A strain of Lactococcus lactis and its application in the fermentation of Ganoderma lucidum spores

Abstract

The present invention discloses a strain of Lactococcus lactis and its application in the fermentation of ganoderma lucidum spores, and the strain deposit number is CGMCC No.30210. The strain is separated and screened from the intestinal contents of the red-chain Chinese snake, grows well on MRS agar medium, is non-hemolytic, is sensitive to a variety of common antibiotics, has strong adaptability to the intestinal environment of humans and animals, has strong lactic acid production ability, strong inhibition ability to common intestinal pathogens, strong adhesion ability, growth inhibition ability and apoptosis-promoting effect on human colon cancer cells, has obvious effects on DSS-induced colitis in mice, and significantly reduces the level of inflammatory factors. The content of bioactive substances such as eicosanoids and steroids can be greatly increased by fermenting the broken wall ganoderma lucidum spore powder with the strain. The strain not only has significant application value in functional foods and biological preparations, but also opens up a new way for the further development of ganoderma lucidum spores.

Description

Technical Field

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

[0002] As a place for exchanging materials and information with the outside world, the intestines of humans and animals play an important role in body functions and immunity. However, the intestinal microbiota and the intestinal system jointly affect the health of the host through complex pathways. More and more studies have shown that probiotics regulate the homeostasis of intestinal microbiota and immune regulation by participating in intestinal metabolic pathways, producing beneficial metabolites, and inhibiting the colonization and proliferation of pathogens, thereby promoting body health.

[0003] Lactococcus lactis is a widely recognized safe bacteria species that plays an important role in regulating the balance of intestinal flora in animals and improving immunity. Our research found that snakes also have abundant microorganisms in their intestines, including a variety of lactic acid bacteria, but there are few reports on the research and utilization of intestinal microorganisms in snakes. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Lactococcus lactis with multiple functions and application thereof.

[0005] The technical solution of the present invention is: a strain of Lactococcus lactis SA0220, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on March 29, 2024, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number being CGMCC No.30210.

[0006] A biological preparation containing the above-mentioned Lactococcus lactis SA0220 or its fermentation broth.

[0007] The use of the Lactococcus lactis SA0220 or biological preparation described above in inhibiting Yersinia enterocolitica, Escherichia coli, Salmonella paratyphi B, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium or Shigella dysenteriae for non-disease treatment purposes.

[0008] The use of the above-mentioned Lactococcus lactis SA0220 in the preparation of a drug for preventing or treating colitis.

[0009] Use of the above-mentioned Lactococcus lactis SA0220 or biological preparation in the preparation of a drug for treating or assisting in treating colon cancer.

[0010] An anti-colon cancer drug or an anti-colon cancer auxiliary drug, comprising the above-mentioned Lactococcus lactis SA0220 or the biological preparation according to claim 2.

[0011] The application of the above-mentioned Lactococcus lactis SA0220 in fermenting broken-wall Ganoderma lucidum spore powder.

[0012] A functional food contains a fermentation liquid of broken-wall ganoderma spore powder, wherein the fermentation liquid of broken-wall ganoderma spore powder is obtained by fermenting the broken-wall ganoderma spore powder with the above-mentioned Lactococcus lactis SA0220.

[0013] Furthermore, the method for fermenting the broken-wall Ganoderma lucidum spore powder is: preparing the broken-wall Ganoderma lucidum spore powder with water into a liquid culture medium, sterilizing and cooling it, inoculating Lactococcus lactis SA0220, and culturing it at 37° C. for 96 hours.

[0014] The use of the above-mentioned Lactococcus lactis SA0220 or biological preparation in the preparation of functional food.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The lactococcus lactis SA0220 provided by the present invention is separated from the intestinal contents of Sinonatrix annularis, grows well on MRS agar medium, is non-hemolytic, is sensitive to a variety of common antibiotics, has strong adaptability to the intestinal environment of humans and animals, has strong lactic acid production ability, has strong inhibition ability to common intestinal pathogens, has strong adhesion ability, growth inhibition ability and apoptosis-promoting effect on human colon cancer cells, has obvious effects on DSS-induced colitis in mice, and significantly reduces the level of inflammatory factors. The fermentation of broken wall ganoderma lucidum spore powder with the strain can greatly increase the content of bioactive substances such as eicosanoids and steroids. The strain not only has significant application value in functional foods and biological preparations, but also opens up a new way for the further development and utilization of ganoderma lucidum spores.

[0017] Collection information:

[0018] Lactococcus lactis SA0220 was deposited at the General Microbiology Center of China Microorganism Culture Collection Administration on March 29, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No.30210. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a phylogenetic relationship diagram of Lactococcus lactis SA0220 and other strains in the present invention.

[0020] Figure 2 It is the number of clones formed by HT-29 cells after being treated with the fermentation supernatant of Lactococcus lactis SA0220 in the present invention, wherein A is the control group and B is the SA0220 treatment group.

