Lactobacillus gasseri strain A21120 with anti-oxidation, anti-inflammatory and anti-tumor functions as well as probiotic agent and application of lactobacillus gasseri strain A21120

Abstract

The invention provides a lactobacillus gasseri strain A21120 with anti-oxidation, anti-inflammatory and anti-tumor functions as well as a probiotic agent and application of the lactobacillus gasseri strain A21120, and belongs to the technical field of functional microorganisms. The strain A21120 is separated and screened from intestinal feces of healthy old people of 100 years old, and belongs to lactobacillus gasseri through morphological identification and molecular identification. The strain has good gastrointestinal fluid resistance and bovine choline resistance and has the potential of colonizing intestinal tracts. The lactobacillus gasseri strain A21120 provided by the invention has bacteriostatic activity, anti-inflammatory activity, anti-oxidation activity and anti-tumor activity, and provides a new way for treatment of clinical related diseases, drug development and preparation of in-vitro preparations or daily necessities.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganisms, and specifically relates to a Lactobacillus gasseri strain A21120 with anti-oxidation, anti-inflammatory and anti-tumor functions, a probiotic agent and an application thereof. Background Art

[0002] Lactobacillus gasseri is a type of probiotic that can be used in food. It can colonize in the human intestine and have a beneficial effect on the host by changing the composition of the host flora. Timely supplementation of probiotics in the diet is extremely important for people who show related symptoms or diseases.

[0003] Nowadays, intestinal microorganisms have played an important role in people's lives. They can not only maintain intestinal health, but also regulate various signal pathways through metabolites, and are closely related to various diseases. For example, Lactobacillus, which coexists with the human intestine, is a beneficial bacteria that plays an important role in inhibiting the invasion of pathogens, alleviating intestinal inflammation and damage, delaying aging, and even resisting the development of tumors. The patent with publication number CN117363524A discloses a strain of Lactobacillus gasseri MY4, which has the effects of anti-oxidation, whitening, and delaying aging. Publication number CN 111518724A discloses a strain of Lactobacillus gasseri HMV18, the exogenous protein of which has antibacterial and anti-tumor effects, and can be used to prepare antibacterial and anti-tumor products. Therefore, probiotics have potential application value and broad market prospects.

[0004] However, there are currently no reports that Lactobacillus gasseri has antioxidant, anti-inflammatory and anti-tumor functions. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a novel Lactobacillus gasseri A21120 having antioxidant, anti-inflammatory and anti-tumor functions.

[0006] The invention provides a Lactobacillus gasseri strain A21120, whose deposit number is GDMCC No: 64676.

[0007] The invention provides a probiotic, wherein the active ingredient comprises the Lactobacillus gasseri strain A21120.

[0008] Preferably, the effective viable count of the Lactobacillus gasseri strain A21120 is 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL.

[0009] Preferably, the dosage form of the probiotic comprises at least one of the following: tablets, drops, capsules and powders.

[0010] The present invention provides the use of the Lactobacillus gasseri strain A21120 or the probiotic agent in preparing a medicine having at least one of the following effects: anti-tumor, anti-inflammatory, anti-oxidation and anti-pathogen.

[0011] Preferably, the tumor or cancer type in the anti-tumor agent includes colon cancer and / or melanoma.

[0012] Preferably, the Lactobacillus gasseri strain A21120 includes live bacteria and inactivated bacteria.

[0013] Preferably, the anti-inflammatory effect is manifested in inhibiting the expression of pro-inflammatory factors;

[0014] The pro-inflammatory factors include IL-6 and / or TNF-α.

[0015] Preferably, the antioxidant activity is manifested in improving the scavenging rate of the following free radicals: DPPH, hydroxyl radicals and superoxide anions.

[0016] Preferably, the pathogens inhibited include Escherichia coli.

