Lactobacillus gasseri strain a21120 with antioxidant, anti-inflammatory and anti-tumor functions, probiotic agent and application thereof

Abstract

The application provides a lactobacillus gasseri strain A21120 with antioxidant, anti-inflammatory and antitumor functions, and a probiotic agent and application thereof, and belongs to the technical field of functional microorganisms. The strain A21120 is obtained by screening from intestinal feces of a centenarian healthy old person, and is identified by morphology and molecules and belongs to lactobacillus gasseri. The strain has good resistance to gastrointestinal fluid and bovine choline capacity, and has the potential to colonize the intestinal tract. The lactobacillus gasseri strain A21120 provided by the application has bacteriostatic, anti-inflammatory, antioxidant and antitumor activities, and provides a new way for the treatment of clinical related diseases, the development of drugs and the preparation of in vitro preparations or daily necessities.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial technology, specifically relating to a type of Lactobacillus gasseri with antioxidant, anti-inflammatory, and anti-tumor functions. Lactobacillus gasseri Strain A21120 and its probiotic agents and applications. Background Technology

[0002] Lactobacillus gasseri is a type of probiotic that can be used in food. It can colonize the human gut and exert beneficial effects on the host by altering the composition of the host microbiota. Timely supplementation of probiotics in the diet is extremely important for people exhibiting related symptoms or diseases.

[0003] Today, gut microbiota play an indispensable role in people's lives. They not only maintain gut health but also regulate various signaling pathways through metabolites, making them closely related to various diseases. For example, lactobacilli, which live symbiotically in the human gut, are beneficial bacteria that play an important role in inhibiting pathogen invasion, alleviating intestinal inflammation and damage, delaying aging, and even resisting the development and progression of tumors. Patent publication number CN117363524A discloses a strain of Lactobacillus gasseri MY4, which has antioxidant, whitening, and anti-aging effects. Patent publication number CN111518724A discloses a strain of Lactobacillus gasseri HMV18, whose exuded proteins have antibacterial and antitumor effects and can be used to prepare antibacterial and antitumor products. Therefore, probiotics have potential application value and broad market prospects.

[0004] However, there are currently no reports on Lactobacillus gasseri possessing antioxidant, anti-inflammatory, and anti-tumor functions simultaneously. Summary of the Invention

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

[0006] This invention provides a Lactobacillus gasseri ( Lactobacillus gasseri The strain is A21120, with the accession number GDMCC No: 64676.

[0007] The present invention provides a probiotic agent, the active ingredient of which includes the Lactobacillus gasseri strain A21120.

[0008] Preferably, the effective viable count of the Lactobacillus gasseri strain A21120 is 1×10⁻⁶. 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 agent includes 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 the preparation of a drug with at least one of the following effects: antitumor, anti-inflammatory, antioxidant and anti-pathogenic.

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

[0012] Preferably, the Lactobacillus gasseri strain A21120 comprises live cells and inactivated cells.

[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 effect 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] This invention provides a strain of *Lactobacillus gasseri*, A21120, with accession number GDMCC No: 64676. Strain A21120 was isolated and screened from the intestinal feces of a healthy centenarian. After morphological and molecular identification, it was determined to belong to *Lactobacillus gasseri*. Lactobacillus gasseri The strain was tested and found to have good resistance to gastrointestinal fluids and bovine bile.

[0018] Meanwhile, in vitro detection results showed that strain A21120 had a good inhibitory effect on the growth of intestinal pathogens, which was stronger than that of the control strain CICC 24878; at the same time, its scavenging rate of DPPH, hydroxyl radicals and superoxide anions was better than that of the control strain CICC 24878, indicating good antioxidant activity. Using lipopolysaccharide (LPS)-induced mouse mononuclear macrophage line RAW264.7 cells as an in vitro inflammatory cell model, anti-inflammatory experiments were conducted. The results showed that, compared with the model group, strain A21120 significantly reduced the expression of pro-inflammatory factors IL-6 and TNF-α, and its anti-inflammatory effect was comparable to that of the control group commercial strain LGG. In vitro anti-tumor experiments showed that both live and inactivated strain A21120 inhibited the proliferation of colon cancer cells and melanoma cells, and the inhibitory activity was comparable to that of the control group commercial strain LGG. It is evident that the Lactobacillus gasseri strain A21120 provided by this invention possesses antibacterial, anti-inflammatory, antioxidant, and antitumor activities, offering a new approach for the treatment of clinically relevant diseases, drug development, and the preparation of in vitro formulations or daily necessities. Attached Figure Description

[0019] Figure 1 Morphological diagram of Lactobacillus gasseri strain A21120;

[0020] Figure 2 The image shows the results of the test for resistance of Lactobacillus gastroenteritis strain A21120 to artificial gastrointestinal fluid.