[0021] Figure 3 The expression level of ROS in HT-29 cells after being treated with the fermentation supernatant of Lactococcus lactis SA0220 in the present invention, wherein A is the control group and B is the SA0220 treatment group.

[0022] Figure 4 The results show that the supernatant of Lactococcus lactis SA0220 fermentation in the present invention promotes apoptosis of HT-29 cells, wherein A is the control group and B is the SA0220 treatment group. DETAILED DESCRIPTION

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

[0024] The standard strain LGG used in the following examples is Lactobacillus rhamnosus ATCC 53103.

[0025] Experimental Example 1 Isolation, Identification and Safety of Lactococcus lactis

[0026] 1.1 Isolation and identification of strains

[0027] The specimens of the red-chained Chinese snake were collected from Yongxing County, Hunan Province, and the strains were isolated and screened from its intestinal contents;

[0028] 16S rRNA universal sequencing primers 27F and 1492R were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd., and the sequences are as follows (5'→3'):

[0029] 27F (SEQ ID No. 1): AGAGTTTGATCMTGGCTCAG

[0030] 1492R(SEQ ID No.2):GGTTACCTTGTTACGACTT

[0031] MRS broth medium formula (per liter): casein digest 10.0g, beef extract powder 10.0g, yeast extract powder 4.0g, triammonium citrate 2.0g, sodium acetate 5.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate tetrahydrate 0.05g, dipotassium hydrogen phosphate 2.0g, glucose 20.0g, Tween-80 1.08g, final pH is about 5.7. Weigh each component according to the above ratio, add distilled water or deionized water to make up to 1L, dispense into conical flasks, and sterilize under high pressure conditions at 121℃ for 20min.

[0032] MRS agar medium formula (per liter): peptone 10.0g, beef powder 5.0g, yeast powder 4.0g, glucose 20.0g, Tween 80 1.0mL, potassium dihydrogen phosphate heptahydrate 2.0g, sodium acetate trihydrate 5.0g, ammonium citrate trihydrate 2.0g, magnesium sulfate heptahydrate 0.2g, manganese sulfate tetrahydrate 0.05g, agar 15.0g, the final pH is about 6.2. Weigh each component according to the above ratio, add distilled water or deionized water to make up to 1L, dispense into conical flasks, and sterilize under high pressure conditions at 121℃ for 20min.

[0033] Screening and separation of strains: Take a small amount of enteric sample, inoculate it in 50mL MRS broth, shake it thoroughly and mix it, and place it in a constant temperature shaker at 37℃ for 24h. Take 1mL of the culture solution, dilute it by 10 times, take 20μL and spread it on MRS agar medium containing 1.5% calcium carbonate, and after culturing at 37℃ for 24h, pick a single colony with a large calcium dissolution circle and separate and purify it continuously for 3 times. Inoculate the purified strain into 600μL MRS broth medium, culture it in a shaker at 37℃ for 24h, add 400μL of 50% (V / V) sterile glycerol, and freeze it in a -80℃ ultra-low temperature refrigerator for later use.

[0034] 10 μL of the frozen bacterial strain was inoculated into MRS liquid medium for proliferation culture, and the strain DNA was extracted using the Tiangen Bacterial Genomic DNA Extraction Kit. PCR amplification was performed using the bacterial 16S rRNA universal primer 27F / 1492R with its total genomic DNA as a template to obtain the target gene sequence (SEQ ID No. 3) consisting of 1043 base pairs (bp).

[0035] The gene sequence obtained by sequencing was input into the NCBI database for comparison. The similarity between the strain and the strain Lactococcus lactis strain HBUR51169 in GenBank was 98.93%. The strain was preliminarily identified as Lactococcus lactis and named Lactococcus lactis SA0220. It was deposited in the General Microbiology Center of China Microbiological Culture Collection on March 29, 2024. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No. 30210. The phylogenetic relationship between strain SA0220 and other strains is as follows Figure 1 shown.

[0036] Lactococcus lactis SA0220 was inoculated on MRS agar medium and cultured at 37°C for 24 hours. It was observed that Lactococcus lactis SA0220 grew well on MRS agar medium, and the colonies were smooth and convex, and the shape was round and milky white. Gram staining kit was used to perform Gram staining on Lactococcus lactis SA0220, and it was observed under a microscope that the Lactococcus lactis SA0220 bacteria were purple, which was consistent with the characteristics of Lactococcus lactis.

[0037] 1.2 Safety of Lactococcus lactis SA0220 strain

[0038] The isolated and identified Lactococcus lactis SA0220 strain was activated and streaked on an MRS agar plate to select a single colony, then streaked on a blood plate medium for 24 hours and observed for hemolysis. The experiment was repeated three times. No hemolysis occurred around the Lactococcus lactis SA0220 colony, indicating that the strain was non-hemolytic.