[0017] The invention provides a Lactobacillus gasseri strain A21120, with a deposit number of GDMCC No: 64676. The strain A21120 is isolated and screened from the intestinal feces of a healthy centenarian, and belongs to Lactobacillus gasseri after morphological identification and molecular identification. The strain is tested to have good resistance to gastrointestinal fluid and bovine choline. At the same time, the in vitro test results show that the strain A21120 has a good effect of inhibiting the growth of intestinal pathogens, and is stronger than the control strain CICC 24878; at the same time, the clearance rate of DPPH, hydroxyl free radicals and superoxide anions is better than that of the control strain CICC 24878, and has good antioxidant properties. Using lipopolysaccharide (LPS)-induced mouse mononuclear macrophage cell line RAW264.7 cells as an in vitro inflammatory cell model, an anti-inflammatory experiment was carried out, and the results showed that compared with the model group, the strain A21120 can significantly reduce the expression of pro-inflammatory factors IL-6 and TNF-α, and the anti-inflammatory effect is equivalent to that of the commercial strain LGG in the control group. The results of in vitro anti-tumor experiments showed that both live and inactivated strains of strain A21120 had an inhibitory effect on colon cancer cells and melanoma cells, and the inhibitory activity was comparable to the anti-tumor activity of the commercial strain LGG in the control group. It can be seen that the Lactobacillus gasseri strain A21120 provided by the present invention has antibacterial, anti-inflammatory, antioxidant and anti-tumor activities, providing a new approach for the treatment of clinically related diseases, drug development, and the preparation of in vitro preparations or daily necessities. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the morphological diagram of Lactobacillus gasseri strain A21120;

[0019] Figure 2 This is a graph showing the test results of the resistance of Lactobacillus gasseri strain A21120 to artificial gastrointestinal fluid;

[0020] Figure 3 This is a graph showing the test results of the resistance of Lactobacillus gasseri strain A21120 to ox bile salt;

[0021] Figure 4 This is a graph showing the anti-inflammatory function test results of Lactobacillus gasseri strain A21120;

[0022] Figure 5 The figure is a test result diagram of the inhibition of the growth of human colon cancer cell HCT116 by Lactobacillus gasseri strain A21120; the left figure is the test result of the live bacteria of Lactobacillus gasseri strain A21120, and the right figure is the test result of the inactivated bacteria of Lactobacillus gasseri strain A21120;

[0023] Figure 6 This is a graph showing the test results of Lactobacillus gasseri strain A21120 inhibiting the growth of mouse colon cancer cell MC38;

[0024] Figure 7 This is a graph showing the test results of Lactobacillus gasseri strain A21120 inhibiting the growth of mouse melanoma cells B16.

[0025] Biomaterial Deposit Information

[0026] Lactobacillus gasseri strain A21120 was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 23, 2024, with the abbreviation of GDMCC. The address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 64676. DETAILED DESCRIPTION

[0027] The invention provides a Lactobacillus gasseri strain A21120, whose deposit number is GDMCC No: 64676.

[0028] In the present invention, the strain A21120 is isolated and screened from the intestinal feces of a healthy centenarian. The morphological characteristics of the strain A21120 on the MRS plate are that the colonies are round, small, with neat edges, translucent, and smooth on the surface, and it belongs to Gram-positive bacteria. The 16SrDNA sequence of the strain A21120 is shown in SEQ ID NO:1. After sequence comparison in the NCBI database, the strain A21120 has a sequence similarity of 99.73% with Lactobacillus gasseri strain 476916S. After morphological identification and molecular identification, it belongs to Lactobacillus gasseri.

[0029] In the present invention, the survival rate of strain A21120 in artificial gastric juice of pH 2.5 for 3 hours is more than 100%, which is better than the control strain Lactobacillus agar CICC 24878, and the survival rate in intestinal juice for 7 hours is almost the same as that of the control strain, indicating that strain A21120 has good resistance to gastrointestinal juice. The survival rate of strain A21120 in 0.3g / L ox bile salt for 24 hours is still 100%, indicating that strain A21120 has excellent ox choline tolerance.