[0021] Figure 3 The image shows the results of bovine bile salt resistance testing for Lactobacillus gasseri strain A21120.

[0022] Figure 4 Figure showing the results of anti-inflammatory function detection of Lactobacillus gasseri strain A21120;

[0023] Figure 5 The image shows the detection results of Lactobacillus gasseri strain A21120 inhibiting the growth of human colon cancer cells HCT116; the left image shows the detection results of live Lactobacillus gasseri strain A21120, and the right image shows the detection results of inactivated Lactobacillus gasseri strain A21120.

[0024] Figure 6 The image shows the detection results of Lactobacillus gasseri strain A21120 inhibiting the growth of mouse colon cancer cells MC38;

[0025] Figure 7 The image shows the detection results of Lactobacillus gasseri strain A21120 inhibiting the growth of mouse melanoma cells B16.

[0026] Information on the preservation of biological materials

[0027] Lactobacillus gasseri ( Lactobacillus gasseriStrain A21120 was deposited on May 23, 2024 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No: 64676. Detailed Implementation

[0028] This invention provides a Lactobacillus gasseri ( Lactobacillus gasseri The strain is A21120, with the accession number GDMCC No: 64676.

[0029] In this invention, strain A21120 was isolated and screened from the intestinal feces of a healthy centenarian. The morphological characteristics of strain A21120 on MRS plates are: small, round colonies with regular edges, semi-transparent, and smooth surfaces, indicating it is a Gram-positive bacterium. The 16S rDNA sequence of strain A21120 is shown in SEQ ID NO:1. Sequence alignment with the NCBI database confirms that strain A21120 is similar to... Lactobacillus gasseri The sequence similarity of strain 476916S was 99.73%. Morphological and molecular identification confirmed it belongs to *Lactobacillus gasseri*. Lactobacillus gasseri ).

[0030] In this invention, strain A21120 exhibited a survival rate exceeding 100% in simulated gastric fluid at pH 2.5 for 3 hours, which was superior to the control strain Lactobacillus additive CICC 24878. Its survival rate in intestinal fluid after 7 hours was nearly identical to the control strain, indicating that strain A21120 possesses excellent resistance to gastrointestinal fluids. Furthermore, strain A21120 maintained a 100% survival rate in 0.3 g / L ox bile salts for 24 hours, demonstrating excellent ox bile alkaloid tolerance.

[0031] In this invention, the method for culturing the *Lactobacillus gasseri* strain A21120 preferably includes the following steps: Inoculating the activated *Lactobacillus gasseri* strain A21120 onto MRS liquid medium and culturing to obtain a culture solution. The culturing temperature is preferably 36-38°C, more preferably 37°C. The culturing time is preferably 12-16 hours, more preferably 14 hours. The culture solution is centrifuged, and the solid phase is collected to obtain *Lactobacillus gasseri* strain A21120 cells. The culture solution is used as a seed culture and inoculated onto fresh MRS liquid medium for culture expansion.

[0032] The present invention provides a probiotic agent, the active ingredient of which includes the Lactobacillus gasseri strain A21120.