[0039] The sensitivity of the strains to common antibiotics was tested by the paper agar diffusion method. The isolated and identified Lactococcus lactis SA0220 strain was activated and cultured, and the concentration of the bacterial solution was adjusted to 1×10 8 CFU / mL, use a sterile cotton swab to evenly smear the bacterial solution on the surface of the MRS medium plate, and after culturing at 37°C for 18 hours, use a vernier caliper to measure the diameter of the inhibition zone around each drug-sensitive paper piece. Repeat each antibiotic three 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). As shown in Table 1, Lactococcus lactis SA0220 showed intermediate (I) to the tetracyclines involved in the test, and was sensitive (S) to ampicillin, ceftriaxone, clarithromycin, chloramphenicol, and ciprofloxacin.

[0040] Table 1 Sensitivity of Lactococcus lactis SA0220 to 6 antibiotics

[0041]

[0042] Experimental Example 2 Adaptability of Lactococcus lactis SA0220 to the gastrointestinal environment of humans and animals

[0043] 2.1 Acid resistance and bile salt resistance evaluation:

[0044] The Lactococcus lactis SA0220 strain isolated and identified in Experimental Example 1 was revived and purified for 3 generations on MRS agar plates. MRS broth medium with a pH of 3.0 and a bile salt concentration of 0.3% was prepared with hydrochloric acid and porcine bile salt, respectively. The revived strain SA0220 was inoculated into the broth medium with a pH of 3.0 and a bile salt concentration of 0.3%, and the initial concentration of the strain was adjusted to 1×10 8 CFU / mL, after culturing at 37℃ for 18h, 20μL of bacterial solution was spread on MRS agar plate medium, and after culturing at 37℃ for 18h, the surface of the MRS plate was observed to see whether there was colony growth. The experiment was repeated 3 times. According to the results of the acid resistance and bile salt resistance test, Lactococcus lactis SA0220 can still grow normal colonies on MRS agar plate after being treated in MRS broth medium with pH 3.0 and bile salt concentration of 0.3% for 18h, and the strain has strong acid resistance and bile salt resistance.

[0045] 2.2 Simulated gastric juice and intestinal juice tolerance test:

[0046] Simulated gastric juice and intestinal juice were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; the main components of artificial gastric juice simulation fluid are sodium chloride, dilute acid and pepsin, and the final pH is 2.5; the main components of artificial intestinal juice simulation fluid are phosphate and trypsin, and the final pH is 6.8. In addition to the acidic and bile salt environments in the human and animal intestines, there are also various digestive enzymes, so it is necessary to further test their adaptability in the gastrointestinal environment. The Lactococcus lactis SA0220 strain isolated and identified in Experimental Example 1 was revived and activated, washed 2-3 times with artificial gastric juice after centrifugation, and the bacteria were resuspended, and the concentration of the bacteria was adjusted to 1×10 8CFU / mL, 300 μL of bacterial suspension was added to 2.7 mL of artificial simulated gastric fluid, and 20 μL of the mixture was diluted 10 times and then counted as the initial value of tolerance to artificial gastric fluid; the simulated gastric fluid with live bacteria was cultured at 37 ° C for 3 hours and then the live bacteria were counted by the same method as the final value of tolerance to artificial gastric fluid. At the same time, 300 μL of the above mixture was added to 2.7 mL of simulated intestinal fluid, and the initial live bacteria were counted by the above method. After 3 hours of culture at 37 ° C, the live bacteria were counted again. The experiment was repeated 3 times, with LGG as the control. As shown in Table 2, the survival rate of Lactococcus lactis SA0220 in artificial simulated gastric fluid and simulated intestinal fluid was significantly higher than that of LGG, and it had good tolerance to artificial simulated gastric and intestinal fluid, which was conducive to its survival in the human and animal gastrointestinal tract.

[0047] Table 2 Tolerance of Lactococcus lactis SA0220 strain to artificial simulated gastric and intestinal fluids (%)

[0048]

[0049] Experimental Example 3 Lactic acid production ability and antibacterial activity of Lactococcus lactis SA0220

[0050] 3.1 Lactic acid production capacity of Lactococcus lactis SA0220

[0051] After the Lactococcus lactis SA0220 strain was activated and cultured, it was inoculated into MRS broth medium, cultured at 37°C for 24h, and centrifuged at 4°C and 12000g for 15min. Take 100μL of the fermentation supernatant and determine the lactic acid content according to the instructions of the lactic acid kit. The results showed that the lactic acid production capacity of the Lactococcus lactis SA0220 fermentation broth was strong, and the average lactic acid content was 80.92μmol / mL (Table 3). Lactic acid, as an important antibacterial compound in the gastrointestinal tract of humans and animals, plays an important role in reducing intestinal pH and changing the permeability of Gram-positive bacterial cell membranes, thereby helping to inhibit the growth of pathogens.

