factor protein of colon cancer cells (gray value)

[0102] Target protein / reference protein Comparison GS0805 P62 19088 31355 Poly ADP-ribose polymerase C-Parp 13507 34456

[0103] (4) Effect of GS0805 fermentation supernatant on reactive oxygen species (ROS) levels in human colon cancer cells

[0104] 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.

[0105] 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 GS0805 fermentation supernatant. 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.

[0106] 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 GS0805 fermentation supernatant 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 10).

[0107] Table 10 Number of cells expressing ROS after treatment with GS0805 fermentation supernatant

[0108]

[0109]

[0110] (5) One-step TUNEL cell apoptosis detection

[0111] When cells undergo apoptosis, some DNA endonucleases are activated, which cut off the genomic DNA between nucleosomes. After catalysis by terminal deoxynucleotidyl transferase (TdT), 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 TU NEL cell apoptosis detection kit. The test results show that after adding 4% (V / V) of the fermentation supernatant of Lactobacillus paracasei GS0805 strain (sterile) to the culture medium, a strong apoptosis signal can be detected. The average red fluorescence intensity of the treatment group is 246±27, while the average red fluorescence intensity of the control group is only 38±4.

[0112] In summary, the Lactobacillus paracasei GS0805 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 MCL-1 and Bcl-2 proteins representing apoptosis inhibition genes, and can increase the content of pro-apoptotic factor poly (ADP-ribose) polymerase Cleave d-parp and P62 protein representing autophagy. These results illustrate the effects of Lactobacillus paracasei GS0805 on the growth inhibition and apoptosis promotion of colon cancer cells from multiple aspects.

[0113] Example 5 Preventive Effect of Lactobacillus paracasei GS0805 on Colitis in Mice

[0114] 5.1 Experimental Methods

[0115] DSS aqueous solution: Dextran sulfate sodium salt (DSS) was used to prepare an aqueous solution with a concentration of 4% (w / v).

[0116] Preparation of bacterial suspension: After culturing probiotics in MRS broth for 24 hours, centrifuge at -4°C and 6000r / min for 5 minutes and discard the supernatant. Resuspend the bacteria in sterile PBS buffer and adjust the bacterial suspension concentration to 5×10 9 CFU / mL, stored in refrigerator for future use.

[0117] Experimental animals: 27 4-week-old SPF male Kunming mice were provided by Chengdu Dashuo Experimental Animal Co., Ltd. and randomly divided into 9 cages, 3 mice in each cage. After adaptive feeding, they were randomly divided into 3 groups, 3 cages in each group, including a blank control group (CK), a DSS model group, and a GS0805 treatment group. The experimental treatments are shown in Table 11.

[0118] Table 11DSS-induced colitis experiment in mice

[0119]

[0120] Serum inflammatory factor detection: At the end of the experiment, three mice were randomly killed in each group, and blood was collected from the eye sockets. The blood was centrifuged at 3000 r / min for 15 min to obtain serum. The inflammatory factors (IL-1β, IL-6, IL-8 and IL-10) in the serum were determined using ELISA kits according to the instructions of the kit.

[0121] 5.2 Results Analysis

[0122] Experimental observations showed that the DSS model group showed a more severe inflammatory response, such as mental depression, diarrhea, bloody stools, etc., while the GS0805 treatment group showed milder symptoms. Dissection revealed that the length of the colon and rectum of mice in the DSS model group was significantly shorter than that of the control group, while the length of the colon and rectum of the GS0805 treatment group was between the two, indicating that the GS0805 strain has a significant alleviating effect on DSS-induced inflammation in mice (Table 12).

[0123] Table 12 Average colorectal length of mice (cm)

[0124]

[0125] The detection of serum inflammatory factors showed that the levels of four serum inflammatory factors in the GS0805 treatment group were lower than those in the DSS model group, further indicating that the GS0805 strain has an alleviating effect on DSS-induced colitis in mice (Table 13).

[0126] Table 13 Detection of serum inflammatory factor levels in mice (x±SD) (unit: pg / mL)

[0127]

[0128] Experimental Example 6 Application of Lactobacillus paracasei GS0805 strain in fermentation of broken wall Ganoderma lucidum spore powder

[0129] Ganoderma lucidum spores have two layers of spore walls (polysaccharide walls) composed of chitin and glucan, and have a concentric layer network structure. They are tough, acid and alkali resistant, and extremely difficult to oxidize and decompose, thus limiting people's digestion and absorption of the effective substances in the spores. Its medicinal value is increasingly valued. Studies have found that Ganoderma lucidum spores have the effects of enhancing immunity, inhibiting tumors, protecting liver damage, and protecting against radiation.