[0033] In this invention, the probiotic agent refers to supplementing the intestinal flora with beneficial bacteria, ensuring a sufficient number of these bacteria to exert unique biological effects, thereby achieving the purpose of health care, disease prevention, and treatment. The *Lactobacillus gasseri* strain A21120 has the morphology of cells and / or spores. The *Lactobacillus gasseri* strain A21120 cells are the solid phase obtained by solid-liquid separation from the culture medium after expanded culture of *Lactobacillus gasseri* strain A21120. The *Lactobacillus gasseri* strain A21120 spores are round or oval, stress-resistant dormant bodies formed within the cells of *Pediococcus pentosaceus* after reaching a certain stage of growth and development. The preferred effective viable count of the *Lactobacillus gasseri* strain A21120 is 1 × 10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL, more preferably 1×10⁻⁶ 10 CFU / mg or 1×10 10 CFU / mL. In this invention, the dosage form of the probiotic agent preferably includes at least one of the following: tablets, drops, capsules, and powder. The powder is processed using low-temperature freeze-drying technology to ensure the activity of the probiotics. The excipients of the powder preferably include freeze-drying agents, such as skim milk powder. The powder is suitable for taking with warm water and is suitable for infants. The tablets are prepared by mixing Lactobacillus gasseri strain A21120 with excipients and compressing them into tablets, suitable for chewing. The excipients preferably include sweeteners or cocoa powder. The capsules preferably contain Lactobacillus gasseri strain A21120 alone or mixed with excipients as a core packaged in a capsule shell. The drops are prepared by mixing Lactobacillus gasseri strain A21120 with edible oil to form drops that are isolated from air and moisture; when taking the drops, the dropper is opened and the sealed oily substance is squeezed into the mouth.

[0034] The present invention provides the use of the Lactobacillus gasseri strain A21120 or the probiotic agent in the preparation of a drug with at least one of the following effects: antitumor, anti-inflammatory, antioxidant and anti-pathogenic.

[0035] In this invention, the tumor or cancer type in the antitumor treatment preferably includes colon cancer and / or melanoma. The antitumor effect is preferably manifested in inhibiting the proliferation of tumor cells or cancer cells. The *Lactobacillus gasseri* strain A21120 preferably comprises live and inactivated cells. The inactivation method for the inactivated cells is preferably treating *Lactobacillus gasseri* strain A21120 at 70°C for 30 minutes.

[0036] In one embodiment of the present invention, human colon cancer cells HCT116 and mouse colon cancer cells MC38 are used as representatives of colon cancer cells to illustrate the inhibitory effect of Lactobacillus gasseri strain A21120 on colon cancer growth. The present invention uses a concentration of 1×10⁻⁶. 6Lactobacillus gasseri strain A21120 (CFU / mL) was used to treat cells for 24–72 h with either live or inactivated bacteria. Results showed that strain A21120 and the commercial strain LGG significantly inhibited colon cancer cell proliferation, with the inhibitory effect of live bacteria becoming more pronounced over time. However, the effect of inactivated bacteria was significantly weaker than that of live bacteria; strain A21120 reached half-inhibition after 48 h of inactivation. In another embodiment of this invention, mouse melanoma cells B16 were used as representatives of melanoma cells to illustrate the melanoma-inhibiting properties of Lactobacillus gasseri strain A21120. This invention uses a concentration of 1 × 10⁻⁶ CFU / mL. 6 Treating cells with CFU / mL of *Lactobacillus gasseri* strain A21120, either live or inactivated, for 24 hours significantly inhibited melanoma cell proliferation, with effects comparable to the control strain LGG. Furthermore, both A21120 and the commercial strain LGG showed weaker effects after inactivation compared to live bacteria. Therefore, *Lactobacillus gasseri* strain A21120 exhibits tumor-suppressive activity and can be used in the preparation of in vitro tumor growth inhibitors.

[0037] In this invention, the anti-inflammatory effect is preferably manifested in inhibiting the expression of pro-inflammatory factors. The pro-inflammatory factors preferably include IL-6 and / or TNF-α. In one embodiment of this invention, mouse mononuclear macrophage cell line RAW264.7 cells were induced with lipopolysaccharide (LPS, 100 ng / ml) as an in vitro inflammation model to verify the anti-inflammatory effect of Lactobacillus gasseri strain A21120. The concentration was 1 × 10⁻⁶. 6 RAW264.7 cells were treated with LPS at a final concentration of 100 ng / ml and CFU / mL of *Lactobacillus gasseri* strain A21120 for 24 h. Cell supernatant was collected, and the expression levels of pro-inflammatory factors were detected using an ELISA kit. The results showed that, compared with the LPS model group, the addition of *Lactobacillus gasseri* strain A21120 under inflammatory conditions significantly reduced the expression of pro-inflammatory factors IL-6 (p<0.001) and TNF-α (p<0.0001), comparable to the anti-inflammatory effect of the control group LGG. This indicates that *Lactobacillus gasseri* strain A21120 has potential anti-inflammatory function. Therefore, *Lactobacillus gasseri* strain A21120 can be used to inhibit inflammatory responses, specifically in anti-inflammatory responses or in the prevention and / or treatment of diseases caused by inflammation.