[0030] In the present invention, the culture method of the Lactobacillus gasseri strain A21120 preferably comprises the following steps: inoculating the activated Lactobacillus gasseri strain A21120 on an MRS liquid culture medium to obtain a culture solution. The culture temperature is preferably 36 to 38° C., more preferably 37° C. The culture time is preferably 12 to 16 hours, more preferably 14 hours. The culture solution is centrifuged, and the solid phase is collected to obtain the bacterial body of the Lactobacillus gasseri strain A21120. The culture solution is inoculated as a seed solution on a fresh MRS liquid culture medium to achieve expanded culture.

[0031] The invention provides a probiotic, wherein the active ingredient comprises the Lactobacillus gasseri strain A21120.

[0032] In the present invention, the probiotic refers to supplementing the beneficial bacteria in the intestine so that the beneficial bacteria have sufficient quantity and exert unique biological effects, thereby achieving the purpose of health care or disease prevention and treatment. The morphology of the Lactobacillus gasseri strain A21120 includes bacterial bodies and / or spores. The bacterial bodies of the Lactobacillus gasseri strain A21120 are the solid phase obtained by solid-liquid separation from the culture medium after the Lactobacillus gasseri strain A21120 is expanded and cultured. The spores of the Lactobacillus gasseri strain A21120 are round or oval stress-resistant dormant bodies formed in the cells after the Pediococcus pentosaceus grows and develops to a certain stage. The effective viable count of the Lactobacillus gasseri strain A21120 is preferably 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL, more preferably 1×10 10 CFU / mg or 1×10 10 CFU / mL. In the present invention, the dosage form of the probiotic preferably includes at least one of the following: tablets, drops, capsules and powders. The powder is processed by low-temperature freeze-drying technology to ensure the activity of the probiotics. The auxiliary materials of the powder preferably include freeze-drying agents, such as skimmed milk powder, etc. The powder is suitable for warm water and is suitable for infants. The tablet is prepared by mixing Lactobacillus gasseri strain A21120 and auxiliary materials and pressing them into tablets, which are suitable for chewing and taking. The auxiliary materials preferably include sweeteners or cocoa powder, etc. The capsule is preferably Lactobacillus gasseri strain A21120 alone or mixed with auxiliary materials as a capsule core in the capsule shell. The drops are made by mixing Lactobacillus gasseri strain A21120 with edible oil to isolate air and moisture. When taking, the dropper port is opened to squeeze the encapsulated oil into the mouth.

[0033] The present invention provides the use of the Lactobacillus gasseri strain A21120 or the probiotic agent in preparing a medicine having at least one of the following effects: anti-tumor, anti-inflammatory, anti-oxidation and anti-pathogen.

[0034] In the present invention, the tumor or cancer type in the anti-tumor preferably includes colon cancer and / or leukoma. The anti-tumor is preferably manifested in inhibiting the proliferation of tumor cells or cancer cells. The Lactobacillus gasseri strain A21120 preferably includes live bacteria and inactivated bacteria. The inactivation method of the inactivated bacteria is preferably to treat the Lactobacillus gasseri strain A21120 at 70°C for 30 minutes.

