A fermented Lactobacillus mucosae strain A21038 with antioxidant, anti-aging and anti-tumor activities and its applications

Abstract

The present invention provides a Limosilactobacillus fermentum strain A21038 with antioxidant, anti-aging and anti-tumor activities and its applications, belonging to the technical field of functional microorganisms. The strain A21038 was isolated and screened from the intestinal feces of centenarian healthy elderly people, and through morphological identification and molecular identification, it belongs to Limosilactobacillus fermentum. The strain A21038 has good abilities of resisting gastrointestinal fluids and taurocholic acid. The Limosilactobacillus fermentum strain A21038 provided by the present invention has antibacterial, anti-inflammatory, antioxidant and anti-tumor activities, providing a new way for the treatment of clinically related diseases, drug development, and the preparation of in vitro preparations or daily necessities.

Description

Technical Field

[0001] The present invention belongs to the technical field of functional microorganisms, and particularly relates to a Limosilactobacillus fermentum strain A21038 with antioxidant, anti-aging and anti-tumor activities and its application. Background Art

[0002] Intestinal microorganisms have played an important role in people's lives. They can not only maintain intestinal health, but also regulate various signaling pathways through metabolites, which is closely related to the occurrence and treatment of various diseases. For example, Lactobacillus, which coexists with the human intestine, is a beneficial bacterium and plays an important role in inhibiting the invasion of pathogenic bacteria, alleviating intestinal inflammation and damage, delaying aging, and even resisting the occurrence and development of tumors. Limosilactobacillus fermentum is a type of probiotic that can be used in food approved by the Ministry of Health. It can colonize in the human intestine, change the composition of the host flora and produce beneficial effects on the host. Timely addition of some active probiotics in the diet is extremely important for people showing related symptoms or diseases. For example, the patent with publication number CN113234612A discloses a Limosilactobacillus fermentum ZS40 with a preventive effect on colitis. This strain can better relieve the occurrence of inflammation by inhibiting the activation of NF-κB and MAPK signaling pathways.

[0003] Redox reactions are constantly occurring in the human body. The skin is the first barrier of the human body, and aging and damage are inevitable. The patent with publication number CN117363524A discloses a Lactobacillus gasseri MY4 and its application in the preparation of sleep-aiding and whitening food and drugs. This strain has the effects of antioxidant, whitening and anti-aging. In addition, the research by Luo et al. reported a Limosilactobacillus reuteri FLRE5K1 strain. The experimental results showed that it could significantly reduce the number of melanoma cells in mice and had no toxic side effects on non-cancerous tissues, with anti-melanoma activity. It can be seen that Lactobacillus, as a probiotic, has extensive applications in antioxidant and anti-tumor aspects. However, there is currently no report on Lactobacillus with both antioxidant, anti-aging and anti-tumor activities. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of Limosilactobacillus fermentum in the preparation of products with antioxidant, anti-aging and anti-tumor effects.

[0005] The purpose of the present invention is also to provide a novel Limosilactobacillus fermentum strain A21038, which has antioxidant, anti-aging and anti-tumor activities at the same time.

[0006] The present invention provides the use of Limosilactobacillus fermentum in the preparation of products having at least two of the following functions: antioxidant, anti-aging, antibacterial, and anti-tumor.

[0007] Preferably, the antioxidant performance is manifested in the scavenging ability for at least one of the following: DPPH radical, hydroxyl radical, superoxide anion, and H2O2.

[0008] Preferably, the anti-aging performance is manifested in having superoxide dismutase activity and / or glutathione peroxidase activity.

[0009] Preferably, when preparing antioxidant and / or anti-aging products, the products include at least one of the following: drugs, health products, cosmetics, and foods.

[0010] Preferably, the bacteria in the antibacterial property include Escherichia coli.

[0011] Preferably, the tumors or cancers in the anti-tumor property include colon cancer and / or melanoma.

[0012] Preferably, when preparing antibacterial and / or anti-tumor products, the products include drugs.

[0013] The present invention provides a Limosilactobacillus fermentum strain A21038 with a deposit number of GDMCC No: 64675.

[0014] The present invention provides a probiotic agent, the active ingredient of which includes the Limosilactobacillus fermentum strain A21038.

[0015] The present invention provides the use of the Limosilactobacillus fermentum strain A21038 or the probiotic agent in the preparation of products having at least one of antioxidant, anti-aging, antibacterial, and anti-tumor properties.