[0038] In this invention, the antioxidant effect 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 this invention, the antioxidant capacity test results show that the *Lactobacillus gasseri* strain A21120 has a DPPH scavenging rate of 88.19%, a hydroxyl radical scavenging rate of 19%, and a superoxide anion scavenging rate of 43%, which is superior to the control strain CICC 24878.

[0039] In this invention, the pathogenic bacteria inhibited preferably include *Escherichia coli*. In one embodiment of this invention, an Oxford cup method antibacterial experiment was conducted to evaluate the antibacterial properties of strain A21120 and the control strain CICC 24878 against *Escherichia coli*. The results showed that *Lactobacillus gasseri* A21120 significantly inhibited the growth of pathogenic bacteria, and its ability was stronger than that of the control strain 24878. Therefore, *Lactobacillus gasseri* strain A21120 has the property of inhibiting intestinal pathogens and can be used in the preparation of formulations that inhibit intestinal pathogens in vitro.

[0040] The following detailed description, in conjunction with embodiments, illustrates a strain of Lactobacillus gasseri A21120 with antioxidant, anti-inflammatory, and anti-tumor functions, its probiotic agent, and its applications, but these should not be construed as limiting the scope of protection of this invention.

[0041] Example 1

[0042] Isolation and identification of Lactobacillus gasseri strain A21120

[0043] Intestinal fecal samples from centenarians were serially diluted and spread onto MRS plates. They were incubated at 37°C for 48 hours to obtain single colonies. Single colonies were picked, inoculated onto MRS liquid medium, and incubated on a shaker at 37°C for 12–16 hours. Bacterial culture was streaked onto MRS plates to continue culturing single colonies. After 24 hours of incubation, single colonies were picked again and streaked repeatedly to obtain pure isolates of *Lactobacillus*. Pure isolates of *Lactobacillus* were selected, Gram-stained, and simultaneously subjected to PCR amplification using universal 16S rDNA primers and 16S rDNA sequencing. The results were then compared to the NCBI database.

[0044] The growth status of the strain in the MRS plate is as follows: Figure 1 As shown, the colonies are round, small, with neat edges, semi-transparent, and smooth, belonging to Gram-positive bacteria.

[0045] The rDNA sequence of strain A2112016S is as follows:

[0046] Lactobacillusgasseri The strain 476916S had a sequence similarity of 99.73% and was identified as *Lactobacillus gasseri*. This invention deposited the strain A21120 at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 64676.

[0047] Example 2

[0048] Lactobacillus gastroenteritis strain A21120 has resistance to artificial gastrointestinal fluids.

[0049] Single colonies of *Lactobacillus gasseri* A21120 and the control strain *Lactobacillus gasseri* CICC 24878 (also known as *Lactobacillus gasseri*, purchased from the China Industrial Microbial Culture Collection Center) were picked and inoculated into MRS liquid medium, respectively, and cultured overnight at 37°C on a shaker. At a 10% inoculation rate, the bacterial suspension was inoculated into artificial gastric fluid (pH 2.5, formulation: 10g pepsin, 16.4ml hydrochloric acid, and distilled water to a final volume of 100ml). Three time points were set (0h, 1h, and 3h), and 100μl of each time was spread onto MRS plates. After incubation at 37°C for 48h, the number of single colonies was counted. The bacterial suspension treated with artificial gastric fluid for 3h was then inoculated into artificial intestinal fluid (pH 6.8, formulation: 10g pancreatin, 6.8g potassium dihydrogen phosphate, and distilled water to a final volume of 100ml) at a 10% inoculation rate.

[0050] 1000ml (pH adjusted with NaOH) was used to set 4 time points (0h, 2h, 4h, 7h). 100μl of each time was spread on MRS plates and incubated at 37℃ for 48h in a biochemical incubator. The number of single colonies was counted and the survival rate was calculated according to Formula I.