[0035] In one embodiment of the present invention, human colon cancer cell HCT116 and mouse colon cancer cell MC38 are used as representatives of colon cancer cells to illustrate the characteristics of Lactobacillus gasseri strain A21120 in inhibiting colon cancer growth. 6 CFU / mL of Lactobacillus gasseri strain A21120 was treated with live or inactivated bacteria for 24 to 72 hours. The results showed that both the strain A21120 and the commercial strain LGG can significantly inhibit the proliferation of colon cancer cells, and the inhibitory effect of live bacteria is more significant as time increases. However, the effect of inactivated bacteria is significantly weaker than that of live bacteria. The strain A21120 reaches a semi-inhibitory effect after 48 hours of inactivation. In another embodiment of the present invention, mouse melanoma cells B16 are used as representatives of melanoma cells to illustrate the characteristics of Lactobacillus gasseri strain A21120 in inhibiting melanoma growth. The present invention uses a concentration of 1×10 6 CFU / mL of Lactobacillus gasseri strain A21120 was treated with live or inactivated bacteria for 24 hours, and the strain A21120 can significantly inhibit the proliferation of melanoma cells, and its effect is equivalent to that of the control strain LGG; at the same time, whether it is A21120 or commercial strain LGG, the effect after inactivation is weaker than that of live bacteria. It can be seen that the Lactobacillus gasseri strain A21120 has a tumor inhibitory effect and can be used in the preparation of an application for inhibiting tumor growth in vitro.

[0036] In the present invention, the anti-inflammatory is preferably manifested in inhibiting the expression of proinflammatory factors. The proinflammatory factors preferably include IL-6 and / or TNF-α. In one embodiment of the present invention, the anti-inflammatory effect of Lactobacillus gasseri strain A21120 was verified by inducing mouse mononuclear macrophage cell line RAW264.7 cells with lipopolysaccharide (LPS, 100ng / ml) as an in vitro inflammation model. Lactobacillus gasseri strain A21120 with a concentration of 1×106CFU / mL and a final concentration of 100ng / ml LPS were treated with RAW264.7 cells for 24h, and the cell supernatant was collected. The expression level of proinflammatory factors was detected using an ELISA kit. The results showed that compared with the LPS model group, the addition of Lactobacillus gasseri A21120 under cell inflammation conditions can significantly reduce the expression of proinflammatory factors IL-6 (p<0.001) and TNF-α (p<0.0001), which is comparable to the anti-inflammatory effect of the control group LGG, indicating that the Lactobacillus gasseri strain A21120 has potential anti-inflammatory function. It can be seen that the Lactobacillus gasseri strain A21120 can be used to inhibit inflammatory response, specifically in anti-inflammatory response or prevention and / or treatment of diseases caused by inflammation.

[0037] In the present invention, the antioxidant is preferably manifested in improving the scavenging rate of the following free radicals: DPPH, hydroxyl radicals and superoxide anions, more preferably DPPH. In one embodiment of the present invention, the antioxidant capacity test results show that the scavenging rate of DPPH by the Lactobacillus gasseri strain A21120 is 88.19%, the scavenging rate of hydroxyl radicals by the Lactobacillus gasseri strain A21120 is 19%, and the scavenging rate of superoxide anions by the Lactobacillus gasseri strain A21120 is 43%, and the antioxidant capacity is better than that of the control strain CICC 24878.

[0038] In the present invention, the pathogens in the inhibited pathogens preferably include Escherichia coli. In one embodiment of the present invention, an Oxford cup antibacterial experiment was carried out to evaluate the antibacterial properties of the A21120 strain and the control strain CICC 24878 against Escherichia coli, and the results showed that the Lactobacillus gasseri A21120 had the ability to significantly inhibit the growth of pathogenic bacteria, and was stronger than the control strain 24878. Therefore, the Lactobacillus gasseri strain A21120 has the property of inhibiting intestinal pathogens and can be used in the preparation of in vitro preparations for inhibiting intestinal pathogens.

[0039] The following is a detailed description of the Lactobacillus gasseri strain A21120 with antioxidant, anti-inflammatory and anti-tumor functions and its probiotic agent and application provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Isolation and identification of Lactobacillus gasseri strain A21120

[0042] The intestinal fecal samples of centenarians were diluted in multiples, spread on MRS plates, and cultured in a biochemical incubator at 37°C for 48 hours to obtain single colonies. Pick a single colony, inoculate it in MRS liquid culture medium, and culture it in a 37°C shaker for 12 to 16 hours. Take the bacterial liquid and streak it on the MRS plate, and continue to culture a single colony. After 24 hours of culture, pick a single colony and streak it again, and continue to culture a single colony, which is the pure isolated lactobacillus obtained. Pick a single colony of pure isolated lactobacillus, perform Gram staining, and perform PCR amplification and 16S rDNA sequencing based on 16S rDNA universal primers, and compare it with the NCBI database.