[0016] The present invention provides the use of Limosilactobacillus fermentum in the preparation of products having at least two of the following functions: antioxidant, anti-aging, antibacterial, and anti-tumor. The present invention respectively carried out antioxidant, anti-aging, antibacterial, and anti-tumor experiments on the Limosilactobacillus fermentum strain in vitro. Among them, the Limosilactobacillus fermentum strain has good inhibitory ability against the pathogenic bacteria Escherichia coli and / or Escherichia coli, indicating that the Limosilactobacillus fermentum strain has good antibacterial property. The present invention respectively uses DPPH radical, hydroxyl radical (OH -) Using DPPH free radicals, superoxide anions, and hydrogen peroxide (H2O2) as indicators to illustrate the antioxidant performance, the results show that the fermented Lactobacillus mucosae has a scavenging rate of more than 98% for DPPH free radicals, and can still grow after being cultured in 0.5 mM high-concentration H2O2 for 12 h, indicating good tolerance to H2O2 and the ability to play an antioxidant role. In the aspect of anti-aging detection, the results show that the fermented Lactobacillus mucosae has superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity, and the effect in delaying skin aging is equivalent to that of the commercial strain Lactobacillus rhamnosus GG. At the same time, using human colon cancer cells HCT116 and mouse colon cancer cells MC38 as experimental objects respectively, the anti-tumor effect of the fermented Lactobacillus mucosae was verified. The results show that both live bacteria and inactivated bacteria have the performance of inhibiting the proliferation of colon cancer cells, and the inhibitory effect is correlated with time. At the same time, using mouse melanoma cells B16 as the experimental object, the anti-tumor effect of the fermented Lactobacillus mucosae was verified, and the result is the same as that of colon cancer inhibition, and it also has the effect of inhibiting the growth of melanoma. Therefore, the present invention proves for the first time that the fermented Lactobacillus mucosae simultaneously has antioxidant, anti-aging, antibacterial, and anti-tumor activities, and has extremely high application value in the preparation of anti-tumor drugs, antibacterial drugs, and anti-aging and / or antioxidant drugs, health products, and cosmetics.

[0017] The present invention also provides a strain of Lactobacillus mucosae A21038, with the preservation number of GDMCC No: 64675. The strain A21038 was isolated from the intestinal fecal samples of centenarian healthy elderly people and belongs to Lactobacillus mucosae through morphological and molecular identification. The strain A21038 has a fast growth rate, has the ability to tolerate gastrointestinal fluids and bile salts, and can colonize the intestine through the gastrointestinal tract to play a role. The strain A21038 has a good inhibitory effect on Escherichia coli pathogenic bacteria, and at the same time has the ability to scavenge free radicals, is tolerant to high-concentration hydrogen peroxide, and has good antioxidant ability. In addition, the strain A21038 has superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity, and has the ability to delay aging. Furthermore, both live bacteria or inactivated bacteria of the strain A21038 have an inhibitory effect on the cell proliferation of colon cancer and melanoma. It can be seen that the strain A21038 simultaneously has antioxidant, anti-aging, antibacterial, and anti-tumor characteristics and has extremely high application value in industrial production. Description of the Drawings

[0018] Figure 1 It is the morphological diagram of the strain Lactobacillus mucosae A21038;

[0019] Figure 2 It is the growth curve diagram of the strain Lactobacillus mucosae A21038;

[0020] Figure 3 Figure showing the test results of the tolerance of Limosilactobacillus fermentum strain A21038 to artificial gastrointestinal fluids;

[0021] Figure 4 Figure showing the test results of the tolerance of Limosilactobacillus fermentum strain A21038 to bovine bile salts;

[0022] Figure 5 Figure showing the test results of the tolerance of Limosilactobacillus fermentum strain A21038 to H2O2;

[0023] Figure 6 Figure showing the test results of the inhibition of the growth of human colon cancer cell line HCT116 by Limosilactobacillus fermentum strain A21038;

[0024] Figure 7 Figure showing the test results of the inhibition of the growth of mouse colon cancer cell line MC38 by Limosilactobacillus fermentum strain A21038;

[0025] Figure 8 Figure showing the test results of the inhibition of the growth of mouse melanoma cell line B16 by Limosilactobacillus fermentum strain A21038.

[0026] Biological material preservation information

[0027] Limosilactobacillus fermentum strain A21038 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on May 23, 2024. The abbreviation of the depository is GDMCC, and the address is the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit number is GDMCC No: 64675. Detailed implementation manners

[0028] The present invention provides the application of Limosilactobacillus fermentum in the preparation of products with at least two of the following functions: antioxidant, anti-aging, antibacterial, and anti-tumor.

[0029] In the present invention, the Limosilactobacillus fermentum preferably includes bacterial cells and / or spores. The bacterial cells of Limosilactobacillus fermentum refer to the solid-phase bacterial cells collected after the activated Limosilactobacillus fermentum is subjected to enlarged culture and solid-liquid separation. The spores of Limosilactobacillus fermentum refer to a round or oval stress-resistant dormant body formed inside the cells after Limosilactobacillus fermentum grows and develops to a certain stage.

[0030] In the present invention, the existing form of the Lactobacillus mucosae fermentum preferably includes at least one of the following: the culture solution of Lactobacillus mucosae fermentum, the culture supernatant of Lactobacillus mucosae fermentum, and the bacterial suspension of Lactobacillus mucosae fermentum. The preparation method of the culture solution of Lactobacillus mucosae fermentum preferably inoculates the activated Lactobacillus mucosae fermentum into MRS liquid medium for culture to obtain the culture solution. The culture temperature is preferably 36-38°C, more preferably 37°C. The culture time is 12-16 h, more preferably 14 h. The inoculation amount of Lactobacillus mucosae fermentum is preferably 5%-10%, more preferably 8%. The preparation methods of the culture supernatant of Lactobacillus mucosae fermentum and the bacterial suspension of Lactobacillus mucosae fermentum preferably carry out solid-liquid separation on the culture solution prepared above, the liquid phase is the culture supernatant, and the solid phase is collected and resuspended to obtain the bacterial suspension.