[0051] Survival rate (%) = (Number of single colonies at different time points / Number of single colonies at 0h) × 100% Formula I

[0052] The results are as follows Figure 2 The results showed that strain A21120 maintained a 100% survival rate in simulated gastric fluid at pH 2.5 for 3 hours, which was superior to the control strain Lactobacillus additive CICC 24878. The survival rate of strain A21120 in simulated intestinal fluid for 7 hours was almost identical to that of the control strain, indicating that strain A21120 has excellent intestinal tolerance and can successfully reach the intestines and exert its effects through stimulation by gastrointestinal fluid.

[0053] Example 3

[0054] Lactobacillus gasseri strain A21120's tolerance to bovine bile salts

[0055] After three MRS streak activation tests, strain A21120 and control strain CICC 24878 were selected and transferred to 5 ml of MRS medium. The cultures were incubated at 37°C for 12-16 h. 1 ml of each medium was then added to MRS medium containing 0.1 g / L and 0.3 g / L ox bile salts, respectively, and cultured anaerobically at 37°C for 18 h. 100 μL of the culture at 0 h, 2 h, and 24 h after addition was serially diluted and plated, and then incubated anaerobically at 37°C for 1-2 days. The viable cell count was then recorded. The bile salt tolerance survival rate of the strain was calculated according to Formula II.

[0056] Bile salt tolerance survival rate = (Nh viable bacteria count / 0h viable bacteria count) × 100%. N represents 2h or 24h. Formula II

[0057] The results are as follows Figure 3 The results showed that strain A21120 still had a 100% survival rate after 24 hours in 0.3 g / L bovine bile salts, indicating that A21120 has excellent bile salt tolerance.

[0058] Example 4

[0059] Lactobacillus gasseri strain A21120 has the function of inhibiting pathogens.

[0060] It will be in the logarithmic growth phase (concentration of 1×10⁻⁶). 9 100 μl of the pathogenic Escherichia coli O157:H7 (ATCC35150) (CFU / mL) was spread on an LB agar plate and placed in an Oxford cup.

[0061] The activated strain A21120 and the control strain CICC 24878 were prepared into bacterial suspensions using MRS liquid medium, and the OD values ​​of the bacterial suspensions were measured. 600 Adjust to 0.5 (concentration of 1×10). 6 (CFU / mL) 200 μl of bacterial suspension was inoculated into Oxford cups and incubated overnight to observe its effect on pathogenic bacteria. A blank control was also set up, inoculated into Oxford cups using only MRS liquid medium and incubated overnight to observe its effect on pathogenic bacteria.

[0062] The results are shown in Table 1 below.

[0063] Table 1 Results of pathogen inhibition detection by bacterial strains

[0064]

[0065] The results showed that the Lactobacillus gasseri strain A21120 had a significant ability to inhibit the growth of pathogenic bacteria, and this ability was stronger than that of the control strain.

[0066] Example 5

[0067] Antioxidant Function of Lactobacillus gasseri strain A21120

[0068] The strain A21120, which had undergone three MRS streak activation tests, and the control strain CICC 24878 were prepared into bacterial suspensions, and the OD values ​​were adjusted. 600 Set to 0.5, for later use.

[0069] DPPH free radicals have a single electron, and their alcoholic solution is purple with strong absorption at 515 nm. In the presence of antioxidants, DPPH free radicals are scavenged, the solution color lightens, and the absorbance at 515 nm decreases. Within a certain range, the change in absorbance is directly proportional to the degree of free radical scavenging. The degree of absorbance decrease reflects the sample's ability to scavenge DPPH free radicals. The strains were tested according to the instructions of the DPPH free radical scavenging kit (Jianglai Biotechnology).

[0070] 2-Deoxyribose in hydroxyl radicals (OH) - In the presence of [a substance], it is oxidized to a malondialdehyde analog, which then condenses with thiobarbituric acid (TBA) to form a colored product. By measuring the maximum absorption peak of this colored product at 532 nm, the hydroxyl radical (OH) can be calculated. - ) content. According to hydroxyl radical (OH) content. - The assay kit (Jianglai Biotechnology) was used to test the two strains.

[0071] Superoxide anion (O2) - ) reacts with hydroxylamine to produce NO2 － NO2 － A pink azo dye is formed by the reaction of p-aminobenzenesulfonic acid and α-naphthylamine. This dye has maximum light absorption at 540 nm. The O2 in the sample can be calculated based on the A540 value. - The content. According to the superoxide anion (O2) content. - The two strains were tested according to the kit (GraceBio) instructions.