[0043] The growth state of the strain in the MRS plate is as follows Figure 1 As shown, the colonies are round, small, with neat edges, translucent, and smooth surface, and are Gram-positive bacteria.

[0044] The strain A2112016SrDNA sequence is:

[0045]

[0046] Example 2

[0047] Resistance of Lactobacillus gasseri strain A21120 to artificial gastrointestinal fluid

[0048] Monoclonal colonies of the Lactobacillus gasseri A21120 and the control strain Lactobacillus gasseri CICC 24878 (also known as Lactobacillus gasseri, purchased from the China Industrial Microbiological Culture Collection Center) were picked and inoculated into MRS liquid culture medium respectively, and cultured at 37° C. in a shaking incubator overnight. The bacterial liquid was inoculated into artificial gastric juice of pH 2.5 (the formula was 10 g of pepsin, 16.4 ml of hydrochloric acid, and the volume was adjusted to 100 ml with distilled water) at an inoculum size of 10%, and 100 μl of each was applied to an MRS plate at three time points (0 h, 1 h, and 3 h). After culturing in a biochemical incubator at 37° C. for 48 h, the number of single colonies was counted. The bacterial liquid treated with artificial gastric juice for 3 h was inoculated into artificial intestinal juice of pH 6.8 (the formula was 10 g of pancreatic enzyme, 6.8 g of potassium dihydrogen phosphate, and the volume was adjusted to 1000 ml with distilled water, and the pH value was adjusted with NaOH) at an inoculum size of 10%, and 100 μl of each was applied to an MRS plate at four time points (0 h, 2 h, 4 h, and 7 h). After culturing in a biochemical incubator at 37° C. for 48 h, the number of single colonies was counted, and the survival rate was calculated according to Formula I.

[0049] Survival rate (%) = (number of single colonies at different time points / number of single colonies at 0 h) × 100% Formula I

[0050] The results are as follows Figure 2 The results show that the survival rate of strain A21120 in artificial gastric juice of pH 2.5 for 3 hours is still 100%, which is better than the control strain Lactobacillus agar CICC 24878. The survival rate of strain A21120 in artificial intestinal juice for 7 hours is almost the same as that of the control strain, indicating that strain A21120 has excellent intestinal tolerance and can successfully pass through gastrointestinal juice stimulation to reach the intestine to exert its effect.

[0051] Example 3

[0052] The tolerance of Lactobacillus gasseri strain A21120 to ox bile salts

[0053] After the strain A21120 and the control strain CICC 24878 were activated by streaking on MRS plates three times, a single clone was selected and transferred to 5 ml of MRS medium, cultured in a 37°C incubator for 12-16 hours, 1 mL was taken and added to MRS medium containing 0.1 g / L and 0.3 g / L ox bile salt, and cultured anaerobically at 37°C for 18 hours, and 100 uL of the culture solution at 0 hours, 2 hours, and 24 hours after addition was taken for gradient dilution and coating, and cultured under anaerobic conditions at 37°C for 1-2 days, and the number of viable bacteria was counted. The survival rate of the strain resistant to bile salt was calculated according to Formula II.

[0054] Bile salt tolerance survival rate = (Nh viable bacteria / 0h viable bacteria) × 100%. N represents 2h or 24h Formula II

[0055] The results are as follows Figure 3 The results showed that the survival rate of the strain A21120 was still 100% after 24 hours in the presence of 0.3 g / L ox bile salt, indicating that A21120 had excellent bile salt tolerance.