[0052] Table 3 Lactic acid content of Lactococcus lactis SA0220 fermentation broth (μmol / mL)

[0053]

[0054] 3.2 Antibacterial activity of Lactococcus lactis SA0220 against common intestinal pathogens

[0055] Escherichia coli (CMCB 44102), Salmonella enterica serovar Paratyphi B (CMCB 50094), Pseudomonas aeruginosa (CMCB 10104), Staphylococcus aureus (CMCCB 26003), Salmonella typhimurium ATCC14028), Shigella dysenteriae (CMCB 51105), and Yersinia enterocolitica subsp enterocolitica (CMCB 52204) were revived and purified three times, and the activated pathogenic bacteria were inoculated into liquid culture medium, and the bacterial solution concentration was adjusted to 1×10 8 CFU / mL. Pipette the above pathogenic bacteria solution at 0.01% and add it to the solid agar medium cooled to 40-45°C after sterilization. Mix thoroughly and dispense into culture dishes at a rate of about 20 mL per dish. After the culture medium cools and solidifies, use a 6mm diameter puncher to punch holes on the plate. After the SA0220 strain isolated and identified in Experimental Example 1 is revived, adjust the bacterial solution concentration to 1×10 8 CFU / mL, the bacterial solution to be tested was added to the wells of the pathogenic bacteria agar plate, and after incubation at 37°C for 18 hours, the diameter of the inhibition zone around the punch point was measured with a vernier caliper (mm) and recorded. The experiment was repeated 3 times.

[0056] The results are shown in Table 4. The Lactococcus lactis SA0220 strain has a strong inhibitory activity against the growth of pathogenic bacteria Escherichia coli, Salmonella paratyphi B, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica.

[0057] Table 4 Antibacterial activity of Lactococcus lactis SA0220 strain (diameter: mm)

[0058]

[0059] Experimental Example 4 Adhesion of Lactococcus lactis SA0220 to Human Colon Cancer HT-29 Cells

[0060] HT-29 cells are often used to simulate the intestinal cell environment for adhesion testing. Resuscitate human colon cancer HT-29 cells, subculture at a cell density of about 80%, remove DMEM medium, rinse with PBS buffer, digest with 0.25% trypsin-EDTA and add DMEM medium to terminate the reaction, and adjust the cell concentration to 5×10 6 cells / mL and dispensed into a twelve-well cell plate. The Lactococcus lactis SA0220 identified and preserved in Example 1 was revived and inoculated into MRS broth medium and cultured at 37°C for 24 hours. After the culture was completed, it was centrifuged at 4°C and 5000r / min for 10 minutes, and washed 1-2 times with sterile PBS buffer and the concentration of the bacterial suspension was adjusted to 1×10 8 CFU / mL and set aside. Aspirate the culture medium in the cell plate, add 500μL bacterial suspension and 500μL DEME culture medium to each well, shake the cell plate gently, and aspirate 20μL bacterial solution in the well for viable bacteria count as the initial viable bacteria count. Incubate the cell plate at 37℃ for 2h, discard the mixed solution in the well and wash 2-3 times with sterile PBS buffer. Digest the cells with 0.5mL 0.25% trypsin for 2min. After the cells are completely detached, add 1mL DMEM culture medium to terminate the digestion and repeat the beating. Collect the culture medium after the experiment and count the viable bacteria as the number of adhered viable bacteria. The experiment was repeated 3 times, with LGG as the control. Adhesion rate (%) = number of viable bacteria after 2h / initial number of viable bacteria × 100%

[0061] The results are shown in Table 5. The average adhesion rate of Lactococcus lactis SA0220 to human colon cancer HT-29 cells was 29.49%, which was higher than that of LGG, which was beneficial to its colonization and function in the intestine.

[0062] Table 5 Adhesion rate of Lactococcus lactis SA0220 to human colon cancer HT-29 cells (%)

[0063]

[0064] Experimental Example 5 Growth inhibition and apoptosis-promoting effects of Lactococcus lactis SA0220 on human colon cancer HT-29 cells

[0065] 5.1 Inhibitory effect of Lactococcus lactis SA0220 fermentation supernatant on HT-29 cell growth:

[0066] HT-29 cell passaging and plating: Preheat PBS, RPMI culture medium and trypsin in a 37°C water bath in advance, take out the HT-29 culture dish cultured in the CO2 incubator, and perform subsequent operations on the clean bench. Aspirate the culture medium in the culture dish, add 1ml PBS to the culture dish for washing, add 1ml trypsin after aspirating, mix gently, and place the culture dish in the incubator for digestion for 2min until the cells become round under an optical microscope. Aspirate the trypsin, add 1ml culture medium, use a pipette to suspend the cells, and transfer them to a 1.5ml EP tube. After centrifugation at 1200rpm / min for 3min, aspirate the supernatant, add 300μL culture medium and mix by blowing, aspirate 150μL into the culture dish containing 4ml culture medium, mix gently, and continue to culture in the CO2 incubator. After 48h, operate according to the cell passaging method until centrifugation. Discard the supernatant, mix the cells with 1 ml of culture medium, and then count them using a hemocytometer. Dilute the cell suspension to 3 × 10 4 CFU / ml. Add 100 μL of cell suspension to each well of the 96-well cell plate, add only culture medium to the blank group, and culture in a CO2 incubator until the cells adhere to the wall and grow well.