[0072] The test results are shown in Table 2.

[0073] Table 2. Results of antioxidant activity tests on the two strains.

[0074]

[0075] The results showed that the Lactobacillus gasseri strain A21120 had good antioxidant capacity, which was better than that of the control strain CICC 24878.

[0076] Example 6

[0077] Anti-inflammatory function detection of Lactobacillus gasseri strain A21120

[0078] According to previous research reports, the commercial strain *Lactobacillus rhamnosus* LGG ( Lacticaseibacillus rhamnosus Lactobacillus gasseri (LGG) possesses various effects and functions, including immunomodulation, anti-inflammatory activity, and inhibition of harmful bacterial overgrowth. However, no reports have been found regarding the anti-inflammatory effects of Lactobacillus gasseri CICC 24878 used in the above examples. Therefore, this example uses LGG as a control strain to study the anti-inflammatory function of strain A21120 described in this invention.

[0079] RAW264.7 mouse mononuclear macrophage cell line was used as an in vitro inflammation model after induction with lipopolysaccharide (LPS). RAW264.7 cells were cultured overnight in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C in a CO2 incubator. The next day, the cells were seeded into 24-well plates at 2 x 10⁶ cells per well. 5 After cell adhesion, four groups were set up: a control group (Con), an LPS modeling group, an A21120 group, and an LGG group, with 3 wells per group. LPS (final concentration 100 ng / ml) and bacterial suspension (OD = 0.5, concentration 1 × 10⁻⁶) were added sequentially. 6 Cells were incubated with ELISA kits containing CFU / mL for 24 hours, with a final volume of 1 mL per well. The cell supernatant was collected by centrifugation at 4°C and 12,000 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 Biotechnology ELISA kit.

[0080] 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 pro-inflammatory 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.

[0081] Example 7

[0082] Detection of antitumor activity of Lactobacillus gasseri strain A21120

[0083] Studies have reported that the commercial strain Lactobacillus rhamnosus LGG has anti-tumor effects, therefore the LGG strain was selected as the positive control in this experiment.

[0084] (1) Inhibitory effect of Lactobacillus gasseri strain A21120 on human colon cancer cells HCT116.

[0085] HCT116 cell line was seeded in RPMI 1640 medium (TransGen) containing 10% fetal bovine serum and cultured. When the cells grew to the logarithmic growth phase, they were digested with trypsin and counted by hemocytometer. The cells were seeded evenly in 96-well plates at 5000 cells per well and cultured overnight in a 37°C tri-gas incubator.

[0086] The strain A21120 and the control strain LGG were removed from the glycerol storage tube, streaked on MRS solid plates, and incubated at 37°C. After 24 hours, single colonies were picked and cultured in 2 ml of MRS liquid medium for 12-16 hours. This step was repeated once to fully activate the strains and obtain a bacterial suspension. The OD of the bacterial suspension was adjusted. 600 =0.5, concentration is 1x10 6 After washing three times with PBS, HCT116 cells were divided into live and inactivated bacteria (70℃ for 30 min) and added sequentially to 96-well plates, 0.2 mL per well. Live bacteria were co-incubated with cells for 2 h, and inactivated bacteria were co-incubated with cells for 48 h after inactivation. HCT116 cell proliferation was detected using CCK-8 reagent (Solepro).

[0087] The results are as follows Figure 5 As shown, strain A21120, whether live or dead, is... Figure 5 (Middle left image) or inactivated bacteria ( Figure 5 (See the right image in the middle). Both strains significantly inhibited the proliferation of human colon cancer cells HCT116, with effects comparable to the control strain LGG. However, the effect of inactivated bacteria was significantly weaker than that of live bacteria.

[0088] (2) Inhibitory effect of Lactobacillus gasseri strain A21120 on mouse colon cancer cells MC38

[0089] MC38 cell lines were seeded in DMEM (TransGen) medium containing 10% fetal bovine serum and cultured. When the cells reached the logarithmic growth phase, they were digested with trypsin and counted using a hemocytometer. Cells were seeded evenly in 96-well plates at a density of 5000 cells per well and cultured overnight in a 37°C tri-gas incubator.