[0056] Example 4

[0057] Lactobacillus gasseri strain A21120 inhibits pathogenic bacteria function

[0058] will be in the logarithmic growth phase (concentration 1×10 9 100 μl of pathogenic bacteria Escherichia coli O157:H7 (ATCC35150) with a concentration of 100 μl CFU / mL was spread on an LB plate and placed in an Oxford cup.

[0059] The activated strain A21120 and the control strain CICC 24878 were prepared into bacterial suspensions using MRS liquid medium, and the OD 600 Adjust to 0.5 (concentration is 1×10 6 CFU / mL), 200 μl of bacterial suspension was inoculated into Oxford cups, overnight, and its effect on pathogenic bacteria was observed. At the same time, a blank control was set up, that is, only MRS liquid culture medium was inoculated into Oxford cups, overnight, and its effect on pathogenic bacteria was observed.

[0060] The results are shown in Table 1 below.

[0061] Table 1 Results of strain inhibition of pathogens

[0062]

[0063] The results showed that the Lactobacillus gasseri strain A21120 had the ability to significantly inhibit the growth of pathogenic bacteria, and was stronger than the control strain.

[0064] Example 5

[0065] Antioxidant function of Lactobacillus gasseri strain A21120

[0066] The strain A21120 and the control strain CICC 24878, which were activated by three MRS plate streaking, were prepared into bacterial suspensions, and the OD 600 To 0.5, set aside.

[0067] The DPPH free radical has a single electron, and its alcohol solution is purple and has strong absorption at 515nm. When an antioxidant is present, the DPPH free radical is scavenged, the color of its solution becomes lighter, and the absorbance at 515nm decreases. Within a certain range, the change in its absorbance is proportional to the degree to which the free radical is scavenged. The ability of the sample to scavenge DPPH free radicals is reflected by the degree of decrease in absorbance. The strain was tested according to the instructions of the DPPH free radical scavenging ability kit (Jianglai Biology).

[0068] 2-Deoxyribose in hydroxyl radical (OH - ) is oxidized to a malondialdehyde analogue in the presence of , and then condenses with thiobarbituric acid (TBA) to form a colored product. The maximum absorption peak of the colored product at 532nm is measured to calculate the hydroxyl radical (OH-) content. - ) content determination kit (Jianglai Biological) instructions for detecting the strain.

[0069] Superoxide anion (O2 - ) reacts with hydroxylamine to produce NO2 － , NO2 － Under the action of p-aminobenzenesulfonic acid and α-naphthylamine, a pink azo dye is generated. The dye has a maximum light absorption at 540nm. The O2 in the sample can be calculated based on the A540 value. - According to the content of superoxide anion (O2 - ) kit (Greith Biotech) instructions were used to detect the strain.

[0070] The test results are shown in Table 2.

[0071] Table 2 Antioxidant test results

[0072]

[0073] The results showed that the Lactobacillus gasseri strain A21120 had good antioxidant capacity, and its effect was better than that of the control strain CICC 24878.

[0074] Example 6

[0075] Detection of anti-inflammatory function of Lactobacillus gasseri strain A21120

[0076] According to previous research reports, the commercial strain Lactobacillus rhamnosus LGG has multiple functions and effects such as immunomodulation, anti-inflammatory effects, and inhibition of excessive growth of harmful bacteria, while the Lactobacillus gasseri CICC 24878 used in the above example has no relevant anti-inflammatory effect reports. Therefore, this example selects LGG as a control strain to study the anti-inflammatory function of the strain A21120 described in the present invention.