[0067] Sample addition treatment: The Lactococcus lactis SA0220 identified and preserved in Example 1 was resuscitated and inoculated into MRS broth medium, cultured at 37°C for 24 hours, and then centrifuged for 10 minutes. After aspirating the supernatant, the fermentation supernatant was collected by filtering again with a sterile filter membrane. The Lactococcus lactis SA0220 fermentation supernatant was diluted with serum-free culture medium at a volume ratio of 1:100. The corresponding diluted SA0220 fermentation supernatant was added to a 96-well plate and cultured for 24 hours, 48 ​​hours and 72 hours respectively. MRS liquid culture medium was used as a negative control. Three replicates were set for each time point.

[0068] CCK-8 detection of cell proliferation: CCK-8: RPMI basic medium was mixed in advance at a volume ratio of 1:9. 100 μL of the mixed solution was aspirated from the 96-well plate at 24h, 48h and 72h of drug treatment, and 100 μL of the mixed CCK-8 solution was added to each well. The plate was placed in the incubator for 1.5h and the OD was measured with an enzyme marker. 560 value.

[0069] The test results are shown in Table 6, and the fermentation supernatant of Lactococcus lactis SA0220 has an inhibitory effect on the growth of human colon cancer HT-29 cells.

[0070] Table 6 Inhibitory effect of fermentation supernatant of Lactobacillus reuteri DA0218 on the growth of HT-29 cells (%)

[0071] 24h 48h 72h Comparison 0.9537±0.1953 1.5827±0.1853 2.1957±0.2105 DA0220 0.8035±0.1053 1.3106±0.1998 1.8903±0.1496 Growth inhibition rate (%) 15.75 17.20 13.91

[0072] 5.2 Effect of Lactococcus lactis SA0220 fermentation supernatant on HT-29 cell colony formation ability:

[0073] The colon cancer cell HT-29 cells were revived and plated in a 6-well plate at a density of 2000 cells per well. The Lactococcus lactis SA0220 strain isolated and identified in Experimental Example 1 was revived and inoculated into MRS liquid culture medium and cultured at 37°C for 24 h to a bacterial concentration of 1×10 8 CFU / mL and collect the fermentation supernatant. Dilute the fermentation supernatant of Lactococcus lactis SA0220 with serum-free culture medium at a volume ratio of 1:100 to a concentration of 1%. Culture HT-29 cells with 1% SA0220 fermentation broth for about 3 weeks. After visible clones are formed in the cell culture plate, discard the culture medium, wash with PBS, and fix with methanol for 30min-2h after washing with PBS. Add 1% crystal violet stain for 30min, wash off the residual crystal violet stain, and dry the culture plate. Use 1% MRS liquid culture medium supernatant as the negative control group, and set up 3 replicates in the experiment.

[0074] The results are shown in Table 7 and Figure 2 As shown, after being treated with SA0220 fermentation broth, the growth density of HT-29 cells was significantly lower than that of the control group, which significantly inhibited the growth of human colon cancer cells HT-29.

[0075] Table 7 Inhibitory effect of SA0220 fermentation supernatant on colony formation of human colon cancer cells

[0076]

[0077] Note: Compared with the control group, **p<0.01

[0078] 5.3 Effect of Lactococcus lactis SA0220 fermentation supernatant on the level of reactive oxygen species (ROS) in HT-29 cells:

[0079] Cell collection: The culture medium of HT-29 cells cultured on the wall was aspirated, and the serum-free culture medium was used to blow repeatedly. The bottom of the well plate (bottom of the bottle) changed from translucent (cell monolayer connected into a sheet) to transparent by naked eye observation, and almost all the cell layer was blown into the culture medium. All the cell suspension was collected into a 1.5ml centrifuge tube. Wash twice with serum-free culture medium, 1000r / min, centrifuge for 5min, aspirate the supernatant, and keep the cell pellet for determination.

[0080] Loading probe: Dilute DCFH-DA with serum-free culture medium at 1:1000 to a final concentration of 10μmol / 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. After the cells are divided into several equal parts, stimulate the cells with SA0220 fermentation supernatant.

[0081] Detection: Use an inverted fluorescence microscope to detect the intensity of fluorescence before and after stimulation in real time or at each time point at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Use the supernatant of MRS liquid culture medium as a negative control group, and set up 3 replicates of the experiment.

[0082] The results showed that after HT-29 cells were treated with SA0220 fermentation supernatant, the number of fluorescently labeled cells increased significantly, and the fluorescence intensity was significantly higher than that of the control group (Table 8, Figure 3 ), indicating that SA0220 fermentation supernatant can promote the expression level of cellular reactive oxygen species (ROS), thereby promoting cell apoptosis.