[0090] The strain A21120 and the control strain LGG described in this invention were removed from the glycerol preservation tube, streaked on MRS solid plates, and incubated at 37°C. After 24 hours, single colonies were picked and cultured in 2 ml of MRS liquid medium for 12-16 hours. This step was repeated once to fully activate the strains and obtain a bacterial suspension. The OD of the bacterial suspension was adjusted. 600 =0.5, concentration is 1×10 6After washing three times with PBS, CFU / mL of the culture was divided into live and inactivated cultures (70℃, 30 min), and 0.2 mL was added to each well of a 96-well plate. The cells were then co-incubated for 24 h, 48 h, and 72 h, respectively. The proliferation of MC38 cells was detected using the CCK-8 reagent (Solepro).

[0091] The results are as follows Figure 6 As shown, both strain A21120 and the commercial strain LGG significantly inhibited the proliferation of colon cancer cells, and the inhibitory effect of live bacteria became more significant with increasing time (P<0.0001). However, the effect of inactivated bacteria was significantly weaker than that of live bacteria; strain A21120 reached half-inhibition effect after 48 hours of inactivation (P<0.0001).

[0092] (3) Inhibitory effect of Lactobacillus gasseri culture on mouse melanoma cells B16 in A21120

[0093] B16 cell lines were seeded in DMEM (TransGen) medium containing 10% fetal bovine serum and cultured. When the cells reached the logarithmic growth phase, they were digested with trypsin and counted using a hemocytometer. The cells were then seeded evenly in 96-well plates at a density of 5000 cells per well and incubated overnight in a 37°C tri-gas incubator.

[0094] The strain A21120 and the control strain LGG described in this invention were removed from the glycerol preservation tube, streaked on MRS solid plates, and incubated at 37°C. After 24 hours, single colonies were picked and cultured in 2 ml of MRS liquid medium for 12-16 hours. This step was repeated once to fully activate the strains and obtain a bacterial suspension. The OD of the bacterial suspension was adjusted. 600 =0.5, concentration is 1×10 6 CFU / mL, after washing three times with PBS, was divided into live and inactivated bacterial cultures (70℃, 30 min), and 0.2 mL was added sequentially to each well of a 96-well plate. The plates were then co-incubated with cells for 24 h. Cell proliferation was assessed using the CCK-8 reagent (Solepro).

[0095] The results are as follows Figure 7 As shown, strain A21120 significantly inhibited melanoma cell proliferation (P<0.0001), with an effect comparable to the control strain LGG. However, the inactivated strains of both A21120 and the commercial strain LGG were less effective than the live strains.

[0096] In summary, strain A21120 possesses potential anti-tumor activity, possibly relying on the live bacteria themselves or their secreted metabolites; the effect of the inactivated bacterial cells is weaker. It is speculated that the inactivated strain's potential anti-tumor cell growth effect may be due to the activity of the inactivated bacterial proteins.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Lactobacillus gasseri ( Lactobacillus gasseri strain A21120, characterized in that, The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the accession number is GDMCC No: 64676.

2. A probiotic agent, characterized in that, The active ingredient includes the Lactobacillus gasseri strain A21120 as described in claim 1.

3. The probiotic agent according to claim 2, characterized in that, The effective viable count of the Lactobacillus gasseri strain A21120 was 1 × 10⁻⁶. 9 CFU / mg ~ 1×10 11 CFU / mg or 1×10 9 CFU / mL ~ 1×10 11 CFU / mL.

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

5. The use of the Lactobacillus gasseri strain A21120 according to claim 1 or the probiotic agent according to any one of claims 2 to 4 in the preparation of an antitumor drug. The tumor is colon cancer and / or melanoma; The Lactobacillus gasseri strain A21120 was a live bacterial cell.

6. The use of the Lactobacillus gasseri strain A21120 of claim 1 in the preparation of a medicament that is anti-inflammatory, antioxidant, and inhibits at least one pathogen, wherein the pathogen is Escherichia coli.

7. The application according to claim 6, 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.

8. The application according to claim 6, characterized in that, The antioxidant effect is manifested in improving the scavenging rate of the following free radicals: DPPH, hydroxyl radicals and superoxide anions.

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