[0031] In the present invention, the strain of Lactobacillus mucosae fermentum preferably includes Lactobacillus mucosae fermentum strain A21038. The preservation number of Lactobacillus mucosae fermentum strain A21038 is GDMCC No: 64675.

[0032] In the present invention, when preparing a product with two functions, the application preferably includes the application in preparing antioxidant and anti-aging products and / or the application in preparing antibacterial and anti-tumor products; or the application in preparing antioxidant and anti-tumor products and / or the application in preparing antibacterial and anti-aging products; or the application in preparing anti-aging and anti-tumor products and / or the application in preparing antioxidant and antibacterial products.

[0033] In the present invention, when preparing a product with three functions, the application preferably includes the application in preparing antioxidant, anti-aging and antibacterial products; or the application in preparing antioxidant, antibacterial and anti-tumor products; or the application in preparing anti-aging, antibacterial and anti-tumor products; or the application in preparing antioxidant, anti-aging and anti-tumor products. When preparing a product with four functions, the application preferably includes the application in preparing antioxidant, anti-aging, antibacterial and anti-tumor products.

[0034] In the present invention, the antioxidant property preferably manifests in the scavenging ability for at least one of the following: DPPH free radicals, hydroxyl free radicals, superoxide anions, and H2O2, more preferably DPPH free radicals and H2O2. The anti-aging property preferably manifests in having superoxide dismutase activity and / or glutathione peroxidase activity. When preparing antioxidant and / or anti-aging products, the products preferably include at least one of the following: pharmaceuticals, health products, cosmetics, and foods. The bacteria in the antibacterial property preferably include Escherichia coli. The tumors or cancers in the anti-tumor property preferably include colon cancer and / or melanoma. When preparing antibacterial and / or anti-tumor products, the products preferably include pharmaceuticals. When preparing antibacterial products, the products can also be used to prepare bacteriostatic agents for use under in vitro conditions. The present invention has no special restrictions on the method for preparing the products with the above functions, and the products can be prepared by using methods well-known in the art. For example, the culture solution, supernatant, or fermentation broth of fermented Lactobacillus mucosae is used as an active ingredient and mixed with excipients to prepare various forms of products.

[0035] The present invention provides a strain of Lactobacillus mucosae A21038 with a deposit number of GDMCC No: 64675.

[0036] In the present invention, the strain A21038 was isolated from the intestinal fecal samples of centenarian healthy elderly people. The colonies of the strain A21038 on the MRS plate are circular, with neat edges, opaque, convex in the middle, smooth on the surface, and light yellow, and it belongs to Gram-positive bacteria. The nucleotide sequence of the 16S rDNA of the strain A21038 is as shown in SEQ ID NO: 1. Through morphological identification and molecular identification, the strain A21038 belongs to Lactobacillus mucosae. From the growth curve of the strain A21038, it is found that the strain A21038 has the characteristic of fast growth rate, entering the logarithmic phase in 2 h, being in the stationary phase from 6 to 24 h, and no decline phase appears within 24 h.

[0037] In the present invention, when the strain A21038 is treated in artificial gastric juice for 3 h, the survival rate is 100%. When treated in artificial intestinal juice for 7 h, the survival rate remains above 70%, and the effect is better than that of the control strain A20014 screened in the same batch. The survival rate of the strain A21038 is 100% at 24 h in 0.5 g / L bile salt. The above results indicate that the strain A21038 has the ability to resist gastrointestinal fluids and bile salts and can colonize in the intestine through the gastrointestinal tract to play a role.

[0038] In the present invention, the strain A21038 has a good inhibitory effect on pathogenic Escherichia coli. The pathogenic Escherichia coli preferably includes Escherichia coli and / or Escherichia coli. In the embodiments of the present invention, Escherichia coli strain O157:H7 (ATCC 35150) and Escherichia coli (ATCC 25922) are used as representatives of pathogenic bacteria to illustrate the antibacterial characteristics of the strain A21038. The results show that the antibacterial zone of the strain A21038 against Escherichia coli strain O157:H7 is 21.33 mm, and the antibacterial effect is better than that of the control strain A20014 screened in the same batch; the antibacterial zone of the strain A21038 against Escherichia coli strain is 25.00 mm, and the antibacterial effect is equivalent to that of the control strain A20014 screened in the same batch.