[0077] Mouse mononuclear macrophage cell line RAW264.7 cells were used as an in vitro inflammatory model after being induced by lipopolysaccharide (LPS). RAW264.7 cells were cultured in a 37°C, CO2 incubator overnight using DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody. The next day, cells were plated into 24-well plates with 2x10 cells per well. 5 After the cells were attached to the wall, four groups were set up, namely the control group Con, LPS model group, A21120 group, and LGG group, with 3 wells / group. LPS model (final concentration of 100 ng / ml), bacterial suspension (OD = 0.5, concentration of 1×10 6 CFU / mL), incubated with cells for 24 h, and the final volume of each well was 1 mL. The cell supernatant was collected by centrifugation at 4°C and 12000 rpm for 10 min, and the expression levels of inflammatory factors IL-6 and TNF-α were detected according to the instructions of the Jianglai Biological ELISA kit.

[0078] The results are as follows Figure 4 As shown, compared with the LPS model group, the addition of Lactobacillus gasseri A21120 under cellular inflammation conditions can significantly reduce the expression of proinflammatory factors IL-6 (P<0.001) and TNF-α (P<0.0001), which is equivalent to the anti-inflammatory effect of the control group LGG, indicating that the strain Lactobacillus gasseri strain A21120 has potential anti-inflammatory function.

[0079] Example 7

[0080] Detection of antitumor effect of Lactobacillus gasseri strain A21120

[0081] Some studies have reported that the commercial strain Lactobacillus rhamnosus LGG has anti-tumor effects, so the LGG strain was selected as the positive control in this experiment.

[0082] (1) Inhibitory effect of Lactobacillus gasseri strain A21120 on human colon cancer cells HCT116

[0083] HCT116 cell line was inoculated and cultured in RPMI1640 medium (TransGen) containing 10% fetal bovine serum. When the cells grew to the logarithmic phase, they were digested with trypsin and counted using a hemocytometer. The cells were evenly inoculated in a 96-well plate at 5,000 cells per well and cultured in a 37°C tri-gas incubator overnight.

[0084] The strain A21120 and the control strain LGG were taken out from the glycerol storage tube, streaked on the MRS solid plate, and cultured in a 37°C incubator. After 24 hours, a single clone was picked and cultured in 2 ml MRS liquid culture medium for 12 to 16 hours. This step was repeated once to fully activate the strain and obtain a bacterial suspension. The OD value of the bacterial suspension was adjusted. 600 =0.5, concentration is 1x10 6 CFU / mL, after washing with PBS three times, it was divided into live bacteria and inactivated bacteria (70℃30min), and added to 96-well plates in turn, 0.2mL per well. Live bacteria were incubated with cells for 2h, and inactivated bacteria were incubated with cells for 48h after inactivation. CCK-8 reagent (Solabo) was used to detect the proliferation of HCT116 cells.

[0085] The results are as follows Figure 5 As shown, the strain A21120 is viable ( Figure 5 Middle left picture) or inactivated bacteria ( Figure 5 The results of the two experiments were compared with those of the control strain LGG. However, the effect of inactivated bacteria was significantly weaker than that of live bacteria.

[0086] (2) Inhibitory effect of Lactobacillus gasseri strain A21120 on mouse colon cancer cells MC38

[0087] MC38 cell line was inoculated in DMEM medium (TransGen) containing 10% fetal bovine serum and cultured. When the cells grew to the logarithmic phase, they were digested with trypsin and counted using a hemocytometer. The cells were evenly inoculated in a 96-well plate at 5,000 cells per well and cultured in a 37°C tri-gas incubator overnight.

[0088] The strain A21120 of the present invention and the control strain LGG were taken out from the glycerol storage tube, streaked on the MRS solid plate, and cultured in a 37°C incubator. After 24 hours, a single clone was picked and cultured in 2 ml of MRS liquid culture medium for 12 to 16 hours. This step was repeated once to fully activate the strain to obtain a bacterial suspension. The OD value of the bacterial suspension was adjusted. 600 =0.5, concentration is 1×10 6CFU / mL, washed with PBS three times, divided into live bacteria and inactivated bacteria (70℃, 30min), and added to 96-well plates in sequence, 0.2mL per well. Incubated with cells for 24h, 48h, and 72h respectively. CCK-8 reagent (Solebio) was used to detect MC38 cell proliferation.