[0083] Table 8 Effect of SA0220 fermentation supernatant on ROS expression in human colon cancer cells

[0084]

[0085] Note: Compared with the control group, **p<0.01

[0086] 5.4 Pro-apoptotic ability of Lactococcus lactis SA0220 on human colon cancer HT-29 cells:

[0087] One-step TUNEL cell apoptosis detection was used. After incubating the SA0220 fermentation supernatant with adherent cultured HT-29 cells for 48 hours, the fermentation supernatant was aspirated and washed once with PBS. The cells were fixed with 4% paraformaldehyde for 30 minutes and washed again with PBS. Add PBS containing 0.3% Triton X-100 and incubate at room temperature for 5 minutes. After preparing the TUNEL detection solution, mix it thoroughly, add 50μL TUNEL detection solution to the HT-29 cells, and incubate at 37℃ in the dark for 60min. Wash 3 times with PBS, then seal the slides with anti-fluorescence quenching sealing solution and observe under an inverted fluorescence microscope, with an excitation wavelength of 550nm and an emission wavelength of 570nm. The supernatant of MRS liquid culture medium was used as the negative control group, and the experiment was repeated 3 times.

[0088] After being treated with the fermentation supernatant of the SA0220 strain, the number of apoptotic HT-29 cells increased significantly ( Figure 4), the fluorescence intensity was significantly higher than that of the control group (Table 9), indicating that its fermentation supernatant has a significant apoptosis-promoting effect on human colon cancer cells.

[0089] Table 9 Pro-apoptotic ability of SA0220 strain fermentation supernatant on human colon cancer cells (TDT fluorescence intensity)

[0090]

[0091] Note: Compared with the control group, **p<0.01

[0092] Experimental Example 6 Preventive Effect of Lactococcus lactis SA0220 on Mouse Inflammation Model

[0093] 6.1 Experimental methods

[0094] Lactococcus lactis SA0220 was cultured in MRS broth for 24 h and then centrifuged for 10 min. The supernatant was discarded. The cells were resuspended in saline buffer and the concentration of the bacterial solution was adjusted to 1×10 9 CFU / mL, a 4% (w / v) aqueous solution was prepared with dextran sulfate sodium salt (DSS). 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 probiotic (Lactococcus lactis SA0220) treatment group. The experimental treatments are shown in Table 10.

[0095] Table 10DSS-induced colitis experiment in mice

[0096]

[0097] Organ index, colorectal length and tissue sections: After the modeling period, 3 mice in each group were killed and dissected, and their spleen, liver and colorectum were quickly taken out, rinsed with physiological saline, dried with filter paper, weighed and measured. Organ index = spleen or liver mass (g) / mouse body weight (g) × 100%

[0098] 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, IL-10 and TNF-α) in the serum were determined using ELISA kits according to the instructions of the kit.

[0099] 6.2 Results Analysis

[0100] Organ index and colorectal length: The autopsy showed that the organ index of the DSS-treated group increased and the colorectal length was significantly shortened compared with the CK group. The colorectal length of the mice treated with Lactococcus lactis SA0220 was close to that of normal mice and significantly different from that of the DSS-treated mice (Table 11).

[0101] Table 11 Changes in organ index and colorectal length in mice

[0102]

[0103] Note: Compared with CK group, *p<0.05; compared with DSS group, #p<0.05

[0104] Blood inflammatory factor detection: At the end of the experiment, four inflammatory factor indicators, TNF-α, IL-1β, IL-6 and IL-8, were measured. The results showed that the levels of inflammatory factors in the serum of mice in the DSS group were significantly increased, and the levels of the four inflammatory factors in the serum of mice in the SA0220 strain treatment group were significantly lower than those in the DSS model group, indicating that Lactococcus lactis SA0220 has a significant alleviating effect on DSS-induced colon inflammation in mice (Table 12).

[0105] Table 12 Changes in the concentration of various inflammatory factors in the serum of mice in each group (Unit: pg / mL)

[0106]

[0107] Note: Compared with CK group, **p<0.01; compared with DSS group, ##p<0.01

[0108] Example 7 Application of Lactococcus lactis SA0220 strain in fermentation of broken wall Ganoderma lucidum spore powder

[0109] 7.1 Fermentation and analytical methods

[0110] Fermentation method: 3 grams of broken wall Ganoderma lucidum spore powder and 20 mL of water were prepared into a culture medium. After sterilization and cooling, Lactococcus lactis SA0220 was inoculated and cultured at 37°C 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 control group was not inoculated with Lactococcus lactis SA0220, and the culture and analysis methods were the same as those of the treatment group.

[0111] Analytical methods: Based on high-resolution mass spectrometry (HRMS) detection technology, the high-quality mzCloud database constructed by standards is combined with the mzVault and MassList databases to match and identify molecular characteristic peaks. The raw data is preprocessed using CD3.3 data processing software. First, simple screening is performed by parameters such as retention time and mass-to-charge ratio, and the peak area is quantified at the same time. Then, the high-resolution secondary spectrum databases mzCloud and mzVault and the MassList primary database are compared (searched) to identify metabolites. Using CD3.3 data processing software, the chromatographic peaks detected in the sample are integrated, where the peak area of ​​each characteristic peak represents the relative quantitative value of a metabolite. The total peak area is used to standardize the quantitative results, and finally the quantitative results of the metabolites are obtained. The results are expressed as the multiple increase in the content of the corresponding component in the sample compared with the control.