[0039] In the present invention, the strain A21038 has a good scavenging ability for free radicals, and at the same time has tolerance to high-concentration hydrogen peroxide and good antioxidant ability. The free radicals preferably include DPPH free radicals, hydroxyl free radicals (OH - ) and superoxide anions (O2 - ). The DPPH free radical is a very stable nitrogen-centered free radical and is one of the important indicators of the antioxidant capacity of a sample. It is widely used in the research of antioxidant foods, health products and drugs. Hydroxyl free radicals (OH-) are a type of reactive oxygen species. Hydroxyl free radicals can kill red blood cells, degrade DNA, cell membranes and polysaccharide compounds. Subsequently, it was found that many harmful effects caused by it would be significantly reduced after adding scavengers of hydroxyl free radicals. Superoxide anions are also detection indicators of antioxidant properties. When an organism is subjected to external stress, a large amount of reactive oxygen species such as superoxide anions in the organism are generated and accumulated, which can be used as a signal of oxidative stress in the organism. Therefore, the generation of superoxide anion free radicals in the organism under adverse conditions can indirectly reflect the damage status of tissue cells and the strength of resistance. Hydrogen peroxide (H2O2) is the most common reactive oxygen molecule in the organism and is a by-product of reactive oxygen metabolism. It is mainly catalyzed by superoxide dismutase (SOD) and xanthine oxidase (XOD) and degraded by catalase (CAT) and active enzyme (POD). The concentration of H2O2 in the normal human body is at a relatively low level, generally 10-100 μM. High-concentration H2O2 will induce an increase in the oxidative stress response in the body, directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells, and damage the cell membrane, thereby accelerating cell aging and disintegration, producing toxic effects on human cells, and even causing abnormal cell functions and organ damage, leading to the occurrence of related diseases.

[0040] In one embodiment of the present invention, the clearance rate of the strain A21038 against DPPH is more than 98%, and the effect is significantly better than that of the control strain A20014 (15%). The clearance rate of the strain A21038 against hydroxyl radicals is 20%, and the effect is significantly better than that of the control strain A20014 (10%). The clearance rate of the strain A21038 against superoxide anions is 33%, and the effect is significantly better than that of the control strain A20014 (24%). In another embodiment of the present invention, the tolerance of the strain A21038 to high-concentration hydrogen peroxide was measured. The strain A21038 can still grow after being treated in 0.5 mM H2O2 for 12 h, and has a good tolerance to H2O2, while the control strain A20014 cannot reproduce under the same conditions and has no tolerance to H2O2.

[0041] In the present invention, the strain A21038 has superoxide dismutase activity and glutathione peroxidase (GSH-Px) activity, and has the ability to delay aging. Superoxide dismutase can catalyze the dismutation of superoxide anions to generate hydrogen peroxide (H2O2) and oxygen (O2). It is an important antioxidant enzyme in organisms and plays a role in preventing skin aging and damage. Glutathione peroxidase specifically uses glutathione (GSH) to reduce peroxides, especially hydrogen peroxide. The glutathione is composed of glutamic acid, cysteine and glycine, and has antioxidant and integrative detoxification effects. Glutathione can not only be used in medical drugs, but also has the whitening effect of reducing melanin growth and delaying aging. In one embodiment of the present invention, it is proved that the strain A21038 has the ability to secrete superoxide dismutase and glutathione peroxidase. After measurement, the activity of superoxide dismutase is 0.27 U / 10 4 CFU, and the activity of glutathione peroxidase is 0.77 nmol / min / mL.

[0042] In the present invention, both the live bacteria and inactivated bacteria of the strain A21038 have inhibitory effects on the cell proliferation of colon cancer and melanoma. The preferred preparation method of the inactivated bacteria of the strain A21038 is to treat the bacteria at 70 °C for 30 min. In one embodiment of the present invention, the live bacteria of the strain A21038 are superior to the inactivated bacteria in inhibiting the growth of tumor cells, and the same result exists in the commercial strain LGG.

[0043] The present invention provides a probiotic agent, and the active ingredient includes the fermented Lactobacillus mucosae strain A21038.

[0044] In the present invention, the probiotic agent refers to supplementing beneficial bacteria in the intestine, enabling the beneficial bacteria to have a sufficient quantity and exerting unique biological effects, thereby achieving the purpose of health care or disease prevention and treatment. The viable cell concentration of the fermented Lactobacillus mucosae strain A21038 is preferably 1×10 9 CFU / mg to 1×10 11 CFU / mg or 1×10 9 CFU / mL to 1×10 11 CFU / mL, more preferably 5×10 9 CFU / mg to 5×10 10 CFU / mg or 5×10 9 CFU / mL to 5×10 10 CFU / mL, still more preferably 8×10 9 CFU / mg to 2×10 11 CFU / mg or 8×10 9 CFU / mL to 2×10 11 CFU / mL, most preferably 1×10 10 CFU / mg or 1×10 10 CFU / mL. The dosage form of the probiotic agent is preferably at least one of the following: tablets, drops, capsules, and powders. The powder is processed by low-temperature freeze-drying technology, which ensures the activity of the probiotics. The excipients of the powder preferably include cryoprotectants, such as skimmed milk powder, etc. The powder is suitable for taking with warm water and is suitable for infants. The tablets are prepared by mixing the fermented Lactobacillus mucosae strain A21038 and excipients and tabletting, and are suitable for chewing. The excipients preferably include sweeteners or cocoa powder, etc. The capsules are preferably prepared by filling the fermented Lactobacillus mucosae strain A21038 alone or in combination with excipients as the core into the capsule shell. The drops are prepared by mixing the fermented Lactobacillus mucosae strain A21038 with edible oil to form drops that isolate air and moisture. When taking, open the dropper mouth and squeeze the encapsulated oily substance into the mouth.