[0089] The results are as follows Figure 6 As shown, the strain A21120 and the commercial strain LGG can significantly inhibit the proliferation of colon cancer cells, and the inhibitory effect of live bacteria is more significant as time increases (P<0.0001). However, the effect of inactivated bacteria is significantly weaker than that of live bacteria, and the strain A21120 reaches a half-inhibitory effect at 48 hours after inactivation (P<0.0001).

[0090] (3) Inhibitory effect of Lactobacillus gasseri cultured in A21120 on mouse melanoma B16 cells

[0091] B16 cell line was inoculated in DMEM medium (TransGen) containing 10% fetal bovine serum and cultured. When the cells grew to the logarithmic phase, they were digested with trypsin and counted using a hemacytometer. The cells were evenly inoculated in a 96-well plate at 5,000 cells per well and cultured in a 37°C tri-gas incubator overnight.

[0092] The strain A21120 of the present invention and the control strain LGG were taken out from the glycerol storage tube, streaked on the MRS solid plate, and cultured in a 37°C incubator. After 24 hours, a single clone was picked and cultured in 2 ml of MRS liquid culture medium for 12 to 16 hours. This step was repeated once to fully activate the strain to obtain a bacterial suspension. The OD value of the bacterial suspension was adjusted. 600 =0.5, concentration is 1×10 6 CFU / mL, washed with PBS three times, divided into live bacteria and inactivated bacteria (70℃, 30min), added to 96-well plates in sequence, 0.2mL per well. Incubated with cells for 24h. CCK-8 reagent (Solabo) was used to detect cell proliferation.

[0093] The results are as follows Figure 7 As shown, the strain A21120 can significantly inhibit the proliferation of melanoma cells (P<0.0001), and its effect is comparable to that of the control strain LGG. Whether it is A21120 or the commercial strain LGG, the effect after inactivation is weaker than that of live bacteria.

[0094] In summary, the strain A21120 has potential anti-tumor ability, which may be dependent on the live bacteria themselves or the metabolites secreted by them, and the effect of the inactivated bacterial cells is weaker. The reason why the strain can play a potential anti-tumor cell growth effect after inactivation may be due to the effect of the inactivated bacterial protein.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus gasseri strain A21120, characterized in that The deposit number is GDMCC No:64676.

2. A probiotic, characterized in that: The active ingredient comprises the Lactobacillus gasseri strain A21120 described in claim 1.

3. The probiotic according to claim 2, characterized in that: The effective viable count of the Lactobacillus gasseri strain A21120 is 1×10 9 CFU / mg~1×10 11 CFU / mg or 1×10 9 CFU / mL~1×10 11 CFU / mL.

4. The probiotic according to claim 2 or 3, characterized in that: The dosage form of the probiotics includes at least one of the following: tablets, drops, capsules and powders.

5. Use of the Lactobacillus gasseri strain A21120 according to claim 1 or the probiotic according to any one of claims 2 to 4 in the preparation of a medicament having at least one of the following effects: anti-tumor, anti-inflammatory, anti-oxidation and inhibition of pathogens.

6. The use according to claim 5, characterized in that: The tumor or cancer type described herein includes colon cancer and / or melanoma.

7. The use according to claim 6, characterized in that: The Lactobacillus gasseri strain A21120 includes live bacteria and inactivated bacteria.

8. The use according to claim 5, characterized in that: The anti-inflammatory effect is manifested in inhibiting the expression of pro-inflammatory factors; The pro-inflammatory factors include IL-6 and / or TNF-α.

9. The use according to claim 5, characterized in that: The antioxidant effect is manifested in improving the scavenging rate of the following free radicals: DPPH, hydroxyl radical and superoxide anion.

10. The use according to claim 5, characterized in that: The pathogens inhibited include Escherichia coli.

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