[0112] 8.2 Analysis results

[0113] (1) Eicosanoid compounds:

[0114] Eicosanoids, also known as eicosanoids, are a large class of biologically active unsaturated fatty acids produced by the oxidation of twenty-carbon polyunsaturated fatty acids, including prostaglandins, thromboxanes and leucotriene. They are important inflammatory factors that are widely present in body fluids and tissues, regulating many physiological and pathological processes in the body.

[0115] There are two main functions of prostaglandins. One is to affect the contraction of smooth muscles, and it has a strong effect on the intestines, blood vessels, bronchi, uterus, etc. The other is to change the activity of adenylate cyclase, which is generally promoted, but inhibited in adipose tissue, so it has an anti-lipolysis effect, participates in the regulation and control of many physiological processes, promotes inflammatory responses, participates in reproductive processes (such as ovulation, conception and uterine contraction during childbirth), and digestion. The analysis results showed that after the broken spore powder of Ganoderma lucidum was fermented with Lactococcus lactis SA0220 strain for 96 hours, a variety of prostaglandin compounds were detected in the supernatant, and the content was greatly increased compared with the control, especially the 11-deoxy prostaglandin F1β (11-Deoxy prostaglandin F1β) content was 11.97 times that of the control, and the contents of dihydroprostaglandin E1 (6.32 times), prostaglandin E2 (4.03 times), isoprostanes (3.62 times), etc. also increased significantly. Dihydroprostaglandin E1 is a prostate drug, the main function of which is to promote blood circulation, increase tissue metabolism, and also have an analgesic effect (Table 12).

[0116] Thromboxanes are also derivatives of arachidonic acid. Unlike other eicosanoids, thromboxanes have a cyclic ether structure and can promote vasoconstriction and platelet aggregation, so they are also called thromboxanes or thromboxanes. The analysis results showed that after the broken wall Ganoderma lucidum spore powder was fermented by Lactococcus lactis SA0220 strain for 96 hours, the content of thromboxane B3 was significantly increased in the supernatant (Table 13).

[0117] Leucotriene is a component of the slow-reacting substance of allergic reaction. It plays an active role in inflammatory reaction and promotes the tendency of white blood cells to destroy tissues. It has a strong vasoconstriction effect, and can also promote increased vascular permeability and promote smooth muscle spasm of blood vessels and bronchial tubes. The analysis results show that after the broken wall Ganoderma lucidum spore powder was fermented by Lactococcus lactis SA0220 strain for 96 hours, the content of 11,12-epoxyeicosatrienoic acid was detected in the supernatant. Studies have shown that this compound is an inhibitor of NLRP3 inflammasomes and can be used for anti-inflammatory, angiogenesis and cardioprotection research. It can inhibit NLRP3 protein expression, significantly reduce the expression of pro-IL-1β in cells and supernatant, and reduce intracellular ROS.

[0118] Table 13 Changes in the content of eicosanoids in the supernatant of the broken-walled Ganoderma lucidum spore powder fermented by strain SA0220 (fold)

[0119]

[0120] (2) Steroid compounds

[0121] Steroid compounds are a general term for a large class of cyclopentyl perhydrophenanthrene derivatives widely distributed in the biological world, also known as steroids and steroid compounds. Steroids include sterols (such as cholesterol, stigmasterol), bile acid and bile alcohol, steroid hormones (such as adrenocortical hormones, androgens, estrogens), etc. The analysis results show that after the broken wall Ganoderma lucidum spore powder was fermented by Lactococcus lactis SA0220 strain for 96 hours, the content of various steroid compounds in the supernatant increased significantly, especially the content of 2-Methoxyestradiol (2-Methoxyestradiol) was 22.92 times that of the control. 2-Methoxyestradiol is a metabolite of estrogen. Studies have shown that it inhibits angiogenesis by reducing endothelial cell proliferation and inducing endothelial cell apoptosis. It can also destroy the stability of microtubules. It is an angiogenesis inhibitor and apoptosis inducer with anti-tumor activity.

[0122] The content of sodium dehydrocholate in the fermentation supernatant also increased significantly (10.71 times). This substance is a bile excretion enhancer, and its main function is to increase the output of bile to remove the increased bile acid load in the body, which plays an important role in maintaining the balance of bile acid and the normal function of the digestive system.

[0123] Hydroxypregnenolone (9.03 times) plays an important role in steroid biosynthesis and hormone synthesis, and is a precursor of many steroid hormones. It is a prohormone that forms dehydroepiandrosterone (DHEA). DHEA and its metabolite dehydroepiandrosterone sulfate (DHEAS) can be converted into estradiol and testosterone, and participate in female fertility, metabolism, and nervous system function.

[0124] In addition, the contents of vitamin D2 (4.05 times), glycolithocholic acid (3.49 times), bile acid (2.78 times), estrone (2.12 times), and androstenone (2.04 times) in the fermentation supernatant also increased significantly, all of which have important biological functions.