[0045] In view of the multiple functions of the fermented Lactobacillus mucosae strain A21038, the present invention provides the use of the fermented Lactobacillus mucosae strain A21038 or the probiotic agent in the preparation of a product having at least one of antioxidant, anti-aging, antibacterial, and anti-tumor properties.

[0046] The present invention does not impose special restrictions on the specific types of the products, and the product types with well-known specific effects in the art can be used.

[0047] The following examples are used to illustrate in detail a fermented Lactobacillus mucosae strain A21038 having antioxidant, anti-aging, and anti-tumor activities and its application provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0048] Example 1

[0049] Isolation and Identification of Lactobacillus mucosae Strain A21038

[0050] The intestinal fecal samples of centenarian healthy elderly were serially diluted and spread on MRS plates, and cultured in a biochemical incubator at 37 °C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium, and cultured on a shaker at 37 °C for 12 - 16 h. The bacterial solution was streaked on MRS plates, and single colonies were continuously cultured. After 24 h, single colonies were picked again and streaked, and single colonies were continuously cultured, which was the obtained pure isolated Lactobacillus. Single colonies of the pure isolated Lactobacillus were picked, subjected to Gram staining, 16S rDNA PCR amplification and 16S rDNA sequencing, and the results were compared with the NCBI database.

[0051] The growth state of strain A21038 in MRS plates is as Figure 1 shown. The colonies are round, with neat edges, opaque, convex in the middle, smooth on the surface, light yellow, and belong to Gram-positive bacteria.

[0052]

[0053] Example 2

[0054] Growth curve of Lactobacillus mucosae fermentum strain A21038

[0055] Take out the strain from the glycerol preservation tube, streak it on the MRS solid plate, place it in an incubator at 37 °C for cultivation. After 24 h, pick a single colony and culture it in 2 ml of MRS liquid medium for 12 - 16 h. Repeat this step once to fully activate the strain. Then, pipette 0.2 mL of the bacterial suspension into a 96-well plate, make 3 replicate wells, and use a microplate reader to detect its growth within 24 h. The detection wavelength is 600 nm. After the measurement, perform statistics and plot the graph.

[0056] Figure 2 The growth curve of Lactobacillus mucosae fermentum A21038 is shown. Strain A21038 has a relatively fast growth rate. It enters the logarithmic growth phase after 2 h, and the stable phase is from 6 - 24 h. The growth of this bacterium is stable and there is no decline phase within 24 h.

[0057] Example 3

[0058] Function of Lactobacillus mucosae fermentum strain A21038 against artificial gastrointestinal fluid

[0059] After thawing the glycerol preservation tubes of strain A21038 and another strain of Lactobacillus mucosae fermentum A20014 isolated from the feces of centenarians (used as a control strain) respectively, streak them on the MRS solid medium plate and place them in an incubator at 37 °C for overnight cultivation. After 24 h, pick single colony inoculations and inoculate them into MRS liquid medium, and culture them on a shaker at 37 °C for 12 - 16 h. Repeat this step once to fully activate the strain to obtain the bacterial liquid. According to an inoculation amount of 10%, inoculate the bacterial liquid into artificial gastric juice with a pH of 2.5 (formula: 10 g of pepsin, 16.4 ml of hydrochloric acid, made up to 100 ml with distilled water), set 3 time points (0 h, 1 h, 3 h), take 100 μl each and spread it on the MRS petri dish, and culture it in a biochemical incubator at 37 °C for 48 h, then count the number of single colonies; inoculate the bacterial liquid after being treated with artificial gastric juice for 3 h into artificial intestinal juice with a pH of 6.8 (formula: 10 g of pancreatin, 6.8 g of potassium dihydrogen phosphate, made up to 1000 ml with distilled water, adjust the pH value with NaOH) according to an inoculation amount of 10%, set 4 time points (0 h, 2 h, 4 h, 8 h), take 100 μl each and spread it on the MRS petri dish, and culture it in a biochemical incubator at 37 °C for 48 h, then count the number of single colonies, and calculate the survival rate according to formula I.

[0060] Survival rate (%) = (Number of single colonies at different time points / Number of single colonies at 0 h) × 100% Formula I

[0061] The results are as Figure 3The results showed that the survival rate of the control strain A20014 in artificial gastric juice at pH 2.5 reached 100%, while its survival rate in intestinal juice decreased with the prolongation of time, and the survival rate was less than 20% at 7 h in intestinal juice. Compared with the control strain A20014, the survival rate of the strain A21038 reached 100% at 3 h in artificial gastric juice, and still maintained a survival rate of more than 70% after 7 h in intestinal juice, which was significantly better than that of the control strain A20014. It indicated that the strain A21038 had excellent gastrointestinal tolerance and could reach the intestine through the stimulation of gastrointestinal juice to play a role.