[0130] Fermentation method: 10 grams of broken wall Ganoderma lucidum spore powder, 3 grams of oligofructose, and 100 mL of water were prepared into a culture medium. After sterilization and cooling, Lactobacillus paracasei GS0805 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 with three replicates, and the control group without probiotics was used. The culture and analysis methods were the same as those of the treatment group.

[0131] 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.

[0132] The analysis results showed that after the broken wall Ganoderma lucidum spore powder was fermented by Lactobacillus paracasei GS0805 strain, the contents of fatty acids, prostaglandins, amino acids and flavonoids in the fermentation supernatant increased significantly. In particular, some short-chain fatty acids, such as 2-hydroxyvaleric acid and 2-hydroxycaproic acid, increased by 115.939 times and 83.996 times respectively (Table 14); the contents of many prostaglandin compounds also increased significantly, such as 11-deoxy prostaglandin F2β, which increased by 113.219 times; and many amino acid compounds also increased significantly, such as 8-aminooctanoic acid, which increased by 16 times. It is worth noting that the content of 3-O-methylated flavonoids, a flavonoid compound, increased by 45.145 times (Table 14). The massive increase of these bioactive substances has opened up a new path for the further in-depth development of Ganoderma lucidum spores.

[0133] A large number of previous studies have shown that fatty acids, prostaglandins, amino acids and flavonoids are all bioactive substances with important physiological functions. For example, after binding to specific receptors, prostaglandins play a key role in mediating a series of cell activities such as cell proliferation, differentiation, apoptosis, and regulating female reproductive function and childbirth, platelet aggregation, and cardiovascular system balance, and are involved in the pathological processes of inflammation, cancer, and various cardiovascular diseases. There are many compounds with medicinal value in flavonoids, which are used to prevent and treat cardiovascular and cerebrovascular diseases, such as reducing vascular fragility, improving vascular permeability, lowering blood lipids and cholesterol, preventing and treating hypertension in the elderly, cerebral hemorrhage, coronary heart disease, angina pectoris, dilating coronary blood vessels, and increasing coronary flow. Many flavonoid components have antitussive, expectorant, antiasthmatic and antibacterial activities, and also have liver protection, liver detoxification, antifungal, treatment of acute and chronic hepatitis, cirrhosis, and anti-free radical and antioxidant effects.

[0134] Table 14 Metabolomics analysis of broken-wall Ganoderma lucidum spore powder fermented by Lactobacillus paracasei GS0805

[0135]

[0136]

Claims

1. A strain of Lactobacillus paracasei ( Lactobacillus paracasei ) GS0805, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 22, 2023, with the deposit number CGMCC No.27394.

2. Lactobacillus paracasei according to claim 1 ( Lactobacillus paracasei ) Use of GS0805 in the inhibition of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Shigella dysenteriae for non-disease treatment purposes.

3. Lactobacillus paracasei according to claim 1 ( Lactobacillus paracasei ) Use of GS0805 in the preparation of drugs for treating diseases caused by Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Salmonella paratyphi B and / or Shigella dysenteriae.

4. Lactobacillus paracasei according to claim 1 ( Lactobacillus paracasei ) Application of GS0805 in the preparation of anti-colon cancer drugs or anti-colon cancer auxiliary drugs.

5. An anti-colon cancer drug or an anti-colon cancer auxiliary drug, characterized in that: Containing the Lactobacillus paracasei described in claim 1 ( Lactobacillus paracasei )GS0805.

6. Lactobacillus paracasei according to claim 1 ( Lactobacillus paracasei )Application of GS0805 in fermenting Ganoderma lucidum spore powder to increase the content of bioactive substances, wherein the bioactive substances are: 2-Hydroxyvaleric acid, 2-Hydroxycaproic acid, (S)-Leucic acid, Heptadecanoic Acid, (+ / -)9,10-dihydroxy-12Z-octadecenoic acid, Nonanoic acid, 15-OxoEDE, 2-Ethylhexanoic acid, Undecanedioic acid, Ethylmalonate, Octanediol acid, Azelaic acid, 11-Deoxyprostaglandin F2β, 15-Deoxy-Δ12,14-prostaglandin J2-2-glycerol ester, ent-8-iso Prostaglandin F2α, Prostaglandin D1, Prostaglandin E2, 6β-Prostaglandin I1, 8-Aminooctanoic One or more of acid, L-Hydroxyproline, N-lactoyl-phenylalanine, N-OleoylGlycine, Proline or 3-O-methylated flavonoids.

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