[0125] Table 14 Changes in steroid content in the supernatant of broken wall Ganoderma lucidum spore powder fermented by strain SA0220 (fold)

[0126]

[0127] (3) Other compounds

[0128] 2-Arachidonoylglycerol: The analysis results showed that the content of 2-Arachidonoylglycerol (2-AG) in the fermentation supernatant of the broken wall Ganoderma lucidum spore powder fermented by Lactococcus lactis SA0220 strain increased significantly, which was 96.85 times that of the control (Table 15). 2-Arachidonoylglycerol is an endogenous cannabinoid ligand with multiple biological functions. It is involved in multiple biological processes, including neural signal transmission, pain perception, and mood regulation. 2-AG, as a full agonist, can activate neuronal cannabinoid receptors and prevent the induction of long-term potentiation in CA3-CA1 synapses. It plays an important role in anti-inflammatory, immunomodulatory, anti-tumor, neuroprotection, etc., and is involved in the regulation of lipid metabolism and energy balance. In addition, 2-AG is also related to the occurrence and development of various diseases, so its research has important medical and biological significance.

[0129] Table 15 Changes in the content of other compounds in the supernatant of the broken wall Ganoderma lucidum spore powder fermented by strain SA0220 (fold)

[0130]

[0131]

[0132] Pyrimidine 2'-deoxyribonucleoside monophosphate (dUMP) and 2'-deoxyuridine-5'-monophosphate: The content of these two compounds increased significantly in the fermentation supernatant. As important precursors for DNA synthesis, they participate in the transmission and expression of genetic information in cells and are of great significance for maintaining normal life activities of cells. At the same time, they also play an important role in nucleotide metabolism, providing cells with necessary energy and material sources, and play an important role in organisms.

[0133] Receptor protein kinase (RLK): The RLK content in the supernatant is 39.25 times that of the control. RLK has signal perception and transmission functions, and can sense extracellular signals such as hormones and growth factors, and exert its effects by phosphorylating downstream target proteins. It is mainly involved in physiological processes such as plant growth, signal transduction, abiotic stress and biotic stress response. RLK receptor protein kinase plays multiple roles in plants, including signal perception and transmission, growth and development regulation, stress response, and disease resistance.

[0134] Phellamurin (Phellamurin) is a plant flavonoid glycoside that has the activity of inducing cell apoptosis and anti-tumor activity. Studies have shown that it can exert anti-tumor effects by reducing the ratios of p-PI3K / PI3K, p-AKT / AKT and p-mTOR / mTOR in cells.

[0135] Trans-cinnamic acid: Trans-cinnamic acid has certain anti-inflammatory, antioxidant and antibacterial effects, and can be used to treat certain inflammatory and infectious diseases. It has also been found to have an inhibitory effect on certain tumor cells, providing a new idea for tumor treatment.

[0136] Acetylcarnitine: It can accelerate the decomposition and oxidation of fat in the human body by affecting the fat metabolism pathway, thereby reducing the accumulation of fat in the body. Acetylcarnitine is an endogenous substrate for the synthesis of acetylcholine and participates in the synthesis of neurotransmitters. Therefore, acetylcarnitine has a positive effect on improving brain nerve function and preventing cognitive and memory function decline.

Claims

1. A strain of Lactococcus lactis ( Lactococcus lactis )SA0220, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.30210.

2. Containing the Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Biological preparation of SA0220 or its fermentation broth.

3. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Use of SA0220 or the biological agent described in claim 2 for the purpose of inhibiting Yersinia enterocolitica, Escherichia coli, Salmonella paratyphi B, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium or Shigella dysenteriae for non-disease treatment purposes.

4. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Use of SA0220 in the preparation of drugs for preventing or treating colitis.

5. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Use of SA0220 or the biological agent described in claim 2 in the preparation of a drug for the treatment or adjuvant treatment of colon cancer.

6. An anti-colon cancer drug or an anti-colon cancer auxiliary drug, characterized in that: Containing the Lactococcus lactis according to claim 1 ( Lactococcus lactis ) SA0220 or the biological agent described in claim 2.

7. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Application of SA0220 in fermentation of broken-wall Ganoderma lucidum spore powder.

8. A functional food, characterized in that: Contains a fermentation liquid of broken spore powder of Ganoderma lucidum, wherein the fermentation liquid of broken spore powder of Ganoderma lucidum is prepared from the Lactococcus lactis according to claim 1 ( Lactococcus lactis )SA0220 is obtained by fermenting broken-wall Ganoderma lucidum spore powder.

9. A functional food according to claim 8, characterized in that: The method for fermenting the broken wall ganoderma spore powder is as follows: the broken wall ganoderma spore powder is prepared into a liquid culture medium with water, sterilized and cooled, inoculated with Lactococcus lactis SA0220, and cultured at 37°C for 96 hours.

10. The Lactococcus lactis according to claim 1 ( Lactococcus lactis ) Use of the biological preparation described in SA0220 or claim 2 in the preparation of functional foods.

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