[0062] Example 4

[0063] Determination of the ability of Lactobacillus mucosae A21038 to tolerate bile salts

[0064] After thawing the glycerol preservation tubes of the strain A21038 and other strains of Lactobacillus mucosae A20014 (used as the control strain) isolated from the feces of centenarians respectively, streak plating was carried out on MRS solid medium and placed in an incubator at 37 °C for overnight culture. After 24 h, single colony colonies were picked and inoculated into MRS liquid medium respectively, and cultured on a shaker at 37 °C for 12 - 16 h. This step was repeated once to fully activate the strain and obtain bacterial solutions. 1 mL was taken and added to MRS medium containing 0.3 g / L or 0.5 g / L bile salts respectively, and anaerobically cultured at 37 °C for 18 h. 100 μL of the culture solutions at 0 h, 2 h, and 24 h after addition were taken for gradient dilution coating respectively, and cultured under anaerobic conditions at 37 °C for 1 - 2 days, and the viable bacteria count was statistically analyzed. The bile salt survival rate of the strain was calculated according to formula II:

[0065] Bile salt survival rate = (Number of viable bacteria at N h / Number of viable bacteria at 0 h) × 100% Formula II

[0066] Among them, N represents 2 h or 24 h.

[0067] The results were as Figure 4 shown that compared with the control strain A20014, the survival rate of the strain A21038 was still more than 100% at 24 h in bile salts with a concentration of 0.5 g / L, indicating that the strain A21038 had excellent bile salt tolerance.

[0068] Example 5

[0069] Function of Lactobacillus mucosae strain A21038 in inhibiting pathogenic bacteria

[0070] 0.1 mL of the pathogenic bacteria Escherichia coli O157:H7 (ATCC35150) and Escherichia coli (ATCC 25922) in the logarithmic growth phase (concentration of 1 × 10 9 CFU / mL) was taken and spread on an LB plate, and Oxford cups were placed.

[0071] According to the method for activating strains in Example 2, an activated bacterial suspension was obtained, and the OD 600 was adjusted to 0.5 (concentration: 1×10 6 CFU / mL). 200 μl of the bacterial suspension was taken and inoculated into Oxford cups, with 3 replicates, incubated overnight, and its effect on pathogenic bacteria was observed. The method for counting the size of the inhibition zone was to measure the size of the inhibition zone with a ruler and take the average value of the 3 replicates. The results are shown in Table 1 below.

[0072] Table 1 Detection results of strain inhibition of pathogenic bacteria

[0073]

[0074] The results showed that the fermented Lactobacillus mucosae strain A21038 had the ability to significantly inhibit the growth of pathogenic bacteria, and was superior to the control strain A20014.

[0075] Example 6

[0076] Antioxidant function of fermented Lactobacillus mucosae strain A21038

[0077] According to the method for activating strain A21038 in Example 2, an activated bacterial suspension and a bacterial suspension of the control strain A20014 were obtained. The OD of the bacterial suspension was adjusted 600 to 0.5 (concentration: 1×10 6 CFU / mL) and reserved for use.

[0078] When there is an antioxidant present, DPPH free radicals are scavenged, the color of its solution becomes lighter, and the absorbance at 515 nm decreases. Within a certain range, the change in absorbance is proportional to the degree of free radical scavenging. The ability of the samples to scavenge DPPH free radicals was reflected by the degree of decrease in absorbance. The two strains were detected according to the instructions of the DPPH free radical scavenging ability kit (Jianglai Biotech).

[0079] 2-Deoxyribose is oxidized to a malondialdehyde analogue in the presence of hydroxyl radicals (OH - ), and then condenses with thiobarbituric acid (TBA) to form a colored product. By measuring the maximum absorption peak of the colored product at 532 nm, the content of hydroxyl radicals (OH - ) can be calculated. The two strains were detected according to the instructions of the kit for measuring the content of hydroxyl radicals (OH - ) (Jianglai Biotech).

[0080] Superoxide anions (O2 - ) react with hydroxylamine to produce NO2 － , and NO2 －In the presence of sulfanilic acid and α-naphthylamine, a pink azo dye is formed. This dye has a maximum light absorption at 540 nm, and the content of O2 in the sample can be calculated based on the A540 value. - The content of - - was detected for the two strains according to the instructions of the superoxide anion (O2

[0081] ) kit (Grees Biological).

[0082] The detection results are shown in Table 2.

[0083]

[0084] The results showed that the fermented Lactobacillus mucosae strain A21038 had good antioxidant ability and was significantly superior to the control strain A20014.

[0085] Example 7

[0086] Determination of the tolerance of fermented Lactobacillus mucosae strain A21038 to H2O2

[0087] Hydrogen peroxide (H2O2) is the most common reactive oxygen molecule in living organisms and is a by-product of reactive oxygen metabolism. It is mainly catalyzed by SOD and XOD and degraded by CAT and POD. In normal human bodies, the concentration of H2O2 is at a relatively low level, generally 10 - 100 μM. High concentrations of H2O2 can induce an increase in oxidative stress reactions in the body, directly or indirectly oxidize biological macromolecules such as nucleic acids and proteins in cells, and damage cell membranes, thereby accelerating cell aging and disintegration, having a toxic effect on human cells, and even causing abnormal cell functions and organ damage, leading to the occurrence of related diseases.

[0088] The strain A21038 and the control strain A20014 were taken out from the glycerol preservation tube and streaked and activated 3 times. Single colonies were picked and cultured in MRS liquid medium. Different concentrations of H2O2, namely 0.5 mM, 1.0 mM, and 2.0 mM, were added, and their growth was detected using a microplate reader. After the determination, a graph was plotted.

[0089] The results are as Figure 5 shown. Fermented Lactobacillus mucosae A21038 ( Figure 5 A in it) could still grow after 12 h in 0.5 mM high-concentration H2O2 and had good tolerance to H2O2, while the control strain A20014 could not reproduce under the same conditions ( Figure 5 B in it) and had no tolerance to H2O2. This indicates that the strain A21038 can still grow stably and play an antioxidant role in a highly oxidative stress environment in the body.

[0090] Example 8

[0091] Anti-aging function of Lactobacillus mucosae fermentum strain A21038

[0092] Superoxide dismutase (SOD) can catalyze the disproportionation of superoxide anions to generate hydrogen peroxide (H2O2) and oxygen (O2). It is an important antioxidant enzyme in organisms and plays a role in preventing skin aging and damage. Glutathione (GSH) is composed of glutamate, cysteine, and glycine, and has antioxidant and integrative detoxification effects. Glutathione can not only be used in medical drugs, but also has the whitening effect of reducing melanin growth and delaying aging.

[0093] The commercial strain Lactobacillus rhamnosus strain LGG, as a beneficial bacterium in the human intestine, can enhance the body's immunity, improve gastrointestinal function, and delay the process of aging of the body. According to the research results of the above embodiments, the performance of strain A21038 of the present invention is significantly better than that of the control strain A20014 of the same genus. Therefore, in the following embodiments, LGG is selected as the control strain to study the anti-aging function of strain A21038 of the present invention.

[0094] (1) Detection of superoxide dismutase (SOD) activity of strain A21038

[0095] Adjust the bacterial liquid concentrations of strain A21038 and the commercial strain Lactobacillus rhamnosus LGG to 5×10 6 CFU / mL respectively, centrifuge to discard the supernatant, and determine the SOD activity of the strains according to the operation procedure of the SOD activity detection kit (Jianglai Bio). The result is defined as the amount of SOD corresponding to 50% SOD inhibition rate in the reaction system per 10 4 CFU single colony as one SOD activity unit.

[0096] (2) Detection of glutathione peroxidase (GSH-Px) activity of strain A21038

[0097] Adjust the bacterial liquid concentrations of strain A21038 and the control strain LGG to 5×10 6 CFU / mL respectively, centrifuge to discard the supernatant, and determine the GSH-Px content in the strains according to the operation procedure of the GSH-Px detection kit (provided by Grees Biological). The result is defined as 1 enzyme activity unit for every 10 4 cells oxidizing 1 nmol GSH per minute under the reaction condition of 25°C.

[0098] The enzyme activities contained in Lactobacillus mucosae fermentum A21038 are shown in Table 3 below.

[0099] Table 3 Detection Results of Enzyme Activity

[0100] Strain <![CDATA[SOD activity (U / 10 4 CFU)]]> GSH-Px enzyme activity (nmol / min / mL) A21038 0.27 0.77 LGG 0.51 0.77

[0101] The results showed that the fermented Lactobacillus mucosae strain A21038 had good glutathione peroxidase activity and could play a role in delaying skin aging, and its effect was comparable to that of the control strain LGG.

[0102] Example 9

[0103] Inhibitory Effect of Fermented Lactobacillus mucosae Strain A21038 on Human Colon Cancer Cell Line HCT116

[0104] The HCT116 cell line was inoculated in RPMI1640 medium (TransGen) containing 10% fetal bovine serum and cultured. When the cells grew to the logarithmic phase, they were digested with trypsin and counted by a hemocytometer. The cells were evenly inoculated into a 96-well plate at 5000 cells per well and cultured overnight in a 37°C triple-gas incubator.

[0105] It has been reported in the prior art that the commercial strain Lactobacillus rhamnosus LGG has antitumor effects. Therefore, the LGG strain was selected as the positive control for this example. The strain A21038 and the control strain LGG were taken out from the glycerol preservation tube, streaked on an MRS solid plate, and cultured in a 37°C incubator. After 24 h, single colonies were picked and cultured in 2 ml of MRS liquid medium for 12 - 16 h. This step was repeated once to fully activate the strain and obtain a bacterial suspension. The OD of the bacterial suspension was adjusted 600 = 0.5, and the concentration was 1×10 6 CFU / mL. After washing 3 times with PBS, they were divided into live bacteria and heat-inactivated bacteria (70°C for 30 min). 0.2 mL of the bacterial suspension was added to the 96-well plate in sequence. The live bacteria were co-incubated with the cells for 2 h, and the heat-inactivated bacteria were co-incubated with the cells until 72 h after inactivation. The CCK-8 reagent (Solarbio) was used to detect the proliferation of HCT116 cells.

[0106] The results were as Figure 6 shown. The live bacteria of the strain A21038 could significantly inhibit the proliferation of HCT116 cells (P < 0.0001), and its effect was comparable to that of the control strain LGG. The strain A21038 showed a significant inhibitory effect within 48 h after inactivation (P < 0.001), and was superior to the control strain LGG. The effect after strain inactivation was significantly weaker than that of live bacteria. The reason why the heat-inactivated bacteria could play a potential role in anti-tumor cell growth might be that the inactivated bacterial proteins played a role, which remained to be further studied.

[0107] Example 10

[0108] Inhibitory effect of Lactobacillus mucosae fermentum strain A21038 on mouse colon cancer cell line MC38

[0109] The MC38 cell line was cultured in DMEM 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 seeded into 96-well plates at 5000 cells per well and cultured overnight in a 37°C triple-gas incubator.

[0110] The strain A21038 and the control strain LGG were taken out from the glycerol preservation tubes respectively, streaked on MRS solid plates, and cultured in a 37°C incubator. After 24 h, single colonies were picked and cultured in 2 ml of MRS liquid medium for 12 - 16 h. This step was repeated once to fully activate the strains and obtain bacterial suspensions. The OD of the bacterial suspensions was adjusted 600 = 0.5, and the concentration was 1×10 6 CFU / mL. After washing 3 times with PBS, they were divided into live bacteria and heat-inactivated bacteria (70°C for 30 min). 0.2 mL of the bacterial suspension was added to each well of the 96-well plates in sequence and co-incubated with the cells for 24 h, 48 h, and 72 h respectively. The CCK-8 reagent (Solarbio) was used to detect the proliferation of MC38 cells.

[0111] The results were as Figure 7 shown. Both the strain A21038 and the commercial strain LGG could significantly inhibit the proliferation of colon cancer cells. Moreover, as the time increased, the inhibitory effect of live bacteria was more significant (P < 0.0001). The heat-inactivated strain A21038 reached the semi-inhibitory effect at 72 h, which was significantly weaker than that of live bacteria. Therefore, it was speculated that the potential anti-colon cancer cell growth ability of the strain A21038 might mainly depend on the live bacteria themselves or their metabolites, and the role played by the inactivated bacterial cells was weaker.

[0112] Example 11

[0113] Inhibitory effect of Lactobacillus mucosae fermentum strain A21038 on mouse melanoma cell line B16

[0114] The B16 cell line was cultured in DMEM 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 seeded into 96-well plates at 5000 cells per well and cultured overnight in a 37°C triple-gas incubator.

[0115] Take out the strain A21038 and the control strain LGG from the glycerol preservation tube, streak them on the MRS solid plate, and place them in an incubator at 37°C for cultivation. After 24 hours, pick monoclonal colonies and culture them in 2 ml of MRS liquid medium for 12 - 16 hours. Repeat this step once to fully activate the strain and obtain a bacterial suspension. Adjust the OD of the bacterial suspension 600 to 0.5 (concentration is 1×10 6 CFU / mL). After washing 3 times with PBS, divide it into live bacteria and heat-inactivated bacteria (70°C for 30 minutes). Add 0.2 mL of the bacterial suspension to each well of a 96-well plate in sequence and co-incubate with cells for 24 hours. Use the CCK-8 reagent (Solarbio) to detect cell proliferation.

[0116] The results are as Figure 8 shown. The strain A21038 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 A21038 or the commercial strain LGG, the effect after inactivation is weaker than that of live bacteria. Therefore, it is speculated that the potential anti-melanoma cell growth ability of the strain A21038 may depend on the live bacteria themselves or the metabolites they produce, and the role played by the inactivated bacterial cells is weaker.

[0117] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A fermented mucus lactobacillus ( Limosilactobacillus fermentum ) strain A21038, characterized in that The deposit number is GDMCC No:64675.

2. A probiotic, characterized in that: The active ingredient comprises the fermented mucus lactobacillus strain A21038 according to claim 1.

3. Use of the fermented Lactobacillus mucosa strain A21038 according to claim 1 or the probiotic according to claim 2 in the preparation of antibacterial and / or anti-tumor drugs; The bacteria in the antibacterial property is Escherichia coli; The tumor or cancer in the anti-tumor agent is colon cancer and / or melanoma.

4. Use of the fermented Lactobacillus mucilaginosus strain A21038 according to claim 1 or the probiotic according to claim 2 in the preparation of antibacterial and anti-tumor drugs or products having at least one of the following functions: anti-oxidation and anti-aging; The bacteria in the antibacterial property is Escherichia coli; The tumor or cancer in the anti-tumor agent is colon cancer and / or melanoma.

5. The application according to claim 4, characterized in that: The antioxidant is manifested in the ability to scavenge at least one of the following: DPPH free radical, hydroxyl free radical, superoxide anion and H2O2.

6. The use according to claim 4, characterized in that: The anti-aging effect is manifested in having superoxide dismutase activity and / or glutathione peroxidase activity.

7. The use according to claim 4, characterized in that: When preparing an antioxidant product, the product comprises at least one of the following: medicine, health care product and cosmetic.

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